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Rethinking Biosynthesis of Aclacinomycin A

Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Author to whom correspondence should be addressed.
Molecules 2023, 28(6), 2761;
Submission received: 4 February 2023 / Revised: 1 March 2023 / Accepted: 6 March 2023 / Published: 18 March 2023


Aclacinomycin A (ACM-A) is an anthracycline antitumor agent widely used in clinical practice. The current industrial production of ACM-A relies primarily on chemical synthesis and microbial fermentation. However, chemical synthesis involves multiple reactions which give rise to high production costs and environmental pollution. Microbial fermentation is a sustainable strategy, yet the current fermentation yield is too low to satisfy market demand. Hence, strain improvement is highly desirable, and tremendous endeavors have been made to decipher biosynthesis pathways and modify key enzymes. In this review, we comprehensively describe the reported biosynthesis pathways, key enzymes, and, especially, catalytic mechanisms. In addition, we come up with strategies to uncover unknown enzymes and improve the activities of rate-limiting enzymes. Overall, this review aims to provide valuable insights for complete biosynthesis of ACM-A.

1. Introduction

Cancer poses a severe threat to humans, and its resulting economic burden may increase in the future [1,2]. Although cancer incidence and fatality rates may recede due to early diagnosis, the treatment cost remains unaffordable for most patients [3]. To address this issue, researchers are gearing up to develop efficient antitumor medicines, and impressive advancements have been achieved in this regard. Extant antitumor medicines are roughly divided into eight categories, including alkylating agents [4], hormone antagonists [5], antimetabolites, cytotoxic antibiotics [6], plant alkaloids [7], platinum compounds [8], protein kinase inhibitors [9], and monoclonal antibodies [10]. Belonging to cytotoxic antibiotics, anthracycline antitumor agents have been widely used for the clinical treatment of cancer for half a century. Of the reported anthracycline antitumor agents, aclacinomycin (ACM), also known as aclarubicin (ACL), is actually the mixture of ACM-A, ACM-B, and ACM-Y, typically synthesized by Streptomyces galilaeus ATCC 31133 [11,12]. ACM-A manifests a long side-chain structure, in which three sugar residues, including L-rhodosamine, 2-deoxyfucose, and L-cinerulose A, are tethered to aklavinone. As an anticancer medicine used in Japan and China, ACM-A exhibits remarkable inhibitory activities against leukemias, gastric cancer, and lung cancer [13,14,15,16]. In principle, anthracyclines exhibit antitumor activity due to the targeting of the minor groove of DNA to form a stable complex [17] (Figure 1). This reversible targeting can repress topoisomerase II and therefore hamper the transcription and replication of DNA in this complex. Consequently, double-stranded DNA unwinds and the accumulated DNA fragments, triggering programmed cell death [18]. ACM-A was also found to evict histones and damage chromatin [19,20]. Compared to doxorubicin (DXR) and daunorubicin (DNR), the two anthracyclines isolated from Streptomyces peucetius ATCC 27952, ACM-A exhibits higher antitumor activity but much lower cardiotoxicity [21].
The chemical synthesis of ACM-A is challenging due to its bulky and complex structure. Aklavinone is an important member of tetrahydronaphthol whose benzylic hydroxy group can be easily eliminated. In previously reported synthesis, Diels–Alder reactions were found to be crucial for the synthesis of aklavinone, which raised the issue of regioselectivity [22,23]. It was reported that refinement led to only a 68% yield ratio of oligosaccharides in ACM-A [24,25]. Clearly, high stereoselectivity is desired, but the modulation of stereoselectivity is laborious and troublesome [26]. Although strategies have been developed (e.g., donor-controlled stereoselectivity), the multi-step syntheses of ACM-A is tedious and seems to be inevitable [27,28]. In addition, the organic synthesis of ACM-A shows shortcomings such as the need for toxic catalysts, low yield, and severe pollution [29]. In contrast, the bioproduction of ACM-A shows merits such as low cost, easy manipulation, and less environmental pollution. ACM-A is mainly obtained by the fermentation of Streptomyces sp.NO.MA144-MI ATCC 31133 or its mutants. Of reported Sreptomyces species, Streptomyces lavendofoliae is the most employed for the industrial production of ACM-A [30]. Since ACM-A is a secondary metabolite and indirectly produced from the core metabolic pathways in Streptomyces, its production is therefore low and needs to be substantially enhanced. Synthetic biology and metabolic engineering raise the hope for the high-level production of ACM-A based on rational design and the subsequent construction of biological systems. In this review, we not only outline the reported biosynthetic pathways of ACM-A, but also discuss the catalytic mechanisms of key enzymes. Furthermore, we come up with protocols for the overproduction of this economically important medicine.

2. Biosynthesis of ACM-A

The history of deciphering ACM-A biosynthesis dates back to the 1970s. In 1975, ACM-A and ACM-B were first isolated from the fermentation broth of S. galilaeus MA144-M1(ATCC 31133) [11]. In 1979, ACM-A was extracted using organic solvents. After undergoing silicic acid column chromatography, the extracts from the fermentation broth were condensed into crystal or microcrystalline powder [31]. In 1981, partial biosynthesis pathways of ACM-A were identified through the genetic loss of glycosylation and isotope labeling [32]. Afterwards, the polyketide synthase (PKS) genes from S. galilaeus ATCC 31615 were cloned and sequenced [33]. So far, a partial ACM-A pathway has been unraveled (Acc. NO. AF264025.1; Acc. NO. AF257324.2) (Figure 2). With one molecule of propionyl-CoA as a starter unit and nine molecules of malonyl-CoA as extender units, the type Ⅱ PKS catalyzes the formation of aklavinone through nine cycles of reactions, involving the incorporation of malonyl-CoA, cyclization, and oxidation. ACM-A is generated upon specific glycosylation and modification (Figure 3) [34,35,36]. ACM-A can be converted to ACM-Y by oxidoreductase [36]. The oxidoreductase located in cytoplasm catalyzes ACM-A to ACM-B, while the oxidoreductase in periplasm catalyzes ACM-B to ACM-A [37,38].

2.1. Gene Cluster for Biosynthesis of Aklavinone in S. galilaeus

Although anthracyclines vary in structure, their biosynthesis pathways are similar. The iterative Claisen condensation reaction is accomplished by type Ⅱ PKSs which are encoded by a gene cluster in the Streptomyces genome. In this cluster, the polyketide assembly requires a minimal set of iteratively used enzymes. This minimal PKS (min PKS) consists of a ketosynthase (KSα), a chain length factor (CLF or KSβ), and an acyl carrier protein (ACP) [39]. In general, KSα shows high sequence similarity to KSβ [40]. However, KSβ is catalytically inactive due to the lack of active-site cysteine. Instead, KSβ carries a highly conserved glutamine [41]. The malonyl-CoA: ACP acyltransferase (MAT) activity is usually crucial for PKS [42]. The biosynthesis of polyketide starts with iterative action of the min PKS complex to form poly-β-ketone intermediates, which are subsequently folded into distinct aromatic compounds by ketoreductases (KR), cyclases (CYC), and aromatases (ARO) [43].
Anthracyclines are assembled by tetracyclic aglycones, 7,8,9,10-tetrahydro-5,12-naphthacene-quinone, and various L-deoxysugar moieties. Aklavinone is the most common aglycones for the biosynthesis of anthracyclines. Sequence analysis reveals that a total of 12 genes participate in the biosynthesis of aklavinone in S. galilaeus ATCC 31615 (Table 1). Briefly, one molecule of propionyl-CoA and nine molecules of malonyl-CoAs are catalyzed into one molecule of 21-carbon decaketide nascent chain (15) by min PKS which contains AknB (KSα, EC:2.3.1.-), AknC (KSβ, EC:2.3.1.-), AknD (ACP), AknE2 (EC:4.2.1.-), and AknF (malonyl-CoA: ACP transacylase, MAT) [33]. The growing polyketide chain accepts one 2-carbon unit from one molecule of malonyl-CoA. The interactions between AknD, AknE2, and AknF are essential for the choosing of the starter unit [44]. Only when all aforementioned enzymes are available is propionyl-CoA then chosen as the starter unit. AknA (EC1.1.1-) and AknE1(EC:4.2.1.-) are NADPH-dependent reductase and aromatase, respectively, which jointly convert the linear skeleton to 12-deoxyaklanoate (17) [45]. The cyclization of the second and third ring is catalyzed by AknW, leading to the formation of 12-deoxyalkalonic acid (18) [33,46]. This intermediate is subsequently converted to aklanonate (19) by mono-oyxgenase AknX-mediated oxidation. This mono-oyxgenase AknX (EC1.13.12.22) was successfully purified and biochemically verified [47]. Native AknX was suggested to manifest homotrimeric subunit structure [47]. Aklanonate (19) is then catalyzed into methyl aklanonate (20) by aknG-encoded (formerly acmA) acid methyltransferase (EC2.1.1.288) [48,49]. AknG is a SAM-dependent O-methyltransferase capable of transferring a methyl group from the donor to receptor using the ubiquitous methyl donor SAM as the cosubstrate. Aklaviketone (21) is formed by the closure of the fourth ring, which is catalyzed by cyclase AknH (EC5.5.1.23) [50,51]. This process is designated as intramolecular aldol condensation [52]. Lastly, reductase AknU (EC1.1.1.362) converts the 7-oxo moiety of aklaviketone into a hydroxyl group to form aklavinone (22) [48,53,54].
The structure analysis of PKSs not only reveals substrate specificity and protein interaction but also provides insights for combinatorial biosynthesis of derivatives based on tailored genes. However, it is challenging to determine crystal structures [44]. Of the PKSs participating in ACM-A biosynthesis, only AknH has been investigated for its structure [50]. AknH shares 61.91% identity with SnoaL, which catalyzes the ring closure in the biosynthesis of nogalamycin [52]. In addition, AknH (PDB: 2F98) forms a tetramer with 222 symmetry, like SnoaL (PDB: 1SJW) (Figure 4A). Both AknH and SnoaL utilize the same substrate due to highly similar crystal structures, but their catalytic products are different due to the difference in stereoselectivity (Figure 4B). The stereoselectivity of AknH is mainly ascribed to amino acid residues Tyr15 and Asn51, whose counterparts in SnoaL are phenylalanine and leucine [50].

2.2. Biosynthesis of TDP-Sugars

In the biosynthesis of antibiotics, sugar donors are tethered to aglycones, which are catalyzed by glycosyltransferases (GTs). This process is crucial for the pharmacological and pharmacokinetic properties of medicines [55,56]. By the introduction of amino groups, the modified sugars provide hydrogen bonds to the functional groups of protein or DNA targets [57]. Dedicated glycosylation leads to the formation of products with diverse recognition elements, and sugar conjugation usually improves the solubility, chemical stability, and polarity of aglycone [57].
The sugar moieties biosynthesis of ACM-A in S. galilaeus ATCC 31615 is mainly executed by a gene cluster consisting of aknY, aknR, aknN, aknQ, aknP, aknZ, aknL, aknM, and aknX2 (Figure 3A) [58]. In other Streptomyces species, the biosynthesis of dTDP-deoxyfucose (11) and dTDP-rhodinose (14) of ACM-A is accomplished by dnm or spn clusters (Table 2) [59,60]. For sugar moieties biosynthesis in S. galilaeus, thymidylyltransferase converts glucose-1-phosphate (1) to TDP-D-glucose (2), which is subsequently converted to TDP-4-keto-6-deoxy-D-glucose (3) by TDP-D-glucose 4,6-dehydratase. The two steps are likely catalyzed by AknY (EC2.7.7.24) and AknR (EC4.2.1.46), respectively. AknN (EC4.2.1-) is a 2,3-dehydratase catalyzing the corresponding C-2 deoxygenation into TDP-3,4-diketo-2,6-dideoxy-D-glucose (4). AknZ is a putative 3-aminotransferase which transfers the amino group to the C-3 position, thereby generating TDP-3-amino-4-keto-2,3,6-trideoxy-D-glucose (5). AknL is proposed to show dTDP-4-dehydrorhamnose 3,5-epimerase activity and catalyze TDP-3-amino-4-keto-2,3,6-trideoxy-D-glucose into TDP-3-amino-4-keto-6-deoxy-L-glucose (6). AknM (EC4.2.1.164) may serve as an aknM-encoded 4-ketoreductase catalyzing C-4 ketone into TDP-L-daunosamine (7). AknX2 is an N-methyltransferase which transfers the methyl group at C-3 amine to TDP-rhodosamine (8) [61]. AknQ (EC1.1.1.384) is a putative 3-ketoreductase catalyzing the C-2 deoxygenation of TDP-3,4-diketo-2,6-dideoxy-D-glucose and thereby the generation of TDP-4-keto-2,6-dideoxy-D-glucose (9), which is then converted into TDP-4-keto-2,3,6-trideoxy-D-glucose (12) by 3-dehydratase AknP (EC4.2.1.164) [60]. d-TDP-rhodinose (14) is presumably synthesized from TDP-4-keto-2,3,6-trideoxy-D-glucose through two steps catalyzed by dTDP-4-dehydrorhamnose 3,5-epimerase and 4-keto reductase. TDP-4-keto-2,6-dideoxy-D-glucose is probably converted into dTDP-4-keto-2,6-dideoxy-beta-L-galactose by 3,5-epimerase. Unfortunately, the genes encoding these enzymes in S. galilaeus have not been completely elucidated.

