Recent Advances and Challenges in the Production of Hydroxylated Natural Products Using Microorganisms

Abstract

Hydroxylation reaction is a significant source of structural diversity in natural products (NPs), playing a crucial role in improving the bioactivity, solubility, and stability of natural product molecules. This review summarizes the latest research progress in the field of natural product hydroxylation, focusing on several key hydroxylases involved in the biosynthesis of NPs, including cytochrome P450 monooxygenases, α-ketoglutarate-dependent hydroxylases, and flavin-dependent monooxygenases. These enzymes achieve selective hydroxylation modification of various NPs, such as terpenoids, flavonoids, and steroids, through different catalytic mechanisms. This review systematically summarizes the recent advances on the hydroxylation of NPs, such as amino acids, steroids, terpenoids, lipids, and phenylpropanoids, demonstrating the potential of synthetic biology strategies in constructing artificial biosynthetic pathways and producing hydroxylated natural product derivatives. Through metabolic engineering, enzyme engineering, genetic engineering, and synthetic biology combined with artificial intelligence-assisted technologies, a series of engineered strains have been successfully constructed for the efficient production of hydroxylated NPs and their derivatives, achieving efficient synthesis of hydroxylated NPs. This has provided new avenues for drug development, functional food, and biomaterial production and has also offered new ideas for the industrial production of these compounds. In the future, integrating artificial synthetic pathway design, enzyme directed evolution, dynamic regulation, and artificial intelligence technology is expected to further expand the application of enzyme-catalyzed hydroxylation reactions in the green synthesis of complex NPs, promoting research on natural product hydroxylation to new heights.

1. Introduction

Natural products (NPs) are organic compounds produced by living organisms in nature [1], including but not limited to secondary metabolites found in plants, animals, and microorganisms [2,3]. NPs range from simple small molecules to complex macromolecules, including alkaloids, amino acids, peptides, fatty acids, polyketides, carbohydrates, terpenoids, phenylpropanoids, and other families of compounds, constituting a chemically diverse resource base [4,5]. NPs have a wide range of biological functions, including antibacterial, anti-inflammatory, antioxidant, and anticancer properties [6,7,8,9], and their unique bioactivities have attracted much attention in the field of medicine. They are not only used directly as drugs but also often serve as lead compounds in the development of new drugs [10]. For example, artemisinin, a natural product derived from the Artemisia annua plant, has become a standard drug for the treatment of malaria due to its excellent anti-malarial effects [11]. In addition, NPs play an important role in the food sector, where ingredients such as vitamins and amino acids [12] are essential for maintaining human health. In the cosmetic industry, NPs, such as plant essential oils and polyphenols, are favored for their mildness and skin friendliness and are used in a wide range of skincare and beauty products [13,14,15].

Despite the wide range of biological activities and medicinal values of NPs, their development and application face significant challenges, such as scarce resources, high extraction and isolation costs, chemical instability, and low bioavailability, which severely limit the large-scale production and clinical application of these compounds [10,16]. On the one hand, the availability of NPs is often limited, which may be due to their rare distribution in nature or technical difficulties in the extraction and isolation process. In addition, over-harvesting may lead to resource depletion of plant species, making access to NPs even more difficult [17,18]. On the other hand, some NPs are limited in pharmaceutical and industrial applications due to low solubility, poor bioavailability, or possible toxicity at high concentrations [19,20]. Various modification methods have been developed to improve the solubility, biocompatibility [21], stability, and pharmacological activity of NPs [22]. Chemical modification methods have been developed, such as esterification [21], acylation [22], reduction [23], and oxidation reactions [24], as well as biological modifications, such as enzymatic modifications and microbial transformation [25]. Much progress has also been made in physical modifications, such as radiation or ultrasonic treatments [26,27]. Zhuang et al. [28] mentioned that the esterification of epigallocatechin gallate can increase its solubility in a lipid environment, which directly affects its bioactivity and bioavailability. Gong et al. [29] investigated the effects of esterification and enzymatic modifications on the properties of wheat starch and its doughs and found that these modifications significantly improved the functionality of the starch, including its heat resistance and antioxidant properties. Lo et al. [30] discussed the synthesis of mono-acylated luteolin derivatives and demonstrated that acylation could enhance the bioactivity and bioavailability of luteolin, thereby enhancing its pharmacological effects.

Hydroxylation is a common method of structural modification of NPs, significantly altering their physicochemical properties and biological activities. Many NPs with medicinal value have active components that are usually hydroxylated derivatives [31,32]. Through chemical synthesis or biotransformation methods, hydroxyl groups can be introduced into the natural product backbone to obtain a range of structurally diverse hydroxylated derivatives. This provides an important structural basis for the study of the conformational relationship of NPs, the research and development of new drugs, and the development of green synthetic processes [33,34]. The introduction of hydroxyl groups can improve the solubility and bioavailability of NPs and change their binding ability to target proteins and, thus, enhance or modify their pharmacological effects [35,36,37,38]. For example, docetaxel, a hydroxylated derivative of paclitaxel, exhibits stronger antitumor activity than paclitaxel itself [39]. Certain hydroxylated flavonoid compounds exhibit superior antioxidant properties compared to their parent flavonoids [40] and can inhibit UV-B-induced skin cancer [41]. Hydroxylation of steroids by microbial methods allows for the production of bioactive hydroxysteroids [38]. Sun et al. [42] achieved efficient stereoselective hydroxylation of deoxycholic acid (DCA) to 6β-OH DCA by constructing and optimizing a whole-cell cytochrome P450 CYP107D1 biocatalyst, which significantly improved the solubility and pharmacological properties of the product. Nguyen et al. [43] utilized the CYP102A1 enzyme for the regioselective hydroxylation of phlorizin from apples, converting it into 3-OH phlorizin, which possesses higher bioavailability and solubility.

Biotechnological approaches are essential for the efficient biomanufacturing of hydroxylated natural products. Metabolic engineering alters the metabolic pathways of host cells and optimizes carbon flux to enhance the yield and productivity of target compounds. For instance, Song et al. [44] applied metabolic engineering to modify Escherichia coli, optimizing the biosynthetic pathways of 3-hydroxypropionic acid and malonic acid, thereby significantly increasing the fermentation yields of these hydroxylated compounds. Enzyme engineering utilizes techniques such as directed evolution and rational design to enhance the catalytic efficiency and specificity of hydroxylases. Cheng et al. [45] improved the catalytic performance of decarboxylase Chitinophage pinensis towards 3-hydroxylysine through semi-rational design and directed evolution, thereby enhancing the biosynthesis of the chiral amino alcohol 2-hydroxycadaverine. Genetic engineering techniques, such as heterologous expression and promoter engineering, enable precise control over key enzymes and regulatory elements. Lu et al. [46] enhanced the expression and activity of nicotinate dehydrogenase in non-model bacteria through heterologous expression and optimization of the expression system, achieving efficient biosynthesis of 6-hydroxy-3-cyanopyridine. Synthetic biology employs a modular and standardized approach to redesign biological systems, constructing efficient pathways for hydroxylation biosynthesis. Zhang et al. [47] employed synthetic biology to modularly assemble key metabolic modules, including tryptophan hydroxylation, tryptophan biosynthesis, and cofactor regeneration, successfully constructing an artificial biosynthetic pathway from glucose to L-5-hydroxytryptophan (5-HTP). Additionally, emerging artificial intelligence technologies such as machine learning are being used to guide enzyme and metabolic engineering, accelerating the construction of hydroxylation cell factories [48,49]. The integrated application of biotechnological methods represents a vital pathway to achieving efficient biomanufacturing of hydroxylated natural products (as shown in

Table 1. Overview of the biosynthesis of hydroxylated NPs from different microbial strains.