2.3. Transfer of TDP-Sugars

GTs transfer glycosylated moieties from activated sugar donors to receptors. Such enzymes effectively mediate the formation of glycosidic linkage of natural oligosaccharides, glycoconjugates, and their analogues in a stereospecific and regiospecific way [62]. In view of sequence similarity and conserved motifs, the reported GTs can be classified into a total of 114 families which are deposited in the Carbohydrate-Active enzyme database (CAZy) (, accessed on 22 October 2021). Of these GTs, the GT1 family is most common due to its excellent glycosylation capacity [63].
In view of structures, the reported GTs can be divided into three superfamilies: GT-A, GT-B, and GT-C folds [64]. In general, the GT1 family exhibits GT-B fold. GT-B glycosyltransferases harbor two distinct β/α/β Rossmann domains which are flexibly connected [65]. The active site generally locates in the cleft between two domains [66]. The C-terminal donor binding motif is relatively conserved, whereas the N-terminal domain shows high divergence and considerable topological plasticity [67]. The binding sites mainly consist of hydrophobic amino acid residues, while the gateway to ligand entry and departure is generally rich in charged amino acid residues [68,69]. In glycosylation, GT-B proteins generally alter their conformation and shape of binding pocket when binding to the sugar donor, leading to a slight rotation of the N-terminal domain toward the C-terminal domain [70].
GTs can be divided into two types: inverting GT and retaining GT, depending on whether the configuration of the transferred sugars in end-product is inverted or retained (Figure 5A) [64,71]. It was reported that the GT1 family undergoes an inversion of glycosylation and demonstrates a direct-displacement SN2-like mechanism: the acceptor nucleophilic hydroxyl attacks the anomeric carbon of the sugar donor and displaces the leaving nucleotide portion on the opposite face [64,72,73]. Compared with standard glycosylation, the inverting GTs catalyze the formation of products with reversal anomeric configuration (Figure 4B).

2.4. Transfer of TDP-β-L-Rhodosamine

Once the polyketide stage is completed, the skeleton at the hydroxy group is glycosylated by specific GTs. In the presence of its auxiliary protein partner AknT, AknS (EC2.4.1.326) is an aklavinone 7-β-L-rhodosaminyltransferase which attaches TDP-β-L-rhodosamine to the C7-OH of aklavinone, resulting in the formation of rhodosaminyl-aklavinone (ACM-T). AknS promiscuously accepts diverse sugar donors and receptors. Consistent with this attribute, the recombinant S. venezuelae produces a range of DXR analogues which contain diverse deoxysugar moieties and AknS/AknT in complex with other enzymes [74]. AknS/AknT can also efficiently transfer TDP-L-daunosamine to exogenous DXR aglycone ε-rhodomycinone [61]. The above studies indicate the wide substrate scope of AknS, which provides insights for the combinatorial biosynthesis of unnatural antibiotics. It is challenging to visualize the transient interaction between AknS and AknT, which jointly form a complex, and their crystallization as well as isolation methods remain poorly understood [75]. Fortunately, AknS belongs to the GT1 family, whose comparable architectures allow for the characterization of congeneric proteins. Of the activator-dependent GTs, SpnP and EryCIII have been investigated for their crystal structures [76,77]. The above structural studies have uncovered key residues for their reactions, such as residue H13 deprotonating the corresponding hydroxy group in aglycone substrates, and residues D356 and E357 contributing to the formation of a two-residue motif (D/E-Q) which mediates the binding of sugar moieties [78]. Interestingly, a three-helix motif was recognized as a common feature and used as an identification tag of GTs that require auxiliary proteins [76]. Moreover, two amino acids (N230 and T335) were found to be crucial for the formation of a hydrogen bond with a TDP unit [76]. AlphaFold is a protein structure prediction tool with high accuracy [79], and the 3D structure of an AknS monomer predicted by the AlphaFold Monomer v2.0. Homologous analysis with other GTs reveals that AknS is arguably a homodimer. This AknS monomer contains a parallel β-sheet surrounded by α-helices, which is consistent with GT-B proteins (Figure 6A). The key residues H13 and D356 are labeled. The three-helix motif is found in the AknS sequence and comprises three helices (Figure 6B).
P450-like enzymes are fundamental for the functions of GTs. Located in the upstream of aknS, the gene aknT encodes an enzyme (AknT) which exhibits oxidoreductase activity. AknS exhibits low activity in the absence of AknT. When binding to AknS at a molar ratio of 3:1, AknT, as a regulator subunit, may optimize the fitness and productive orientation of akalvinone and thereby increase the catalytic constant of AknS by 40-fold [34]. A BLAST search using AknT sequence as a query revealed a plethora of homologous proteins which are putative P450-derived enzymes from Streptomyces. The predicted structure of AknT is shown in Figure 7A. AknT shows a typical cytochrome P450 fold and the conserved amino acid sequence is only limited to the C-terminal [77,80]. Notably, AknT lacks the conserved cysteine binding to heme group, indicating its difference relative to the authentic cytochrome P450 family. Such a difference presumably makes the conformation of AknT more dynamic than typical P450 [77]. Indeed, AknT transiently forms a ternary compound with AknS and substrate, which stimulates the conformational change in AknS and facilitates the transfer of sugar moieties [81]. A previous study indicated that the complex of EryCIII with its auxiliary protein EryCII manifests an α2β2 heterodimer, which is an elongated quaternary organization when the auxiliary protein resides in the periphery of the GT homodimer (Figure 7B) [77]. In this complex, EryCII may serve as a scaffold to provide an α-helix for linking EryCIII [77]. These GT-auxiliary protein pairs may assemble in the same way. It is thus speculated that the interaction surface of the AknS/AknT heteromer is different from that of the AknS homomer.

2.5. Transfer of 2-Deoxy-β-L-Fucose and L-Rhodinose

There exist trisaccharide chains adhered to the skeleton of ACM-A. However, only AknS and AknK have been identified from the ACM-A-producing strain, meaning that one of the two GTs (AknS and AknK) accomplishes two glycosylation reactions [34,35,81]. LanGT1 and LanGT4 are the first discovered iterative GTs [82]. AknK (EC2.4.1.327) is an L-2-deoxyfucosyltransferase transferring two L-2-deoxysugars to the axial 4-OH of anthracycline monosaccharides. In the first reaction, AknK transfers 2-deoxy-β-L-fucose from the activated donor dTDP-2-deoxy-β-L-fucose to mono-glycosylated ACM-T, leading to the generation of di-glycosylated ACM-S [35,81]. L-rhodinose is considered a terminal sugar initially added to ACM-T. Next, L-rhodinose is converted to L-cinerulose (a 2,3,6-trideoxy-4-keto-L-hexose) to form ACM-N [35]. AknK shows lower activity in catalyzing the second transfer reaction compared to the first, indicating the specificity of AknK to dTDP-rhodinose.
AknK exhibits substrate promiscuity and accepts various TDP-sugars as donors and different aglycones as receptors. In addition, its catalytic activity varies in different reactions [35]. For instance, AknK shows the highest activity in the conversion of TDP-L-daunosamine to ACM-T, followed by the conversion of TDP-l-2-deoxyfocus. Most GTs function in a regio- and stereospecific manner. SpnG is a spinosyn rhamnosyltransferase which only accepts TDP substituent in an axial position to catalyze the nucleophilic displacement reaction [78]. AknK requires a receptor with an axial hydroxyl at the nucleophilic attacking site [35].
The TDP-binding active pockets are usually conserved, and the structural specificity of GTs relies on a receptor-binding region [83]. ProteinsPlus (, accessed on 3 October 2022) can detect binding pockets for further protein–ligand interaction analyses [84,85]. Based on the structure information given by the AlphaFold Monomer v2.0 pipeline, a binding pocket was recognized (Figure 8A). A docking analysis of AknK was carried out to elucidate the binding pockets of sugar donors by building a grid covering the deep pocket consisting of the aforementioned key residues and predicted pocket. A total of 100 dockings were performed by the genetic algorithm. Results show that two binding pockets of dTDP-2-deoxyfucose and dTDP-rhodinose are located in the cleft between the N-terminal and C-terminal domains. The energy of docking for dTDP-2-deoxyfucose is 4.74 kcal/mol, but that for dTDP-rhodinose is only 2.27 kcal/mol. The interaction residues in the binding pockets are labeled in Figure 8C,E.

2.6. Optimization of the Catalytic Activity of GTs

As a key enzyme for the biosynthesis of ACM-A, GT can be modified to show appropriate catalytic activity. However, its substrate specificity restricts applications. Although a one-pot multi-enzyme cascade system can yield natural or unnatural compounds, the natural GTs show drawbacks such as relatively low activity, cumbersome purification, and a short half-life [86,87,88]. In addition, heterologous bacterial hosts may generate inclusion bodies [89,90]. To improve the solubility of GTs, a plethora of protein fusion partners and solubility tags have been successfully utilized [89,91,92].
The GT-catalyzed glycosidic bond is formed in a high regio- and stereo-manner. That is, promiscuous GTs may fail to recognize nonnative receptors [93]. The sugar donor versatility of GTs has been investigated and improved. For example, a study examined UrdGT1b and UrdGT1c, which involve in urdamycin A biosynthesis. Mutations of amino acids led to the altered substrate selectivity of GTs [94]. Since members of the GT-B fold family are structurally modular, GT chimeras may be generated by domain swapping, leading to the alternation of sugar donor specificity, especially when parent templates are highly identical [95]. Moreover, the domain exchange of AtUGT78D2 and AtUGT78D led to the generation of fusion proteins. Three of them demonstrated expanded sugar-donor range and enhanced catalytic activity [96].
The rational modification of the GTs related to ACM-A biosynthesis is constrained by the lack of knowledge on crystal structure. To bypass this obstacle, high-throughput screening-dependent directed evolution could be implemented. For instance, a fluorescence-based high-throughput screening (HTS) together with error-prone PCR/saturation mutagenesis was conducted to modify GTs, leading to a 200–300-fold increase in catalytic activity [97]. A high-throughput colorimetric screen in conjunction with saturation mutagenesis was also conducted to generate GT variants, which showed enhanced catalytic activity [98]. In general, rational design is a prerequisite for the molecular modification of enzymes [99]. In this regard, SEARCHGTr is a web tool for GT analysis, which provides information on donor/receptor specificity and putative substrate-binding residues [100]. The increasing crystal structures of GTs enable this web tool to accurately predict substrate-binding pockets and facilitate their redesigning. Indeed, relying on docking analysis and in silico mutagenesis, the candidate key residues in active sites can be genetically modified to improve substrate specificity and enzyme activity [101,102,103]. The screening of novel enzymes from nature is an alternative to conventional chemical synthesis by which chemoselectivity, regioselectivity, and enantioselectivity are hard to control. Indeed, extensive studies on GTs and the burgeoning transcriptome data allow not only the mining of new GTs with substrate promiscuity but also the enriching of the glycosylation toolkit for diverse sugar molecules [104,105,106]. No matter which strategies are adopted, the donor/receptor promiscuity of enzymes is required for the development of versatile biocatalysts.

2.7. Oxidation of Terminal Sugar Residue

Aclacinomycin oxidoreductase (AknOx) was first isolated from S. galilaeus ATCC 31133 [107]. Later, the AknOx secreted from S. galilaeus ATCC 31615 was purified to determine its crystal structure [36,108]. AknOx catalyzes two steps of modifications of terminal sugar residue, namely the four-electron oxidation of rhodinose to L-aculose. The first step is the conversion of rhodinose to cinerulose A, i.e., oxidation of the hydroxyl at C4 to the keto group (EC1.1.3.45). The second step is the elimination of two hydrogen atoms, leading to a double bond between C2 and C3 (EC1.1.3.14) [36]. That is, AknOx firstly serves as an oxidase to transfer hydride from rhodinose to FAD. In the second step, AknOx still acts on the terminal sugar as a proton/hydride redox catalyst. The cells lacking AknOx accumulate various ACM-A analogues [107]. Unlike AknOx, which catalyzes the formation of cinerulose A, GcnQ is a flavin-dependent oxidoreductase catalyzing the formation of L-aculose. In the latter reaction, no intermediate cinerulose A was detected [109].
AknOx is one member of vanillyl-alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family, whose catalytic motif consists of two tyrosine residues [110]. The proton abstraction in the first step catalyzed by AknOx is usually executed by Tyr-493, and Tyr-421 mediates the second reaction to generate L-aculose [36]. Clearly, AknOx is an unusual flavoenzyme, as it harnesses one active site but two different sets of catalytic residues. When tyrosine is mutated into phenylalanine, AknOx non-covalently binds to flavin and shows reduced activity [111].
AknOx is an oxygen-dependent FAD-linked oxidoreductase. Cofactor reduction is implemented by the substrate. The subsequent regeneration of flavin by reacting with molecular oxygen leads to the formation of hydrogen peroxide and the reoxidation of FAD [112]. BLASTp analysis reveals the high identity of AknOx amino acid sequences, and most of them come from the Streptomyces species. A large proportion of these proteins were annotated as FAD-dependent oxidoreductases or dehydrogenases containing the berberine bridge enzyme (BBE) sequence motif. In such enzymes, highly conserved His and Cys residues are characteristically linked to cofactor FAD via 8α-histidylation and 6-S-cysteinylation [113]. Of the FAD-dependent enzymes, Dbv29 is a flavin mononucleotide-dependent hexose oxidase catalyzing a four-electron oxidation reaction [114]. The isoalloxazine ring of flavin is covalently linked to the side-chains of His91 and Cys151. In AknOx, the two amino acid residues are replaced by His70 and Cys130, respectively (Figure 9C) [36,114]. This double-anchor allow enzymes showing an open active site to bind to bulky substrates and maintain architecture, thereby preventing the dissociation of the cofactor [111]. In addition, covalent attachment enhances the oxidation capacity of cofactors and remarkably improves the reduction potential [112]. AknOx harbors a Rossmann fold-binding motif (GXXGXXXG) that covalently binds to the BBE motif and adenosine moiety of FAD [115]. The solution of purified enzymes containing a flavin-like cofactor was bright yellow. Conversely, when His and Cys were mutated, AknOx did not manifest bright yellow and therefore lost catalytic activity, while Dbv29 retained the original yellow color [116].
Secretory expression is important for the large-scale production of proteins. The evolved Twin-Arginine Translocation (Tat) system can translocate fully-folded and cofactor-binding proteins across the cytoplasmic membrane [117]. The endogenous Tat system in refined model organisms benefits the large-scale production of secreted proteins and enzymes [118]. Bioinformatics analysis reveals that the N-terminal of AknOx carries a Tat signal peptide. This signal peptide benefits large-scale protein secretion in heterologous hosts.
AknOx is one of the few structurally identified enzymes participating in ACM-A biosynthesis. The crystal structure of AknOx was determined by multiwavelength anomalous diffraction [108]. In addition, its crystal structure was reported by a group from the University of Turku [36]. In its crystal, each stable unit contains four subunits of AknOx that constitute two dimers, and the AknOx harbors two domains: one is the F domain binding to FAD, the other is S domain carrying the majority of residues for interplay with the substrate (Figure 9A,B). FAD interacts with binding sites through several main- and side-chain hydrogen bonds [36]. The side-chain of ACM-A inserts into the pocket of the S domain, resulting in van der Waals between the atoms of the ligand and several hydrophobic residues [36].

2.8. Regulatory Genes for ACM-A Biosynthesis

In the genera of Streptomyces, the biosynthesis of secondary metabolites usually undergoes complicated hierarchical regulation [119,120]. First, signals are usually sensed by global and/or pleiotropic regulators. Next, signals are transferred to pathway-specific regulators of corresponding compounds and, in turn, activate biosynthesis. The Streptomyces Antibiotic Regulatory Protein (SARP) is a specific regulator influencing the gene clusters for the biosynthesis of secondary metabolites. SARP activates the ACM-A biosynthesis gene clusters, mainly by binding to DNA recognition sequences [121]. AknI and AknO are homologous to the SARP family which regulates gene expression. The inactivation of AknO blocks the formation of ACM-A [48]. Both AknO and AknI belong to the AfsR/DnrI/RedD regulatory family, which carries several highly conserved residues essential for DNA binding to the N-terminal [122]. AntiSMASH analysis of biosynthetic gene clusters led to the identification of other regulators such as LuxR and TetR family regulators, which are adjacent to ACM-A biosynthetic genes in S. galilaeus [123,124]. The regulation of ACM-A biosynthesis requires in-depth study. In Streptomyces, two-component signal transduction systems (TCSs) serve as a primary mechanism influencing metabolite production, virulence factors, and quorum sensing (QS). The regulatory proteins in TCSs respond to the environmental cues captured by the sensor kinase in the cell membrane. Then, kinase phosphorylates itself to respond to the signal and transfers the phosphoryl group to a regulator [125]. TCSs were mainly identified from the model strain S. coelicolor [126]. However, the complicated regulatory network in S. galilaeus remains to be unraveled.

3. Strategies for Improving ACM-A Production

ACM-A and its analogues were originally isolated from S. galilaeus ATCC 31133 broth [11]. It is challenging to separate ACM-A from its analogues due to similar structures. To increase ACM-A and accordingly reduce analogues, S. lavendofoliae ATCC 15872 was mutated, leading to S. lavendofoliae DKRS, which produced 125 mg/L ACM-A [127]. Adjusting the pH value at the late stage of fermentation could also promote ACM-B conversion to ACM-A and thereby facilitate the purification of ACM-A [128]. Interestingly, the mutants of S. galilaeus H026 caused by the treatment of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) could convert AcmB to AcmA, but could not catalyze AcmA to AcmB, which is conducive to the industrial production of AcmA [129]. In an effort to advance aklavinone biosynthesis, a group from Stanford University leveraged bimodular PKSs to produce hexaketides and octaketides [130]. Recently, by optimizing promoters, enzymes, and chassis cells, a recombinant Streptomyces strain was engineered, which produced 15–20 mg/L aklavinone [131]. More importantly, a total of 37 genomes were shown to harbor the gene clusters for the biosynthesis of aclacinomycin, indicating the feasibility of screening strains for the high-level production of ACM-A [132,133].
How to improve ACM-A production may also draw lessons from the biosynthesis of other antibiotics in Streptomyces. The biosynthesis pathway is strictly controlled by regulatory cascades mainly consisting of pleiotropic regulators, global regulators, and feedback regulation [134]. Signal-transduction-mediated pleiotropic regulation works at the beginning of antibiotics biosynthesis. A previous study identified a γ-butyrolactone (GBL) named autoregulatory factor (A-Factor) which could promote the production of streptomycin in S. griseus [135]. Other GBLs can be categorized based on structure differences in their side-chain. The A-Factors bind to cytoplasmic receptors (e.g., TetR family regulators) and activate gene transcription [136]. Relying on this principle, the levels of secondary metabolites in Streptomyces could be enhanced by the exogenous addition of A-Factor analogues or the manipulation of signal-molecule-related genes [137,138,139]. Since SARP family proteins are pathway-specific regulators, the overexpression of SARP regulator DnrI and AfsR lead to improved production of DNR and DXR [140,141].
WblA and BldA are two highly homologous global regulators affecting antibiotic biosynthesis in Streptomyces [142,143]. While the deletion of the negative regulator WblA led to a 70% increase in DXR production in S. peucetius, the heterologous expression of bldA resulted in a 45.7% increase in DNR production in the same strain [140,144]. The Streptomyces TCSs participate in the global regulation of secondary metabolisms and presumably are related to QS [145]. The discovery of QS inducers and related mechanisms suggests the essential roles of QS in metabolic regulation of Streptomyces [146,147,148,149]. When an endogenous QS system was combined with CRISPR interference, the resulting circuits precisely regulated gene expression and resulted in a 560% increase in rapamycin production in S. rapamycinicus [150]. A Gram-negative bacterial QS system was also engineered in Streptomyces, leading to the improved production of oxazolomycin [151]. In particular, gene expression was regulated by DNA methylation, which hampered the binding of RNA polymerases to promoters or transcription factors to their recognization sites at a pretranscriptional level in bacteria [152,153]. Unlike DNA methylation, small regulatory RNAs (sRNAs) control gene expression at posttranscriptional level [154]. It was reported that paired-termini antisense RNAs were used to inhibit DoxR and overproduce doxorubicin [155]. Compared to sRNAs, DNA methylation seems to be more attractive in strain improvement, as it is heritable. Unfortunately, so far, DNA methylation in Streptomyces remains poorly understood, and in-depth study is needed to determine the epigenetic variation loci affecting metabolite formation. We believe epigenome research will offer valuable insights into the high-level production of ACM-A and beyond.
The biosynthesis of antibiotics leads to feedback inhibition, which halts cell growth and constrains their further accumulation due to toxicity and the presence of pathway-specific negative regulators [156,157]. Negative feedback can be minimized by the overexpression of resistance genes [157,158], transporters [159], and efflux-pump-coding genes [160,161]. Ribosome engineering is also effective to improve cell resistance to metabolites, especially when in conjunction with conventional mutagenesis or genome shuffling [162,163,164,165,166]. Compared with genetic modification strategies for screening secondary metabolites, small-molecule perturbation seems to be more applicable to generate mutants. For example, a group identified four molecules designated as ACR2 (antibiotic remodeling compound) which were able to inhibit the enoyl reductase activity of FabI. The inhibition of FabI blocked fatty acid biosynthesis, as it catalyzes the final and rate-limiting step [167]. While this inhibition reduced the precursors toward fatty acid biosynthesis, it increased those toward secondary metabolites, especially for CoA-dependent metabolite formation. In fact, this inhibition led to the improved production of doxorubicin, baumycin, and desferrioxamine B and E [168].
Apart from aforementioned strategies, ACM-A could also be overproduced by the overexpression of rate-limiting enzymes [169] or the genes for the glycosylation and biosynthesis of sugar moieties [170]. Incorporating a heterologous metabolic pathway into host cells may lead to the increased production of desired metabolites if the host cells provide sufficient precursors and energy and show genetic tractability [171,172]. Synthetic biology provides feasible strategies to create efficient chassis cells for heterologous protein expression [173,174]. For the overproduction of ACM-A, one challenge is how to express a functional min PKS which is insoluble in some prokaryotes [175]. The solution to this challenge may be the employment of a molecular chaperon [176] or an appropriate promoter, because the former helps proper protein folding and the latter controls transcription speed. Another issue is the perturbation of the host metabolism. The sigma factor (σs) could be leveraged to stimulate the production of the desired metabolites, as σs can allocate metabolic fluxes [177,178]. Indeed, σ-based regulation has been utilized to intensify gene expression [179,180]. In recent years, orthogonal gene circuits and modular devices are considered effective to coordinate product formation and cell growth [177,181].
One notable challenge for overproducing ACM-A is how to efficiently drive the long biosynthesis pathway. Solutions to this challenge include the coupling of the ACM-A pathway to a core pathway, the timely alleviation of feedback inhibition, the adequate utilization of metabolism [182], and the augmentation of precursor supply [183,184,185,186]. In addition, cluster-free hosts can divert the precursor to the desired chemicals [187]. Although the overexpression of key enzymes is, in most cases, feasible to overproduce desired chemicals, it exerts a metabolic burden on the host cells [188]. Apart from molecular biology approaches, the timely in situ removal of metabolites is actually feasible to minimize metabolic stress and simplify downstream purification [189,190]. Recently, a tRNA from Streptomyces was shown to benefit antibiotic production by circumventing inefficient wobble base-pairing, which is insightful for product formation [191]. Table 3 shows the reported strategies for improving the production of ACM-A and other secondary metabolites in Streptomyces.
Anthracyclines are important anticancer agents; however, their cardiotoxicity limits doses in clinical practice. Hence, it is desirable to seek new anthracycline analogues with high antitumor activity and low cardiotoxicity. 11-hydroxyaclacinomycin A is an analog showing higher in vitro cytotoxicity against melanoma and leukemia compared to ACM-A. 11-hydroxyaclacinomycin A could be produced by transforming S. galilaeus ATCC 31133 with doxorubicin resistance genes drrA and drrB, as well as the aklavinone 11-hydroxylase-coding gene dnrF from the doxorubicin producer [192]. Moreover, the isomerization of the ACM-A hydroxyl region in S. galilaeus mutants led to the production of iso-aclacinomycins, which showed enhanced (1~5 folds) anticancer activity relative to ACM-A [193]. Overall, a lot of approaches could be developed to overproduce ACM-A and its analogues.

4. Conclusions

The antitumor activity of ACM-A has brought a glimmer of hope for cancer patients. This review comprehensively describes the pathways and key enzymes for the biosynthesis of ACM-A. Although the enzymes for the biosynthesis of ACM-A have not been completely unraveled, genome sequencing and bioinformatics analysis hold promise to fasten its annotation and identification. Although current low production constrains its clinical application, a set of strategies have been developed. For instance, constraint-based modeling techniques have been developed for the accurate analysis of metabolic networks [194], and cell systems can be modified at DNA, RNA, and protein levels. For DNA modification, the genome and gene cluster can be reshaped by DNA editing tools such as Multiplex Automated Genome Engineering (MAGE), Multiplexed Site-specific Genome Engineering (MSGE) [195,196], CRISPR/Cas9, base editing [197,198], and prime editing [199]. CRISPR-Cas9 holds the potential to rewire the metabolic pathways and regulatory networks in Streptomyces [200,201]. Transcription can be modulated by RNA interference, anti-sense technology, and CRISPR-Cas13 technology, especially in eucaryotic host cells [202]. Notably, the combination of CRISPR-dCas9 with DNA methylation seems to be a promising strategy for targeted gene knockdown [203]. Apart from the modifications to DNA and RNA, protein engineering is ushering in a new era, as structure prediction tools, especially AlphaFold, help the directed evolution of the enzymes that catalyze the formation of desired metabolites [79]. We envision that the aforementioned strategies and tools will substantially advance the bioproduction of ACM-A and beyond.