Types of Natural Products	Product	Chemical Structure	Host	Enzymes	Engineered Strategy	Titer	Reference
Hydroxylation of amino acids and their derivatives	L-DOPA		E. coli	Fn-TPL	Overexpression of a novel TPL	110 g/L	[50]
	L-DOPA		C. glutamicum	Ralstonia solanacearum tyrosinase	Heterologous expression of Ralstonia solanacearum tyrosinase in C. glutamicum	0.26 g/L	[51]
	L-DOPA		E. coli	Eh-TPL	Enzyme evolution and high-throughput screening method	69.1 g/L	[52]
	5-HTP		E. coli	TPH1	Heterologous expression of human tryptophan hydroxylase (TPH1) in E. coli	0.02 g/L	[53]
	5-HTP		E. coli	TPH1	By protein engineering, manipulation of plasmid copy number, and fine-tuning of transcriptional regulation strategies	5.1 g/L	[54]
	5-HTP		E. coli	TPH1	By rational design and molecular dynamics simulations	0.91 g/L	[55]
	4-HIL		E. coli	IDO	Dynamically regulate ODHC activity	29.16 g/L	[56]
	(2S,3R,4S)-4-HIL		E. coli	IDO	A high-throughput screening method was developed	80.8 g/L/d	[57]
	T-4-HYP		E. coli	P4H	Introduced a NOG pathway and redesigned key regulatory genes	89.4 g/L	[58]
	T-4-HYP		E. coli	P4H	Heterologous expression of P4H from Dactylosporangium sp. strain RH1 in E. coli	41.0 g/L	[59]
	4-HIL		C. glutamicum	IDO	By employing L-isoleucine-responsive transcriptional regulation or attenuation strategies	34.21 g/L	[60]
Hydroxylation of steroids and their derivatives	Testosterone		P. pastoris	17β-HSD3	Optimization of the gene codons of human 17β-HSD3	11.6 g/L	[61]
	2α-Hydroxylated steroids		E. coli	CYP154C2	Rational engineering	15–20 mg/L	[32]
	11α-OH-4AD		S. cerevisiae or Aspergillus oryzae	CYP68N1	Introducing a rapid identification strategy for filamentous fungi P450 enzymes	0.845 g/L	[33]
	14α-OH-AD, 14α-OH-RSS		S. cerevisiae	P-450lun	Increasing the copies of P-450lun and CPRlun	150 mg/L, 64 mg/L	[62]
Hydroxylation of terpenoids and their derivatives	2-Hydroxy-dehydroabietic acid		S. cerevisiae	CYP72D19	Molecular docking and site-directed mutagenesis were used	-	[63]
	Trihydroxylated diterpene cyclooctatin		E. coli	P450s CotB3/CotB4	A new reductase/ferredoxin system was identified and characterized	15 mg/L	[64]
	Ursolic acid, oleanolic acid, asiatic acid, madecassic acid, arjunolic acid		Yarrowia lipolytica	CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p	Reconstructing the metabolic pathways in Y. lipolytica	11.6 mg/g, 10.2 mg/g, 0.12 mg/g, 8.9 mg/g, 4.4 mg/g	[65]
	6β-Hydroxy-α-amyrin, 6β-hydroxy-β-amyrin, (2α,3β)-urs-12-ene-2,3-diol, (2α,3β)-olean-12-ene-2,3-diol, uvaol, erythrodiol		S. cerevisiae	EcOSC, CYP716E60, CYP716C80, CYP716A86, and CYP716A862	Identified a oxidosqualene cyclase (EcOSC) gene and four CYP716 genes	-	[66]
Hydroxylation of lipids and their derivatives	9,10-Dihydroxyhexadecanoic		E. coli	FAD, EH, EPOX	The heterologous expression of fatty acid hydroxylases	-	[67]
	ω-HFAs		S. cerevisiae	CYP52M1	Constructing a self-sufficient cytochrome P450 enzyme system	347 mg/L	[68]
	ω-HPA		E. coli	CYP153A, camA/camB	By enhancing fatty acid synthesis, blocking the β-oxidation pathway, and optimizing NADH supply	610 mg/L	[69]
Hydroxylation of phenylpropanoids and their derivatives	Esculetin, piceatannol		E. coli	HpaBC	Overexpression of HpaBC	2.7 g/L, 1.2 g/L.	[70]
	Caffeic acid		S. cerevisiae	4HPA3H	A functional 4HPA3H was constructed to replace plant-derived cytochrome P450 enzymes	289.4 mg/L	[71]
	Eriodictyol, catechin, caffeic acid		E. coli	HpaBC	Optimization of media, induction temperature, induction point, and substrate delay time	62.7 mg/L, 34.7 mg/L, 3.5 g/L	[72]

).

In this review, we systematically sort out the latest research progress in the field of natural product hydroxylation. Firstly, the application of enzymes, such as cytochrome P450 monooxygenase, α-ketoglutarate-dependent hydroxylase, and flavin-dependent monooxygenase, in the hydroxylation reaction is discussed, which provides a theoretical basis for the design of novel biocatalysts. Progress in the hydroxylation of NPs, such as amino acids, steroids, terpenoids, lipids, and phenylpropanoids, is further discussed, elucidating the potential of synthetic biology strategies in constructing artificial biosynthetic pathways and producing hydroxylated NPs. Finally, we prospect the future development of hydroxylated natural product biosynthesis, especially the application prospects in new drug development, functional food, and biomaterial production (Figure 1). With the continuous progress of synthetic biology technology, biosynthesis of hydroxylated NPs will play a more important role in the future.