Author Contributions

P.T. designed the manuscript. Z.X. wrote the first draft. P.T. polished the manuscript. All authors have read and agreed to the published version of the manuscript.


This study was funded by grants from the National Natural Science Foundation of China (22278022) and the National Key Research and Development Program of China (2018YFA0901800). The APC was funded by the both.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors have no conflicts of interest to declare.


  1. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef]
  3. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2020. CA Cancer J. Clin. 2020, 70, 7–30. [Google Scholar] [CrossRef] [PubMed]
  4. Puyo, S.; Montaudon, D.; Pourquier, P. From old alkylating agents to new minor groove binders. Crit. Rev. Oncol. Hematol. 2014, 89, 43–61. [Google Scholar] [CrossRef] [PubMed]
  5. Parczyk, K.; Schneider, M.R. The future of antihormone therapy: Innovations based on an established principle. J. Cancer Res. Clin. Oncol. 1996, 122, 383–396. [Google Scholar] [CrossRef] [PubMed]
  6. Silvis, N.G. Antimetabolites and cytotoxic drugs. Dermatol. Clin. 2001, 19, 105–118, viii–ix. [Google Scholar] [CrossRef] [PubMed]
  7. Schlager, S.; Drager, B. Exploiting plant alkaloids. Curr. Opin. Biotechnol. 2016, 37, 155–164. [Google Scholar] [CrossRef]
  8. Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573–584. [Google Scholar] [CrossRef]
  9. Johnson, L.N. Protein kinase inhibitors: Contributions from structure to clinical compounds. Q. Rev. Biophys. 2009, 42, 1–40. [Google Scholar] [CrossRef]
  10. Nelson, P.N.; Reynolds, G.M.; Waldron, E.E.; Ward, E.; Giannopoulos, K.; Murray, P.G. Monoclonal antibodies. Mol. Pathol. 2000, 53, 111–117. [Google Scholar] [CrossRef]
  11. Oki, T.; Matsuzawa, Y.; Yoshimoto, A.; Numata, K.; Kitamura, I. New antitumor antibiotics aclacinomycins A and B. J. Antibiot. 1975, 28, 830–834. [Google Scholar] [CrossRef] [PubMed]
  12. Kim, H.S.; Kim, Y.H.; Yoo, O.J.; Lee, J.J. Aclacinomycin X, a novel anthracycline antibiotic produced by Streptomyces galilaeus ATCC 31133. Biosci. Biotechnol. Biochem. 1996, 60, 906–908. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Gao, H.; Wang, J.Y.; Shen, X.Z.; Deng, Y.H.; Zhang, W. Preparation of magnetic polybutylcyanoacrylate nanospheres encapsulated with aclacinomycin A and its effect on gastric tumor. World J. Gastroenterol. 2004, 10, 2010–2013. [Google Scholar] [CrossRef]
  14. Lee, Y.L.; Chen, C.W.; Liu, F.H.; Huang, Y.W.; Huang, H.M. Aclacinomycin A sensitizes K562 chronic myeloid leukemia cells to imatinib through p38MAPK-mediated erythroid differentiation. PLoS ONE 2013, 8, e61939. [Google Scholar] [CrossRef] [Green Version]
  15. Feng, C.; Li, X.; Dong, C.; Zhang, X.; Zhang, X.; Gao, Y. RGD-modified liposomes enhance efficiency of aclacinomycin A delivery: Evaluation of their effect in lung cancer. Drug Des. Devel. Ther. 2015, 9, 4613–4620. [Google Scholar] [PubMed]
  16. Wang, F.; Xie, M.; Chen, P.; Wang, D.; Yang, M. Homoharringtonine combined with cladribine and aclarubicin (HCA) in acute myeloid leukemia: A new regimen of conventional drugs and its mechanism. Oxid. Med. Cell. Longev. 2022, 2022, 8212286. [Google Scholar] [CrossRef]
  17. Moore, M.H.; Hunter, W.N.; d’Estaintot, B.L.; Kennard, O. DNA-drug interactions. The crystal structure of d(CGATCG) complexed with daunomycin. J. Mol. Biol. 1989, 206, 693–705. [Google Scholar] [CrossRef]
  18. Capranico, G.; Binaschi, M.; Borgnetto, M.E.; Zunino, F.; Palumbo, M. A protein-mediated mechanism for the DNA sequence-specific action of topoisomerase II poisons. Trends Pharmacol. Sci. 1997, 18, 323–329. [Google Scholar] [CrossRef]
  19. Pang, B.; Qiao, X.; Janssen, L.; Velds, A.; Groothuis, T.; Kerkhoven, R.; Nieuwland, M.; Ovaa, H.; Rottenberg, S.; van Tellingen, O.; et al. Drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin. Nat. Commun. 2013, 4, 1908. [Google Scholar] [CrossRef] [Green Version]
  20. Qiao, X.; van der Zanden, S.Y.; Wander, D.P.A.; Borras, D.M.; Song, J.Y.; Li, X.; van Duikeren, S.; van Gils, N.; Rutten, A.; van Herwaarden, T.; et al. Uncoupling DNA damage from chromatin damage to detoxify doxorubicin. Proc. Natl. Acad. Sci. USA 2020, 117, 15182–15192. [Google Scholar] [CrossRef]
  21. Hori, S.; Shirai, M.; Hirano, S.; Oki, T.; Inui, T.; Tsukagoshi, S.; Ishizuka, M.; Takeuchi, T.; Umezawa, H. Antitumor activity of new anthracycline antibiotics, aclacinomycin-A and its analogs, and their toxicity. Gan 1977, 68, 685–690. [Google Scholar] [PubMed]
  22. Jung, M.E.; Lowe, J.A. Synthetic approaches to aclacinomycin and pyrromycin antitumour antibiotics via Diels–Alder reactions of 6-alkoxy-2-pyrones: Total synthesis of chrysophanol, helminthosporin and pachybasin. J. Chem. Soc. Chem. Commun. 1978, 3, 95–96. [Google Scholar] [CrossRef]
  23. Krohn, K. Total Synthesis of Anthracyclinone. Angew. Chem. Int. Ed. 1986, 25, 790–807. [Google Scholar] [CrossRef]
  24. Monneret, C.; Martin, A.; Pais, M. Synthesis of the Oligosaccharide moieties of musettamycin, marcellomycin and aclacinomycin A, Antitumor antibiotics. J. Carbohydr. Chem. 1988, 7, 417–434. [Google Scholar] [CrossRef]
  25. Martin, A.; Pais, M.; Monneret, C. Synthesis of a trisaccharide related to the antitumour antibiotic, aclacinomycin A. J. Chem. Soc. Chem. Commun. 1983, 6, 305–306. [Google Scholar] [CrossRef]
  26. Zhu, S.; Liang, R.; Jiang, H.; Wu, W. An efficient route to polysubstituted tetrahydronaphthols: Silver-catalyzed [4+2] cyclization of 2-alkylbenzaldehydes and alkenes. Angew. Chem. Int. Ed. Engl. 2012, 51, 10861–10865. [Google Scholar] [CrossRef]
  27. Yao, H.; Vu, M.D.; Liu, X.W. Recent advances in reagent-controlled stereoselective/stereospecific glycosylation. Carbohydr. Res. 2019, 473, 72–81. [Google Scholar] [CrossRef]
  28. Bennett, C.S.; Galan, M.C. Methods for 2-Deoxyglycoside Synthesis. Chem. Rev. 2018, 118, 7931–7985. [Google Scholar] [CrossRef] [Green Version]
  29. Wander, D.P.A.; van der Zanden, S.Y.; van der Marel, G.A.; Overkleeft, H.S.; Neefjes, J.; Codee, J.D.C. Doxorubicin and aclarubicin: Shuffling anthracycline glycans for improved anticancer agents. J. Med. Chem. 2020, 63, 12814–12829. [Google Scholar] [CrossRef]
  30. Cho, W.T.; Kim, W.S.; Kim, M.K.; Park, J.K.; Kim, H.R.; Rhee, S.K.; Domracheva, A.G.; Panichkina, T.B.; Saburoba, L.A.; Nobikoba, L.M.; et al. Method for producing aclacinomycins A, B, Y using Strepomyces lavendofoliae DKRS. US5484712, 16 January 1996. [Google Scholar]
  31. Oki, T.; Kitamura, I.; Yoshimoto, A.; Matsuzawa, Y.; Shibamoto, N.; Ogasawara, T.; Inui, T.; Takamatsu, A.; Takeuchi, T.; Masuda, T.; et al. Antitumor anthracycline antibiotics, aclacinomycin A and analogues. I. Taxonomy, production, isolation and physicochemical properties. J. Antibiot. 1979, 32, 791–800. [Google Scholar] [CrossRef] [Green Version]
  32. Kitamura, I.; Tobe, H.; Yoshimoto, A.; Oki, T.; Naganawa, H.; Takeuchi, T.; Umezawa, H. Biosynthesis of aklavinone and aclacinomycins. J. Antibiot. 1981, 34, 1498–1500. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Räty, K.; Kantola, J.; Hautala, A.; Hakala, J.; Ylihonko, K.; Mäntsälä, P. Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster. Gene 2002, 293, 115–122. [Google Scholar] [CrossRef] [PubMed]
  34. Lu, W.; Leimkuhler, C.; Gatto, G.J., Jr.; Kruger, R.G.; Oberthur, M.; Kahne, D.; Walsh, C.T. AknT is an activating protein for the glycosyltransferase AknS in L-aminodeoxysugar transfer to the aglycone of aclacinomycin A. Chem. Biol. 2005, 12, 527–534. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Lu, W.; Leimkuhler, C.; Oberthur, M.; Kahne, D.; Walsh, C.T. AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A. Biochemistry 2004, 43, 4548–4558. [Google Scholar] [CrossRef]
  36. Alexeev, I.; Sultana, A.; Mäntsälä, P.; Niemi, J.; Schneider, G. Aclacinomycin oxidoreductase (AknOx) from the biosynthetic pathway of the antibiotic aclacinomycin is an unusual flavoenzyme with a dual active site. Proc. Natl. Acad. Sci. USA 2007, 104, 6170–6175. [Google Scholar] [CrossRef] [Green Version]
  37. Hoshino, T.; Sekine, Y.; Fujiwara, A. Microbial conversion of anthracycline antibiotics. I. Microbial conversion of aclacinomycin B to aclacinomycin A. J. Antibiot. 1983, 36, 1458–1462. [Google Scholar]
  38. Gräfe, U.; Dornberger, K.; Fleck, W.F.; Bormann, E.J.; Ihn, W. Bioconversion of aclacinomycin A to Y and B by an intracellular enzyme of Streptomyces spec. AM 33352. Biotechnol. Lett. 1988, 10, 167–170. [Google Scholar] [CrossRef]
  39. Ridley, C.P.; Lee, H.Y.; Khosla, C. Evolution of polyketide synthases in bacteria. Proc. Natl. Acad. Sci. USA 2008, 105, 4595–4600. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Wang, J.; Zhang, R.; Chen, X.; Sun, X.; Yan, Y.; Shen, X.; Yuan, Q. Biosynthesis of aromatic polyketides in microorganisms using type II polyketide synthases. Microb. Cell Fact. 2020, 19, 110. [Google Scholar] [CrossRef]
  41. Bisang, C.; Long, P.F.; Cortes, J.; Westcott, J.; Crosby, J.; Matharu, A.L.; Cox, R.J.; Simpson, T.J.; Staunton, J.; Leadlay, P.F. A chain initiation factor common to both modular and aromatic polyketide synthases. Nature 1999, 401, 502–505. [Google Scholar] [CrossRef] [PubMed]
  42. Fujii, I. Heterologous expression systems for polyketide synthases. Nat. Prod. Rep. 2009, 26, 155–169. [Google Scholar] [CrossRef] [PubMed]
  43. Metsä-Ketelä, M.; Palmu, K.; Kunnari, T.; Ylihonko, K.; Mäntsälä, P. Engineering anthracycline biosynthesis toward angucyclines. Antimicrob. Agents Chemother. 2003, 47, 1291–1296. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Tsai, S.C. The structural enzymology of iterative aromatic polyketide synthases: A critical comparison with fatty acid synthases. Annu. Rev. Biochem. 2018, 87, 503–531. [Google Scholar] [CrossRef] [PubMed]
  45. Tsukamoto, N.; Fujii, I.; Ebizuka, Y.; Sankawa, U. Nucleotide sequence of the aknA region of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus. J. Bacteriol. 1994, 176, 2473–2475. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Prado, L.; Lombo, F.; Brana, A.F.; Mendez, C.; Rohr, J.; Salas, J.A. Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus. Mol. Gen. Genet. 1999, 261, 216–225. [Google Scholar] [CrossRef]
  47. Chung, J.Y.; Fujii, I.; Harada, S.; Sankawa, U.; Ebizuka, Y. Expression, purification, and characterization of AknX anthrone oxygenase, which is involved in aklavinone biosynthesis in Streptomyces galilaeus. J. Bacteriol. 2002, 184, 6115–6122. [Google Scholar] [CrossRef] [Green Version]
  48. Räty, K.; Kunnari, T.; Hakala, J.; Mantsala, P.; Ylihonko, K. A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin. Mol. Gen. Genet. 2000, 264, 164–172. [Google Scholar] [CrossRef]
  49. Kantola, J.; Kunnari, T.; Hautala, A.; Hakala, J.; Ylihonko, K.; Mäntsälä, P. Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp. Microbiology 2000, 146, 155–163. [Google Scholar] [CrossRef]
  50. Kallio, P.; Sultana, A.; Niemi, J.; Mäntsälä, P.; Schneider, G. Crystal structure of the polyketide cyclase AknH with bound substrate and product analogue: Implications for catalytic mechanism and product stereoselectivity. J. Mol. Biol. 2006, 357, 210–220. [Google Scholar] [CrossRef]
  51. Kendrew, S.G.; Katayama, K.; Deutsch, E.; Madduri, K.; Hutchinson, C.R. DnrD cyclase involved in the biosynthesis of doxorubicin: Purification and characterization of the recombinant enzyme. Biochemistry 1999, 38, 4794–4799. [Google Scholar] [CrossRef]
  52. Sultana, A.; Kallio, P.; Jansson, A.; Wang, J.S.; Niemi, J.; Mäntsälä, P.; Schneider, G. Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation. EMBO J. 2004, 23, 1911–1921. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Dickens, M.L.; Ye, J.; Strohl, W.R. Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5. J. Bacteriol. 1996, 178, 3384–3388. [Google Scholar] [CrossRef] [Green Version]
  54. Torkkell, S.; Kunnari, T.; Palmu, K.; Mäntsälä, P.; Hakala, J.; Ylihonko, K. The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: Characterization of a 20-kb DNA region and generation of hybrid structures. Mol. Genet. Genomics 2001, 266, 276–288. [Google Scholar] [CrossRef] [PubMed]
  55. Thibodeaux, C.J.; Melancon, C.E., 3rd; Liu, H.W. Natural-product sugar biosynthesis and enzymatic glycodiversification. Angew. Chem. Int. Ed. Engl. 2008, 47, 9814–9859. [Google Scholar] [CrossRef] [Green Version]
  56. Salcedo, R.G.; Olano, C.; Fernandez, R.; Brana, A.F.; Mendez, C.; de la Calle, F.; Salas, J.A. Elucidation of the glycosylation steps during biosynthesis of antitumor macrolides PM100117 and PM100118 and engineering for novel derivatives. Microb. Cell Fact. 2016, 15, 187. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Walsh, C.T.; Losey, H.C.; Freel Meyers, C.L. Antibiotic glycosyltransferases. Biochem. Soc. Trans. 2003, 31, 487–492. [Google Scholar] [CrossRef]
  58. Räty, K.; Hautala, A.; Torkkell, S.; Kantola, J.; Mäntsälä, P.; Hakala, J.; Ylihonko, K. Characterization of mutations in aclacinomycin A-non-producing Streptomyces galilaeus strains with altered glycosylation patterns. Microbiology 2002, 148, 3375–3384. [Google Scholar] [CrossRef] [Green Version]
  59. Otten, S.L.; Gallo, M.A.; Madduri, K.; Liu, X.; Hutchinson, C.R. Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine. J. Bacteriol. 1997, 179, 4446–4450. [Google Scholar] [CrossRef] [Green Version]
  60. Hong, L.; Zhao, Z.; Melancon, C.E., 3rd; Zhang, H.; Liu, H.W. In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa. J. Am. Chem. Soc. 2008, 130, 4954–4967. [Google Scholar] [CrossRef] [Green Version]
  61. Han, A.R.; Park, J.W.; Lee, M.K.; Ban, Y.H.; Yoo, Y.J.; Kim, E.J.; Kim, E.; Kim, B.G.; Sohng, J.K.; Yoon, Y.J. Development of a Streptomyces venezuelae-based combinatorial biosynthetic system for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates. Appl. Environ. Microbiol. 2011, 77, 4912–4923. [Google Scholar] [CrossRef] [Green Version]
  62. Palcic, M.M. Glycosyltransferases as biocatalysts. Curr. Opin. Chem. Biol. 2011, 15, 226–233. [Google Scholar] [CrossRef] [PubMed]
  63. Zhang, P.; Zhang, Z.; Zhang, L.; Wang, J.; Wu, C. Glycosyltransferase GT1 family: Phylogenetic distribution, substrates coverage, and representative structural features. Comput. Struct. Biotechnol. J. 2020, 18, 1383–1390. [Google Scholar] [CrossRef] [PubMed]
  64. Lairson, L.L.; Henrissat, B.; Davies, G.J.; Withers, S.G. Glycosyltransferases: Structures, functions, and mechanisms. Annu. Rev. Biochem. 2008, 77, 521–555. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Coutinho, P.M.; Deleury, E.; Davies, G.J.; Henrissat, B. An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 2003, 328, 307–317. [Google Scholar] [CrossRef]
  66. Gloster, T.M. Advances in understanding glycosyltransferases from a structural perspective. Curr. Opin. Struct. Biol. 2014, 28, 131–141. [Google Scholar] [CrossRef] [Green Version]
  67. Chang, A.; Singh, S.; Phillips, G.N., Jr.; Thorson, J.S. Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation. Curr. Opin. Biotechnol. 2011, 22, 800–808. [Google Scholar] [CrossRef] [Green Version]
  68. Brazier-Hicks, M.; Offen, W.A.; Gershater, M.C.; Revett, T.J.; Lim, E.K.; Bowles, D.J.; Davies, G.J.; Edwards, R. Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants. Proc. Natl. Acad. Sci. USA 2007, 104, 20238–20243. [Google Scholar] [CrossRef] [Green Version]
  69. Offen, W.; Martinez-Fleites, C.; Yang, M.; Kiat-Lim, E.; Davis, B.G.; Tarling, C.A.; Ford, C.M.; Bowles, D.J.; Davies, G.J. Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification. EMBO J. 2006, 25, 1396–1405. [Google Scholar] [CrossRef] [Green Version]
  70. Liang, D.M.; Liu, J.H.; Wu, H.; Wang, B.B.; Zhu, H.J.; Qiao, J.J. Glycosyltransferases: Mechanisms and applications in natural product development. Chem. Soc. Rev. 2015, 44, 8350–8374. [Google Scholar] [CrossRef]
  71. Breton, C.; Fournel-Gigleux, S.; Palcic, M.M. Recent structures, evolution and mechanisms of glycosyltransferases. Curr. Opin. Struct. Biol. 2012, 22, 540–549. [Google Scholar] [CrossRef]
  72. Tvaroška, I.; Kozmon, S.; Wimmerova, M.; Koca, J. Substrate-assisted catalytic mechanism of O-GlcNAc transferase discovered by quantum mechanics/molecular mechanics investigation. J. Am. Chem. Soc. 2012, 134, 15563–15571. [Google Scholar] [CrossRef]
  73. Tvaroška, I.; Kozmon, S.; Wimmerova, M.; Koca, J. A QM/MM investigation of the catalytic mechanism of metal-ion-independent core 2 beta1,6-N-acetylglucosaminyltransferase. Chemistry 2013, 19, 8153–8162. [Google Scholar] [CrossRef] [PubMed]
  74. Kim, E.; Song, M.C.; Kim, M.S.; Beom, J.Y.; Jung, J.A.; Cho, H.S.; Yoon, Y.J. One-Pot combinatorial biosynthesis of glycosylated anthracyclines by cocultivation of Streptomyces strains producing aglycones and nucleotide deoxysugars. ACS. Comb. Sci. 2017, 19, 262–270. [Google Scholar] [CrossRef] [PubMed]
  75. Harrus, D.; Kellokumpu, S.; Glumoff, T. Crystal structures of eukaryote glycosyltransferases reveal biologically relevant enzyme homooligomers. Cell. Mol. Life Sci. 2018, 75, 833–848. [Google Scholar] [CrossRef] [Green Version]
  76. Isiorho, E.A.; Jeon, B.S.; Kim, N.H.; Liu, H.W.; Keatinge-Clay, A.T. Structural studies of the spinosyn forosaminyltransferase, SpnP. Biochemistry 2014, 53, 4292–4301. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  77. Moncrieffe, M.C.; Fernandez, M.J.; Spiteller, D.; Matsumura, H.; Gay, N.J.; Luisi, B.F.; Leadlay, P.F. Structure of the glycosyltransferase EryCIII in complex with its activating P450 homologue EryCII. J. Mol. Biol. 2012, 415, 92–101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  78. Isiorho, E.A.; Liu, H.W.; Keatinge-Clay, A.T. Structural studies of the spinosyn rhamnosyltransferase, SpnG. Biochemistry 2012, 51, 1213–1222. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Zidek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  80. Otten, S.L.; Liu, X.; Ferguson, J.; Hutchinson, C.R. Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis. J. Bacteriol. 1995, 177, 6688–6692. [Google Scholar] [CrossRef] [Green Version]
  81. Leimkuhler, C.; Fridman, M.; Lupoli, T.; Walker, S.; Walsh, C.T.; Kahne, D. Characterization of rhodosaminyl transfer by the AknS/AknT glycosylation complex and its use in reconstituting the biosynthetic pathway of aclacinomycin A. J. Am. Chem. Soc. 2007, 129, 10546–10550. [Google Scholar] [CrossRef] [Green Version]
  82. Luzhetskyy, A.; Fedoryshyn, M.; Durr, C.; Taguchi, T.; Novikov, V.; Bechthold, A. Iteratively acting glycosyltransferases involved in the hexasaccharide biosynthesis of landomycin A. Chem. Biol. 2005, 12, 725–729. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Chang, A.; Singh, S.; Helmich, K.E.; Goff, R.D.; Bingman, C.A.; Thorson, J.S.; Phillips, G.N., Jr. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity. Proc. Natl. Acad. Sci. USA 2011, 108, 17649–17654. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Diedrich, K.; Graef, J.; Schoning-Stierand, K.; Rarey, M. GeoMine: Interactive pattern mining of protein-ligand interfaces in the Protein Data Bank. Bioinformatics 2021, 37, 424–425. [Google Scholar] [CrossRef]
  85. Schoning-Stierand, K.; Diedrich, K.; Fahrrolfes, R.; Flachsenberg, F.; Meyder, A.; Nittinger, E.; Steinegger, R.; Rarey, M. ProteinsPlus: Interactive analysis of protein-ligand binding interfaces. Nucleic Acids Res. 2020, 48, W48–W53. [Google Scholar] [CrossRef] [Green Version]
  86. Kim, E.; Moore, B.S.; Yoon, Y.J. Reinvigorating natural product combinatorial biosynthesis with synthetic biology. Nat. Chem. Biol. 2015, 11, 649–659. [Google Scholar] [CrossRef] [PubMed]
  87. Schmölzer, K.; Lemmerer, M.; Nidetzky, B. Glycosyltransferase cascades made fit for chemical production: Integrated biocatalytic process for the natural polyphenol C-glucoside nothofagin. Biotechnol. Bioeng. 2018, 115, 545–556. [Google Scholar] [CrossRef]
  88. Schmölzer, K.; Gutmann, A.; Diricks, M.; Desmet, T.; Nidetzky, B. Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development. Biotechnol. Adv. 2016, 34, 88–111. [Google Scholar] [CrossRef]
  89. Mestrom, L.; Marsden, S.R.; Dieters, M.; Achterberg, P.; Stolk, L.; Bento, I.; Hanefeld, U.; Hagedoorn, P.L. Artificial Fusion of mCherry enhances trehalose transferase solubility and stability. Appl. Environ. Microbiol. 2019, 85, e03084-18. [Google Scholar] [CrossRef] [Green Version]
  90. Yang, Z.; Zhang, L.; Zhang, Y.; Zhang, T.; Feng, Y.; Lu, X.; Lan, W.; Wang, J.; Wu, H.; Cao, C.; et al. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS ONE 2011, 6, e22981. [Google Scholar] [CrossRef]
  91. Shu, W.; Zheng, H.; Fu, X.; Zhen, J.; Tan, M.; Xu, J.; Zhao, X.; Yang, S.; Song, H.; Ma, Y. Enhanced heterologous production of glycosyltransferase UGT76G1 by co-expression of endogenous prpD and malK in Escherichia coli and its transglycosylation application in production of rebaudioside. Int. J. Mol. Sci. 2020, 21, 5752. [Google Scholar] [CrossRef]
  92. Moremen, K.W.; Ramiah, A.; Stuart, M.; Steel, J.; Meng, L.; Forouhar, F.; Moniz, H.A.; Gahlay, G.; Gao, Z.; Chapla, D.; et al. Expression system for structural and functional studies of human glycosylation enzymes. Nat. Chem. Biol. 2018, 14, 156–162. [Google Scholar] [CrossRef] [PubMed]