2. Types of Hydroxylation of NPs

2.1. Cytochrome P450 Monooxygenase-Mediated Hydroxylation

Cytochrome P450 monooxygenases (CYPs) are a superfamily of versatile enzymes that catalyze a wide range of oxidative reactions, including the hydroxylation of various natural products and xenobiotics. They play crucial roles in the biosynthesis of secondary metabolites and the metabolism of drugs and other foreign compounds [73]. CYPs are a class of heme-containing monooxygenases that are widely distributed in nature [73,74]. CYPs are characterized by their substrate adaptability and diverse catalytic reactions, and they play a key role in natural product biosynthesis and drug metabolism [75]. CYPs are involved in the hydroxylation of a wide range of NPs, including terpenoids and steroids among others [43,76,77]. For example, several hydroxylation steps in the biosynthesis of paclitaxel diterpenes are catalyzed by CYPs [78]. Zhang et al. [79] successfully redesigned and expressed a variant of taxane-10β-hydroxylase, a key enzyme in paclitaxel synthesis, in E. coli by means of computationally-assisted protein engineering, M3, and achieved efficient synthesis of paclitaxel intermediates taxadiene-5α-yl-acetoxy-10β-ol in an efficient ab initio biosynthesis with a yield of 3.89 mg/L. Hydroxylation of steroid C14 also involves catalysis by CYPs [80], and Felpeto-Santero et al. [81] introduced synthetic bacterial manipulators constructed from CYP103168 and CPR64795 genes in the fungus Cochliobolus lunatus into the Mycolicibacterium smegmatis strain to achieve direct single-step efficient fermentation production of 14αOH-4-androstene-3,17-dione and 14αOH-1,4-androstadiene-3,17-dione from cholesterol and phytosterols with substrate conversions of 69.3% and 70.9%.

2.2. Hydroxylation Mediated by Non-Heme Iron-Dependent Hydroxylases

Non-heme iron-dependent hydroxylases are another important class of enzymes involved in the hydroxylation of natural products. These hydroxylases feature non-heme iron (II) as a cofactor and α-ketoglutarate as a co-substrate, enabling them to catalyze hydroxylation, cyclization, and desaturation reactions of various substrates [82]. These enzymes are particularly common in microbial secondary metabolism and include mainly non-heme iron oxygenases, dioxygenases, and α-ketoglutarate-dependent hydroxylases [83,84]. They play a crucial role in the biosynthesis of NPs, such as amino acids and terpenoids [85,86].

Non-heme iron-dependent hydroxylases play a crucial role in the hydroxylation of NPs. For instance, tyrosine hydroxylase hydroxylates the 3-position of tyrosine residues to produce 3,4-dihydroxyphenyl-l-alanine (L-DOPA), which is a precursor for catecholamine neurotransmitters, such as dopamine and norepinephrine. This process is essential for maintaining the normal function of the nervous system [87]. Du et al. [88] successfully mimicked the active site of tyrosine hydroxylase using an Fe²⁺ and EDTA redox complex, achieving high-yield, efficient, and specific synthesis of L-DOPA, a key compound for the treatment of Parkinson’s disease, from tyrosine. Proline hydroxylase hydroxylates the 4-position of proline residues in collagen to produce 4-hydroxyproline, which participates in the formation of intramolecular and intermolecular hydrogen bonds, thereby maintaining the stability of the collagen triple helix [89]. Zhang et al. [90] optimized the synthesis pathway of trans-4-hydroxy-L-proline (t4Hyp) in E. coli through modular metabolic engineering, balancing the synthesis of α-ketoglutarate and L-proline and enhancing the expression of proline 4-hydroxylase (P4H). Ultimately, a titer of 8.80 g/L of t4Hyp in shake-flask cultivation was achieved. By employing synthetic biology strategies, artificial biosynthetic pathways involving non-heme iron hydroxylases can be constructed for the directed hydroxylation modification of natural product scaffolds [91].