  93. Yoon, J.A.; Kim, B.G.; Lee, W.J.; Lim, Y.; Chong, Y.; Ahn, J.H. Production of a novel quercetin glycoside through metabolic engineering of Escherichia coli. Appl. Environ. Microbiol. 2012, 78, 4256–4262. [Google Scholar] [CrossRef] [Green Version]
  94. Hoffmeister, D.; Wilkinson, B.; Foster, G.; Sidebottom, P.J.; Ichinose, K.; Bechthold, A. Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity. Chem. Biol. 2002, 9, 287–295. [Google Scholar] [CrossRef] [Green Version]
  95. Kim, H.L.; Kim, A.H.; Park, M.B.; Lee, S.W.; Park, Y.S. Altered sugar donor specificity and catalytic activity of pteridine glycosyltransferases by domain swapping or site-directed mutagenesis. BMB Rep. 2013, 46, 37–40. [Google Scholar] [CrossRef] [Green Version]
  96. Kim, H.S.; Kim, B.G.; Sung, S.; Kim, M.; Mok, H.; Chong, Y.; Ahn, J.H. Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity. Planta 2013, 238, 683–693. [Google Scholar] [CrossRef] [PubMed]
  97. Williams, G.J.; Thorson, J.S. A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution. Nat. Protoc. 2008, 3, 357–362. [Google Scholar] [CrossRef] [PubMed]
  98. Gantt, R.W.; Peltier-Pain, P.; Singh, S.; Zhou, M.; Thorson, J.S. Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis. Proc. Natl. Acad. Sci. USA 2013, 110, 7648–7653. [Google Scholar] [CrossRef] [Green Version]
  99. Li, C.; Zhang, R.; Wang, J.; Wilson, L.M.; Yan, Y. Protein engineering for improving and diversifying natural product biosynthesis. Trends Biotechnol. 2020, 38, 729–744. [Google Scholar] [CrossRef]
  100. Kamra, P.; Gokhale, R.S.; Mohanty, D. SEARCHGTr: A program for analysis of glycosyltransferases involved in glycosylation of secondary metabolites. Nucleic Acids Res. 2005, 33, W220–W225. [Google Scholar] [CrossRef]
  101. Akere, A.; Chen, S.H.; Liu, X.; Chen, Y.; Dantu, S.C.; Pandini, A.; Bhowmik, D.; Haider, S. Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases. Biochem. J. 2020, 477, 2791–2805. [Google Scholar] [CrossRef]
  102. Joshi, R.; Trinkl, J.; Haugeneder, A.; Hartl, K.; Franz-Oberdorf, K.; Giri, A.; Hoffmann, T.; Schwab, W. Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity. Glycobiology 2019, 29, 765–775. [Google Scholar] [CrossRef] [PubMed]
  103. Schmölzer, K.; Czabany, T.; Luley-Goedl, C.; Pavkov-Keller, T.; Ribitsch, D.; Schwab, H.; Gruber, K.; Weber, H.; Nidetzky, B. Complete switch from alpha-2,3- to alpha-2,6-regioselectivity in Pasteurella dagmatis beta-D-galactoside sialyltransferase by active-site redesign. Chem. Commun. 2015, 51, 3083–3086. [Google Scholar] [CrossRef] [PubMed]
  104. Pandey, R.P.; Bashyal, P.; Parajuli, P.; Yamaguchi, T.; Sohng, J.K. Two Trifunctional leloir glycosyltransferases as biocatalysts for natural products glycodiversification. Org. Lett. 2019, 21, 8058–8064. [Google Scholar] [CrossRef]
  105. Chen, L.; Zhang, Y.; Feng, Y. Structural dissection of sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae for substrate specificity. J. Struct. Biol. 2018, 204, 371–379. [Google Scholar] [CrossRef] [PubMed]
  106. Ding, F.; Liu, F.; Shao, W.; Chu, J.; Wu, B.; He, B. Efficient synthesis of crocins from crocetin by a microbial glycosyltransferase from Bacillus subtilis 168. J. Agric. Food Chem. 2018, 66, 11701–11708. [Google Scholar] [CrossRef] [PubMed]
  107. Yoshimoto, A.; Ogasawara, T.; Kitamura, I.; Oki, T.; Inui, T.; Takeuchi, T.; Umezawa, H. Enzymatic conversion of aclacinomycin A to Y by a specific oxidoreductase in Streptomyces. J. Antibiot. 1979, 32, 472–481. [Google Scholar] [CrossRef] [Green Version]
  108. Sultana, A.; Alexeev, I.; Kursula, I.; Mantsala, P.; Niemi, J.; Schneider, G. Structure determination by multiwavelength anomalous diffraction of aclacinomycin oxidoreductase: Indications of multidomain pseudomerohedral twinning. Acta Crystallogr. D Biol. Crystallogr. 2007, 63, 149–159. [Google Scholar] [CrossRef]
  109. Zhang, Y.; Huang, H.; Chen, Q.; Luo, M.; Sun, A.; Song, Y.; Ma, J.; Ju, J. Identification of the grincamycin gene cluster unveils divergent roles for GcnQ in different hosts, tailoring the L-rhodinose moiety. Org. Lett. 2013, 15, 3254–3257. [Google Scholar] [CrossRef]
  110. Leferink, N.G.; Heuts, D.P.; Fraaije, M.W.; van Berkel, W.J. The growing VAO flavoprotein family. Arch. Biochem. Biophys. 2008, 474, 292–301. [Google Scholar] [CrossRef] [Green Version]
  111. Heuts, D.P.; Scrutton, N.S.; McIntire, W.S.; Fraaije, M.W. What’s in a covalent bond? On the role and formation of covalently bound flavin cofactors. FEBS J. 2009, 276, 3405–3427. [Google Scholar] [CrossRef] [Green Version]
  112. Winkler, A.; Kutchan, T.M.; Macheroux, P. 6-S-cysteinylation of bi-covalently attached FAD in berberine bridge enzyme tunes the redox potential for optimal activity. J. Biol. Chem. 2007, 282, 24437–24443. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  113. Winkler, A.; Motz, K.; Riedl, S.; Puhl, M.; Macheroux, P.; Gruber, K. Structural and mechanistic studies reveal the functional role of bicovalent flavinylation in berberine bridge enzyme. J. Biol. Chem. 2009, 284, 19993–20001. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  114. Li, Y.S.; Ho, J.Y.; Huang, C.C.; Lyu, S.Y.; Lee, C.Y.; Huang, Y.T.; Wu, C.J.; Chan, H.C.; Huang, C.J.; Hsu, N.S.; et al. A unique flavin mononucleotide-linked primary alcohol oxidase for glycopeptide A40926 maturation. J. Am. Chem. Soc. 2007, 129, 13384–13385. [Google Scholar] [CrossRef]
  115. Mo, X.; Huang, H.; Ma, J.; Wang, Z.; Wang, B.; Zhang, S.; Zhang, C.; Ju, J. Characterization of TrdL as a 10-hydroxy dehydrogenase and generation of new analogues from a tirandamycin biosynthetic pathway. Org. Lett. 2011, 13, 2212–2215. [Google Scholar] [CrossRef] [PubMed]
  116. Liu, Y.C.; Li, Y.S.; Lyu, S.Y.; Hsu, L.J.; Chen, Y.H.; Huang, Y.T.; Chan, H.C.; Huang, C.J.; Chen, G.H.; Chou, C.C.; et al. Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds. Nat. Chem. Biol. 2011, 7, 304–309. [Google Scholar] [CrossRef] [PubMed]
  117. Berks, B.C.; Sargent, F.; Palmer, T. The Tat protein export pathway. Mol. Microbiol. 2000, 35, 260–274. [Google Scholar] [CrossRef] [PubMed]
  118. Valverde, J.R.; Gullon, S.; Mellado, R.P. Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans. BMC Microbiol. 2018, 18, 59. [Google Scholar] [CrossRef] [Green Version]
  119. Liu, G.; Chater, K.F.; Chandra, G.; Niu, G.; Tan, H. Molecular regulation of antibiotic biosynthesis in streptomyces. Microbiol. Mol. Biol. Rev. 2013, 77, 112–143. [Google Scholar] [CrossRef] [Green Version]
  120. Wei, J.; He, L.; Niu, G. Regulation of antibiotic biosynthesis in actinomycetes: Perspectives and challenges. Synth. Syst. Biotechnol. 2018, 3, 229–235. [Google Scholar] [CrossRef]
  121. Krause, J.; Handayani, I.; Blin, K.; Kulik, A.; Mast, Y. Disclosing the potential of the SARP-type regulator papr2 for the activation of antibiotic gene clusters in Streptomycetes. Front. Microbiol. 2020, 11, 225. [Google Scholar] [CrossRef]
  122. Sheldon, P.J.; Busarow, S.B.; Hutchinson, C.R. Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI. Mol. Microbiol. 2002, 44, 449–460. [Google Scholar] [CrossRef] [PubMed]
  123. Xu, Y.; Ke, M.; Li, J.; Tang, Y.; Wang, N.; Tan, G.; Wang, Y.; Liu, R.; Bai, L.; Zhang, L.; et al. TetR-Type regulator SLCG_2919 is a negative regulator of lincomycin biosynthesis in Streptomyces lincolnensis. Appl. Environ. Microbiol. 2019, 85, e02091-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  124. Brotherton, C.A.; Medema, M.H.; Greenberg, E.P. LuxR homolog-linked biosynthetic gene clusters in proteobacteria. mSystems 2018, 3, e00208-17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  125. Gao, R.; Stock, A.M. Biological insights from structures of two-component proteins. Annu. Rev. Microbiol. 2009, 63, 133–154. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  126. Sánchez de la Nieta, R.; Santamaría, R.I.; Díaz, M. Two-Component Systems of Streptomyces coelicolor: An intricate network to be unraveled. Int. J. Mol. Sci. 2022, 23, 15085. [Google Scholar] [CrossRef]
  127. Kim, W.-S.; Youn, D.J.; Cho, W.; Kim, M.-K.; Kim, H.R.; Rhee, S.K.; Choi, E.-S. Improved production, and purification of aclacinomycin A from Streptomyces lavendofoliae DKRS. J. Microbiol. Biotechnol. 1995, 5, 297–301. [Google Scholar]
  128. Kim, W.S.; Youn, D.J.; Kim, H.R.; Rhee, S.K.; Choi, E.S. Metabolic conversion of aclacinomycins B and Y to A by pH shift during fermentation with Streptomyces lavendofoliae DKRS. Biotechnol. Tech. 1995, 9, 671–676. [Google Scholar] [CrossRef]
  129. Ylihonko, K.; Hakala, J.; Niemi, J.; Lundell, J.; Mantsala, P. Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants. Microbiology 1994, 140, 1359–1365. [Google Scholar] [CrossRef] [Green Version]
  130. Lee, T.S.; Khosla, C.; Tang, Y. Engineered biosynthesis of aklanonic acid analogues. J. Am. Chem. Soc. 2005, 127, 12254–12262. [Google Scholar] [CrossRef] [Green Version]
  131. Wang, R.; Nguyen, J.; Hecht, J.; Schwartz, N.; Brown, K.V.; Ponomareva, L.V.; Niemczura, M.; van Dissel, D.; van Wezel, G.P.; Thorson, J.S.; et al. A biobricks metabolic engineering platform for the biosynthesis of anthracyclinones in Streptomyces coelicolor. ACS Synth. Biol. 2022, 11, 4193–4209. [Google Scholar] [CrossRef]
  132. Belknap, K.C.; Park, C.J.; Barth, B.M.; Andam, C.P. Genome mining of biosynthetic and chemotherapeutic gene clusters in Streptomyces bacteria. Sci. Rep. 2020, 10, 2003. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  133. Bundale, S.; Begde, D.; Pillai, D.; Gangwani, K.; Nashikkar, N.; Kadam, T.; Upadhyay, A. Novel aromatic polyketides from soil Streptomyces spp.: Purification, characterization and bioactivity studies. World J. Microbiol. Biotechnol. 2018, 34, 67. [Google Scholar] [CrossRef] [PubMed]
  134. Xia, H.; Li, X.; Li, Z.; Zhan, X.; Mao, X.; Li, Y. The application of regulatory cascades in Streptomyces: Yield enhancement and metabolite mining. Front. Microbiol. 2020, 11, 406. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  135. Khokhlov, A.S.; Tovarova, I.I.; Borisova, L.N.; Pliner, S.A.; Shevchenko, L.; Kornitskaia, E.I.; Ivkina, N.S.; Rapoport, I.A. The A-factor, responsible for streptomycin biosynthesis by mutant strains of Actinomyces streptomycini. Dok. Akad. Nauk SSSR 1967, 177, 232–325. [Google Scholar]
  136. Horinouchi, S.; Beppu, T. Hormonal control by A-factor of morphological development and secondary metabolism in Streptomyces. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2007, 83, 277–295. [Google Scholar] [CrossRef] [Green Version]
  137. Tan, G.Y.; Bai, L.; Zhong, J.J. Exogenous 1,4-butyrolactone stimulates A-factor-like cascade and validamycin biosynthesis in Streptomyces hygroscopicus 5008. Biotechnol. Bioeng. 2013, 110, 2984–2993. [Google Scholar] [CrossRef]