2.3. Other Enzyme-Mediated Hydroxylation Reactions

In addition to CYPs and non-heme iron-dependent hydroxylases, there are other types of enzymes that can catalyze the hydroxylation of NPs, such as flavin-dependent monooxygenase (FMOs) and copper-dependent monooxygenases [92,93]. These enzymes contribute to the diversification of natural product frameworks, offering more possibilities for structural variation. Wu et al. [94] investigated the substrate specificity of the flavin-dependent monooxygenase PboD from Aspergillus on cyclic dipeptides (CDPs) containing tryptophan. They explored the catalytic efficiency of PboD on various CDPs through in vitro experiments and biotransformation experiments in Aspergillus ustus expressing the pboD gene and increased the structural diversity of hydroxylated pyrroloindoline diketopiperazines. Herrmann et al. [95] rationally engineered the flavin-dependent monooxygenase 4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) from E. coli to convert polycyclic phenolic NPs into the corresponding catechols. This transformation was achieved through in vivo and in vitro methods, exhibiting strict regiospecificity without the formation of by-products. Klinman et al. [96] explored the catalytic mechanisms of dopamine β-monooxygenase and peptidylglycine α-hydroxylating monooxygenase during substrate hydroxylation, with these enzymes catalyzing the conversion of dopamine to norepinephrine and C-terminal glycine-extended peptides to their α-hydroxylated products.

3. Hydroxylation of Different Types of NPs and Their Derivatives

3.1. Hydroxylation of Amino Acids and Their Derivatives

The hydroxylation of amino acids and their derivatives is an important chemical reaction that has a wide range of applications in various industries, including biology, medicine, food, and cosmetics [97]. Hydroxylated amino acids (HAAs) are important biosynthetic precursors of biologically active NPs and are highly valued for their antiviral and anticancer properties [98]. For instance, (S)-4-hydroxyphenylglycine is a vancomycin-class antibiotic [99], 5-HTP serves as a precursor for neurotransmitters and hormones that regulate sleep [100], 4-hydroxyisoleucine (4-HIL) shows promise as a drug for the treatment of type II diabetes [101], the C5-hydroxylation of L-leucine is a precursor in the synthesis of NPs such as griselimycins and amanitin [6], L-DOPA is a key precursor in the biosynthesis of norepinephrine and dopamine and is widely used in medications for various tremor-related diseases [102], and hydroxylated lysine and hydroxyproline are involved in the synthesis of collagen [103,104]. Additionally, some HAAs, due to the presence of chiral carbons, serve as important components of chiral compounds and are extensively used in the synthesis of multi-functional biomolecules, pharmaceuticals, and fine chemicals [105,106,107,108].

The introduction of hydroxyl groups significantly increases the polarity and hydrophilicity of amino acid molecules. This enhancement is attributed to the ability of hydroxyl groups to form intermolecular and intramolecular hydrogen bonds, thereby strengthening the interaction between molecules and water [35,109]. Consequently, the vast majority of HAAs are polar amino acids. In recent years, researchers have successfully developed various efficient methods for the production of HAAs using metabolic engineering and protein engineering strategies. Fordjour et al. [110] knocked out the tyrR, ptsG, crr, pheA, and pykF genes in E. coli BL21(DE3) while overexpressing galP and glk to direct carbon flux and evolved HpaB to optimize its activity, achieving efficient de novo production of L-DOPA from glucose and glycerol, with a titer of 25.53 g/L. Rolf et al. [111] screened and characterized a novel lysine double oxidase (KDO) for whole-cell transformation to synthesize (3S)-3-hydroxy-L-lysine or (4R)-4-hydroxy-L-lysine, achieving a hydroxy-L-lysine (Hyl) titer of 5 g/L after 12 h of biotransformation. These homologs expanded the previously limited spectrum of KDOs (nine wild-type enzymes) and provided an excellent starting point for further biosynthetic processes of Hyl. Prell et al. [112] used a heterologous expression strategy to express the KDOFj lysine hydroxylase from Flavobacterium johnsoniae and the LdcC lysine decarboxylase from E. coli in Corynebacterium glutamicum, successfully converting lysine to 3-hydroxycadaverine, with a maximum titer of 8.6 g/L. Wang et al. [113] semi-rationally designed the enhancement of thermal stability of tryptophan hydroxylase (TPH), guided by the calculation of folding free energy, using the thermostable mutants M1 (S422V) and M30 (V275L/I412K) for the biotransformation of L-tryptophan, with M1 and M30 producing 2.32 g/L and 2.76 g/L of 5-HTP, respectively. Wang et al. [114] constructed an E. coli cell factory expressing Schistosoma mansoni tryptophan hydroxylase (SmTPH) and Harminia axyridis dopa decarboxylase (HaDDC), achieving one-pot synthesis from L-tryptophan to 5-hydroxytryptamine, with a titer of 414.5 mg/L. Sun et al. [6] established an in vitro multi-enzyme cascade catalytic system (MECCS) and a whole-cell catalytic system (WCCS) for the simultaneous production of succinic acid (SA) and 5-hydroxyleucine (5-HLeu), with an SA titer of 15.12 g/L and a 5-HLeu titer of 18.83 g/L. Zhang et al. [60] employed an L-isoleucine-responsive transcriptional or attenuation strategy to dynamically regulate the activity of the α-ketoglutarate dehydrogenase complex (ODHC), thereby enhancing the ability of engineered C. glutamicum to produce. The optimal engineered strain, HIL18, produced 34.21 g/L of 4-HIL. Notably, this dynamic regulation also facilitated the efficient production of α-ketoglutarate and its derivatives, such as L-glutamate, γ-aminobutyric acid, L-glutamine, and L-arginine.