  138. Misaki, Y.; Yamamoto, S.; Suzuki, T.; Iwakuni, M.; Sasaki, H.; Takahashi, Y.; Inada, K.; Kinashi, H.; Arakawa, K. SrrB, a pseudo-receptor protein, acts as a negative regulator for lankacidin and lankamycin production in Streptomyces rochei. Front. Microbiol. 2020, 11, 1089. [Google Scholar] [CrossRef]
  139. Ma, D.; Wang, C.; Chen, H.; Wen, J. Manipulating the expression of SARP family regulator BulZ and its target gene product to increase tacrolimus production. Appl. Microbiol. Biotechnol. 2018, 102, 4887–4900. [Google Scholar] [CrossRef]
  140. Pokhrel, A.R.; Chaudhary, A.K.; Nguyen, H.T.; Dhakal, D.; Le, T.T.; Shrestha, A.; Liou, K.; Sohng, J.K. Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952. Microbiol. Res. 2016, 192, 96–102. [Google Scholar] [CrossRef]
  141. Malla, S.; Niraula, N.P.; Liou, K.; Sohng, J.K. Improvement in doxorubicin productivity by overexpression of regulatory genes in Streptomyces peucetius. Res. Microbiol. 2010, 161, 109–117. [Google Scholar] [CrossRef]
  142. Nah, H.J.; Park, J.; Choi, S.; Kim, E.S. WblA, a global regulator of antibiotic biosynthesis in Streptomyces. J. Ind. Microbiol. Biotechnol. 2021, 48, kuab007. [Google Scholar] [CrossRef] [PubMed]
  143. Merrick, M.J. A morphological and genetic mapping study of bald colony mutants of Streptomyces coelicolor. J. Gen. Microbiol. 1976, 96, 299–315. [Google Scholar] [CrossRef] [Green Version]
  144. Noh, J.H.; Kim, S.H.; Lee, H.N.; Lee, S.Y.; Kim, E.S. Isolation and genetic manipulation of the antibiotic down-regulatory gene, wblA ortholog for doxorubicin-producing Streptomyces strain improvement. Appl. Microbiol. Biotechnol. 2010, 86, 1145–1153. [Google Scholar] [CrossRef] [PubMed]
  145. Zschiedrich, C.P.; Keidel, V.; Szurmant, H. Molecular mechanisms of two-component signal transduction. J. Mol. Biol. 2016, 428, 3752–3775. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  146. Recio, E.; Colinas, A.; Rumbero, A.; Aparicio, J.F.; Martin, J.F. PI factor, a novel type quorum-sensing inducer elicits pimaricin production in Streptomyces natalensis. J. Biol. Chem. 2004, 279, 41586–41593. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  147. Kitani, S.; Miyamoto, K.T.; Takamatsu, S.; Herawati, E.; Iguchi, H.; Nishitomi, K.; Uchida, M.; Nagamitsu, T.; Omura, S.; Ikeda, H.; et al. Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis. Proc. Natl. Acad. Sci. USA 2011, 108, 16410–16415. [Google Scholar] [CrossRef] [Green Version]
  148. Matselyukh, B.; Mohammadipanah, F.; Laatsch, H.; Rohr, J.; Efremenkova, O.; Khilya, V. N-methylphenylalanyl-dehydrobutyrine diketopiperazine, an A-factor mimic that restores antibiotic biosynthesis and morphogenesis in Streptomyces globisporus 1912-B2 and Streptomyces griseus 1439. J. Antibiot. 2015, 68, 9–14. [Google Scholar] [CrossRef]
  149. Takano, E. Gamma-butyrolactones: Streptomyces signalling molecules regulating antibiotic production and differentiation. Curr. Opin. Microbiol. 2006, 9, 287–294. [Google Scholar] [CrossRef] [Green Version]
  150. Tian, J.; Yang, G.; Gu, Y.; Sun, X.; Lu, Y.; Jiang, W. Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces. Nucleic Acids Res. 2020, 48, 8188–8202. [Google Scholar] [CrossRef]
  151. Liu, X.; Li, J.; Li, Y.; Li, J.; Sun, H.; Zheng, J.; Zhang, J.; Tan, H. A visualization reporter system for characterizing antibiotic biosynthetic gene clusters expression with high-sensitivity. Commun. Biol. 2022, 5, 901. [Google Scholar] [CrossRef]
  152. Adhikari, S.; Curtis, P.D. DNA methyltransferases and epigenetic regulation in bacteria. FEMS Microbiol. Rev. 2016, 40, 575–591. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  153. Ghosh, D.; Veeraraghavan, B.; Elangovan, R.; Vivekanandan, P. Antibiotic resistance and epigenetics: More to it than meets the eye. Antimicrob. Agents Chemother. 2020, 64, e02225-19. [Google Scholar] [CrossRef] [PubMed]
  154. Na, D.; Yoo, S.M.; Chung, H.; Park, H.; Park, J.H.; Lee, S.Y. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat. Biotechnol. 2013, 31, 170–174. [Google Scholar] [CrossRef]
  155. Chaudhary, A.K.; Pokhrel, A.R.; Hue, N.T.; Yoo, J.C.; Sohng, J.K. Paired-termini antisense RNA mediated inhibition of DoxR in Streptomyces peucetius ATCC 27952. Biotechnol. Bioprocess Eng. 2015, 20, 381–388. [Google Scholar] [CrossRef]
  156. Galm, U.; Hager, M.H.; Van Lanen, S.G.; Ju, J.; Thorson, J.S.; Shen, B. Antitumor antibiotics: Bleomycin, enediynes, and mitomycin. Chem. Rev. 2005, 105, 739–758. [Google Scholar] [CrossRef] [PubMed]
  157. Malla, S.; Niraula, N.P.; Liou, K.; Sohng, J.K. Self-resistance mechanism in Streptomyces peucetius: Overexpression of drrA, drrB and drrC for doxorubicin enhancement. Microbiol. Res. 2010, 165, 259–267. [Google Scholar] [CrossRef]
  158. Yin, S.; Wang, X.; Shi, M.; Yuan, F.; Wang, H.; Jia, X.; Yuan, F.; Sun, J.; Liu, T.; Yang, K.; et al. Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus. Sci. China Life Sci. 2017, 60, 992–999. [Google Scholar] [CrossRef]
  159. Yu, L.; Yan, X.; Wang, L.; Chu, J.; Zhuang, Y.; Zhang, S.; Guo, M. Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus. BMC Biotechnol. 2012, 12, 52. [Google Scholar] [CrossRef] [Green Version]
  160. Yao, H.; Shen, Z.; Wang, Y.; Deng, F.; Liu, D.; Naren, G.; Dai, L.; Su, C.C.; Wang, B.; Wang, S.; et al. Emergence of a potent multidrug efflux pump variant that enhances campylobacter resistance to multiple antibiotics. mBio 2016, 7, e01543-16. [Google Scholar] [CrossRef] [Green Version]
  161. Nag, A.; Mehra, S. A major facilitator superfamily (MFS) efflux pump, SCO4121, from Streptomyces coelicolor with roles in multidrug resistance and oxidative stress tolerance and its regulation by a MarR regulator. Appl. Environ. Microbiol. 2021, 87, e02238-20. [Google Scholar] [CrossRef]
  162. Ochi, K.; Okamoto, S.; Tozawa, Y.; Inaoka, T.; Hosaka, T.; Xu, J.; Kurosawa, K. Ribosome engineering and secondary metabolite production. Adv. Appl. Microbiol. 2004, 56, 155–184. [Google Scholar] [PubMed]
  163. Hosoya, Y.; Okamoto, S.; Muramatsu, H.; Ochi, K. Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria. Antimicrob. Agents Chemother. 1998, 42, 2041–2047. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  164. Shentu, X.; Liu, N.; Tang, G.; Tanaka, Y.; Ochi, K.; Xu, J.; Yu, X. Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering. J. Antibiot. 2016, 69, 406–410. [Google Scholar] [CrossRef] [PubMed]
  165. Li, D.; Zhang, J.; Tian, Y.; Tan, H. Enhancement of salinomycin production by ribosome engineering in Streptomyces albus. Sci. China Life Sci. 2019, 62, 276–279. [Google Scholar] [CrossRef]
  166. Lv, X.A.; Jin, Y.Y.; Li, Y.D.; Zhang, H.; Liang, X.L. Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin. Appl. Microbiol. Biotechnol. 2013, 97, 641–648. [Google Scholar] [CrossRef]
  167. Payne, D.J.; Warren, P.V.; Holmes, D.J.; Ji, Y.; Lonsdale, J.T. Bacterial fatty-acid biosynthesis: A genomics-driven target for antibacterial drug discovery. Drug Discov. Today 2001, 6, 537–544. [Google Scholar] [CrossRef]
  168. Craney, A.; Ozimok, C.; Pimentel-Elardo, S.M.; Capretta, A.; Nodwell, J.R. Chemical perturbation of secondary metabolism demonstrates important links to primary metabolism. Chem. Biol. 2012, 19, 1020–1027. [Google Scholar] [CrossRef] [Green Version]
  169. Lomovskaya, N.; Otten, S.L.; Doi-Katayama, Y.; Fonstein, L.; Liu, X.C.; Takatsu, T.; Inventi-Solari, A.; Filippini, S.; Torti, F.; Colombo, A.L.; et al. Doxorubicin overproduction in Streptomyces peucetius: Cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene. J. Bacteriol. 1999, 181, 305–318. [Google Scholar] [CrossRef] [Green Version]
  170. Malla, S.; Niraula, N.P.; Liou, K.; Sohng, J.K. Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius. J. Biosci. Bioeng. 2009, 108, 92–98. [Google Scholar] [CrossRef]
  171. Wong, M.; Badri, A.; Gasparis, C.; Belfort, G.; Koffas, M. Modular optimization in metabolic engineering. Crit. Rev. Biochem. Mol. Biol. 2021, 56, 587–602. [Google Scholar] [CrossRef]
  172. Zhao, M.; Zhao, Y.; Yao, M.; Iqbal, H.; Hu, Q.; Liu, H.; Qiao, B.; Li, C.; Skovbjerg, C.A.S.; Nielsen, J.C.; et al. Pathway engineering in yeast for synthesizing the complex polyketide bikaverin. Nat. Commun. 2020, 11, 6197. [Google Scholar] [CrossRef] [PubMed]
  173. Beites, T.; Mendes, M.V. Chassis optimization as a cornerstone for the application of synthetic biology based strategies in microbial secondary metabolism. Front. Microbiol. 2015, 6, 906. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  174. Liu, R.; Deng, Z.; Liu, T. Streptomyces species: Ideal chassis for natural product discovery and overproduction. Metab. Eng. 2018, 50, 74–84. [Google Scholar] [CrossRef] [PubMed]
  175. Zhang, W.; Li, Y.; Tang, Y. Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli. Proc. Natl. Acad. Sci. USA 2008, 105, 20683–20688. [Google Scholar] [CrossRef] [Green Version]
  176. Liu, X.; Hua, K.; Liu, D.; Wu, Z.L.; Wang, Y.; Zhang, H.; Deng, Z.; Pfeifer, B.A.; Jiang, M. Heterologous biosynthesis of type ii polyketide products using E. coli. ACS Chem. Biol. 2020, 15, 1177–1183. [Google Scholar] [CrossRef]
  177. Sun, D.; Liu, C.; Zhu, J.; Liu, W. Connecting metabolic pathways: Sigma factors in Streptomyces spp. Front. Microbiol. 2017, 8, 2546. [Google Scholar] [CrossRef]
  178. Zhuo, Y.; Zhang, W.; Chen, D.; Gao, H.; Tao, J.; Liu, M.; Gou, Z.; Zhou, X.; Ye, B.C.; Zhang, Q.; et al. Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis. Proc. Natl. Acad. Sci. USA 2010, 107, 11250–11254. [Google Scholar] [CrossRef] [Green Version]
  179. Van Brempt, M.; Clauwaert, J.; Mey, F.; Stock, M.; Maertens, J.; Waegeman, W.; De Mey, M. Predictive design of sigma factor-specific promoters. Nat. Commun. 2020, 11, 5822. [Google Scholar] [CrossRef]
  180. Chen, D.; Arkin, A.P. Sequestration-based bistability enables tuning of the switching boundaries and design of a latch. Mol. Syst. Biol. 2012, 8, 620. [Google Scholar] [CrossRef]
  181. Costello, A.; Badran, A.H. Synthetic biological circuits within an orthogonal central dogma. Trends Biotechnol. 2021, 39, 59–71. [Google Scholar] [CrossRef]
  182. Li, W.; Ma, L.; Shen, X.; Wang, J.; Feng, Q.; Liu, L.; Zheng, G.; Yan, Y.; Sun, X.; Yuan, Q. Targeting metabolic driving and intermediate influx in lysine catabolism for high-level glutarate production. Nat. Commun. 2019, 10, 3337. [Google Scholar] [CrossRef] [Green Version]
  183. Zhang, X.; Lu, C.; Bai, L. Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production. Sci. China Life Sci. 2017, 60, 1000–1009. [Google Scholar] [CrossRef] [PubMed]
  184. Lu, C.; Zhang, X.; Jiang, M.; Bai, L. Enhanced salinomycin production by adjusting the supply of polyketide extender units in Streptomyces albus. Metab. Eng. 2016, 35, 129–137. [Google Scholar] [CrossRef] [PubMed]
  185. Ng, I.S.; Ye, C.; Zhang, Z.; Lu, Y.; Jing, K. Daptomycin antibiotic production processes in fed-batch fermentation by Streptomyces roseosporus NRRL11379 with precursor effect and medium optimization. Bioprocess Biosyst. Eng. 2014, 37, 415–423. [Google Scholar] [CrossRef] [PubMed]
  186. Zabala, D.; Brana, A.F.; Salas, J.A.; Mendez, C. Increasing antibiotic production yields by favoring the biosynthesis of precursor metabolites glucose-1-phosphate and/or malonyl-CoA in Streptomyces producer strains. J. Antibiot. 2016, 69, 179–182. [Google Scholar] [CrossRef] [PubMed]
  187. Myronovskyi, M.; Rosenkranzer, B.; Nadmid, S.; Pujic, P.; Normand, P.; Luzhetskyy, A. Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters. Metab. Eng. 2018, 49, 316–324. [Google Scholar] [CrossRef]
  188. Ajikumar, P.K.; Xiao, W.H.; Tyo, K.E.; Wang, Y.; Simeon, F.; Leonard, E.; Mucha, O.; Phon, T.H.; Pfeifer, B.; Stephanopoulos, G. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 2010, 330, 70–74. [Google Scholar] [CrossRef] [Green Version]
  189. Dong, Y.; Li, X.; Duan, J.; Qin, Y.; Yang, X.; Ren, J.; Li, G. Improving the yield of xenocoumacin 1 enabled by in situ product removal. ACS Omega 2020, 5, 20391–20398. [Google Scholar] [CrossRef]
  190. Malla, S.; Niraula, N.P.; Singh, B.; Liou, K.; Sohng, J.K. Limitations in doxorubicin production from Streptomyces peucetius. Microbiol. Res. 2010, 165, 427–435. [Google Scholar] [CrossRef]
  191. Chen, X.; Li, S.; Zhang, B.; Sun, H.; Wang, J.; Zhang, W.; Meng, W.; Chen, T.; Dyson, P.; Liu, G. A new bacterial tRNA enhances antibiotic production in Streptomyces by circumventing inefficient wobble base-pairing. Nucleic Acids Res. 2022, 50, 7084–7096. [Google Scholar] [CrossRef]
  192. Hwang, C.K.; Kim, H.S.; Hong, Y.S.; Kim, Y.H.; Hong, S.K.; Kim, S.J.; Lee, J.J. Expression of Streptomyces peucetius genes for doxorubicin resistance and aklavinone 11-hydroxylase in Streptomyces galilaeus ATCC 31133 and production of a hybrid aclacinomycin. Antimicrob. Agents Chemother. 1995, 39, 1616–1620. [Google Scholar] [CrossRef] [Green Version]
  193. Hu, Y.; Zhang, Z.; Yin, Y.; Tang, G.L. Directed biosynthesis of iso-aclacinomycins with improved anticancer activity. Org. Lett. 2020, 22, 150–154. [Google Scholar] [CrossRef]
  194. Hadicke, O.; von Kamp, A.; Aydogan, T.; Klamt, S. OptMDFpathway: Identification of metabolic pathways with maximal thermodynamic driving force and its application for analyzing the endogenous CO2 fixation potential of Escherichia coli. PLoS Comput. Biol. 2018, 14, e1006492. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  195. Li, L.; Zheng, G.; Chen, J.; Ge, M.; Jiang, W.; Lu, Y. Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes. Metab. Eng. 2017, 40, 80–92. [Google Scholar] [CrossRef]
  196. Wang, H.H.; Kim, H.; Cong, L.; Jeong, J.; Bang, D.; Church, G.M. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 2012, 9, 591–593. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  197. Gaudelli, N.M.; Komor, A.C.; Rees, H.A.; Packer, M.S.; Badran, A.H.; Bryson, D.I.; Liu, D.R. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 2017, 551, 464–471. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  198. Grunewald, J.; Zhou, R.; Lareau, C.A.; Garcia, S.P.; Iyer, S.; Miller, B.R.; Langner, L.M.; Hsu, J.Y.; Aryee, M.J.; Joung, J.K. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat. Biotechnol. 2020, 38, 861–864. [Google Scholar] [CrossRef]
  199. Anzalone, A.V.; Koblan, L.W.; Liu, D.R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 2020, 38, 824–844. [Google Scholar] [CrossRef]
  200. Tong, Y.; Weber, T.; Lee, S.Y. CRISPR/Cas-based genome engineering in natural product discovery. Nat. Prod. Rep. 2019, 36, 1262–1280. [Google Scholar] [CrossRef]
  201. Rogers, J.K.; Taylor, N.D.; Church, G.M. Biosensor-based engineering of biosynthetic pathways. Curr. Opin. Biotechnol. 2016, 42, 84–91. [Google Scholar] [CrossRef] [Green Version]
  202. Kannan, S.; Altae-Tran, H.; Jin, X.; Madigan, V.J.; Oshiro, R.; Makarova, K.S.; Koonin, E.V.; Zhang, F. Compact RNA editors with small Cas13 proteins. Nat. Biotechnol. 2022, 40, 194–197. [Google Scholar] [CrossRef] [PubMed]
  203. Vojta, A.; Dobrinić, P.; Tadić, V.; Bočkor, L.; Korać, P.; Julg, B.; Klasić, M.; Zoldoš, V. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 2016, 44, 5615–5628. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Figure 1. The crystal structure of DNA–daunomycin complex (PDB 1DA0).
Figure 1. The crystal structure of DNA–daunomycin complex (PDB 1DA0).
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Figure 2. Organization of the aclacinomycins biosynthesis gene cluster. Arrows indicate the directions of gene transcription. GT, glycosyltransferase; PKS, polyketide synthase.
Figure 2. Organization of the aclacinomycins biosynthesis gene cluster. Arrows indicate the directions of gene transcription. GT, glycosyltransferase; PKS, polyketide synthase.
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Figure 3. Biosynthesis pathway of aclacinomycin A. (A) Biosynthesis of TDP-sugars; (B) Type II PKS pathway and post-PKS tailoring modifications.
Figure 3. Biosynthesis pathway of aclacinomycin A. (A) Biosynthesis of TDP-sugars; (B) Type II PKS pathway and post-PKS tailoring modifications.
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Figure 4. Structure information of AknH. (A) Structure of AknH; (B) Superimposition built by Pymol. AknH and SnoaL are shown in green and yellow, respectively.
Figure 4. Structure information of AknH. (A) Structure of AknH; (B) Superimposition built by Pymol. AknH and SnoaL are shown in green and yellow, respectively.
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Figure 5. Catalytic mechanisms of glycosyltransferases. (A) Reaction mechanism of inverting and retaining glycosyltransferases; (B) Inverting glycosyltransferase by a direct-displacement SN2-like reaction mechanism.
Figure 5. Catalytic mechanisms of glycosyltransferases. (A) Reaction mechanism of inverting and retaining glycosyltransferases; (B) Inverting glycosyltransferase by a direct-displacement SN2-like reaction mechanism.
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Figure 6. Predicted structure module of AknS. (A) AknS monomer prediction carried by AlphaFold Monomer v2.0; (B) The three-helix motif in AknS with labels of highly conserved residues.
Figure 6. Predicted structure module of AknS. (A) AknS monomer prediction carried by AlphaFold Monomer v2.0; (B) The three-helix motif in AknS with labels of highly conserved residues.
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Figure 7. Structure information of AknT. (A) Predicted structure model of AknT; (B) The cartoon image of ErycIII in complex of EryCII.
Figure 7. Structure information of AknT. (A) Predicted structure model of AknT; (B) The cartoon image of ErycIII in complex of EryCII.
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Figure 8. Homology model of AknK and docking analysis. (A) Homology model of AknK; the close-up view of the profile maps showing the active site binding as well as enlarged views of molecular docking of AknK with (B,C) dTDP-2-deoxyfucose; and (D,E) dTDP-rhodinose, respectively.
Figure 8. Homology model of AknK and docking analysis. (A) Homology model of AknK; the close-up view of the profile maps showing the active site binding as well as enlarged views of molecular docking of AknK with (B,C) dTDP-2-deoxyfucose; and (D,E) dTDP-rhodinose, respectively.
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Figure 9. Structure information of AknOx. (A) Structure of AknOx; (B) The S domain and F domain are shown in yellow and light green, respectively; (C) FAD-binding pocket of AknOx and Dbv29.
Figure 9. Structure information of AknOx. (A) Structure of AknOx; (B) The S domain and F domain are shown in yellow and light green, respectively; (C) FAD-binding pocket of AknOx and Dbv29.
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Table 1. The identified and putative functions of the akn genes.
Table 1. The identified and putative functions of the akn genes.
Gene ProductNumber of Amino Acid ResiduesUniprotFunctions
AknD91Q9L549Acyl carrier protein (ACP)
AknE1450Q9L554Aromatase (ARO)
AknE2368Q9L548Determines the starter unit
AknF347Q9L547Malonyl-CoA: ACP transacylase (MAT)
AknX122Q9L552Mono-oxygenase (OXY)
AknG286Q9L546Methyl transferase (MET)
AknH144O52646Aklanonic acid methyl ester cyclase (AAME-cyclase)
AknU267Q9L4U4Aklaviketone reductase (KRII)
Table 2. The identified and putative functions of akn enzymes and isozymes.
Table 2. The identified and putative functions of akn enzymes and isozymes.
Gene ProductNumber of Amino Acid ResiduesUniprotFunctionsIsozyme
AknY291Q9L4U0Glucose-1-phosphate thymidylyltransferaseDnmL
Table 3. Strategies for improving the desired metabolites in Streptomyces.
Table 3. Strategies for improving the desired metabolites in Streptomyces.
OrganismSecondary MetaboliteStrategyProduction ImprovedRef.
S. lavendofoliaeACM-ANTG mutagenesis300%[127]
S. coelicoloraklavinoneoptimization of promoters, vectors, and chassis strains>100%[131]
S. galilaeusACM-Aadjusting broth pHData not shown[128]
S. galilaeusACM-ANTG mutagenesisData not shown[129]
S. coelicolorDXR/DNR/akavinonedisruption of global downregulator gene30% [144]
S. peucetiusdaunorubicinoverexpression of resistance genes410%[157]
S. peucetiusdaunorubicinoverexpression of global regulator45.7%[140]
S. peucetiusdoxorubicinoverexpression of rate-limiting enzymes86%[169]
S. peucetiusdoxorubicinexpression of structural sugar biosynthesis and glycosyltransferase genes460%[170]
S. avermitilisavermectinribosomal engineering50%[178]
S. rocheiLankacidin/lankamycindeletion of GBL receptors>300%[138]
S. hygroscopicusvalidamycinexogenous addition of A-Factor analogues30%[137]
S. tsukubaensistacrolimusexpression of GBL synthetase36%[139]
S. rimosusoxytetracyclineoverexpression of transporters60%[159]
S. coelicolorgermicidinsexogenous addition of ARC2Data not shown[167]
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Xu, Z.; Tian, P. Rethinking Biosynthesis of Aclacinomycin A. Molecules 2023, 28, 2761.

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Xu Z, Tian P. Rethinking Biosynthesis of Aclacinomycin A. Molecules. 2023; 28(6):2761.

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Xu, Ziling, and Pingfang Tian. 2023. "Rethinking Biosynthesis of Aclacinomycin A" Molecules 28, no. 6: 2761.

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