A small subset of HAAs, despite containing hydroxyl groups, exhibits lower polarity due to their unique cyclic structures and the formation of intramolecular hydrogen bonds [115,116]. A quintessential example is 4-hydroxyproline, which, despite the presence of a hydroxyl group in its molecule, has a special five-membered ring structure that allows for the hydroxyl group on the ring to form intramolecular hydrogen bonds with the main chain’s amino and carboxyl groups, thereby reducing the overall polarity of the molecule [117]. In the study of hydroxyproline biosynthesis, researchers have made a series of significant advances. Zhang et al. [118] biochemically characterized the α-ketoglutarate/Fe²⁺-dependent proline hydroxylase (HtyE) in species of Aspergillus pachycristatus and Aspergillus aculeatus, finding that both Ap-HtyE and Aa-HtyE can convert L-proline to t4Hyp and t3Hyp and that these two enzymes can effectively hydroxylate 4R-methyl-proline. Prier et al. [119] selectively transformed cyclic ketone esters into all isomers of 3-hydroxyproline and 3-hydroxypipecolic acid using a dynamic kinetic resolution catalyzed by ketone reductases, with yields of 3-cis-L-proline analogs and 3-cis-D-proline analogs reaching 93% and 73%, respectively. Klein et al. [120] established an in vivo biosynthetic pathway in E. coli for the preparation of various stereoisomers of hydroxyproline, including c3Hyp, c4Hyp, and t4Hyp, achieving yields of hydroxyproline up to 35–61% (175–305 mg per shake flask) under shake-flask culture conditions. Gong et al. [58] introduced a non-oxidative glycolysis (NOG) pathway and redesigned key regulatory genes to promote NADPH generation and cofactor (O₂, Fe²⁺) supply modules, thereby efficiently producing t4Hyp, with a titer of 89.4 g/L.

3.2. Hydroxylation of Steroids and Their Derivatives

Steroids are a widely occurring class of important NPs characterized by a cyclopentane polyhydrophenanthrene carbon skeleton [121]. In biological systems, steroids play a variety of endocrine roles, including the regulation of metabolism, immunity, and reproduction, while also participating in the composition and fluidity of cell membranes, thereby maintaining membrane protein functionality [122,123,124]. Hydroxylation modifications of steroids can significantly alter their polarity, biological activity, and toxicity [32,33,125]. Therefore, developing efficient methods for regio- and stereoselective hydroxylation of steroids is of great significance. In recent years, CYPs derived from bacteria or fungi have been successfully engineered to achieve regio- and stereoselective hydroxylation of steroids [76]. For instance, He et al. [126] identified a P450 monooxygenase, CYP68JX, in Colletotrichum lini ST-1, which exhibited high inducible steroid hydroxylation activity. They confirmed its ability to hydroxylate dehydroepiandrosterone at the C7α and C15α positions through expression in recombinant yeast. Zhang et al. [127] isolated and characterized a novel bacterial P450 monooxygenase, CYP109B4, and achieved regioselective conversion from 16β to 15β through rational mutagenesis; the study highlighted the critical role of "hotspot" residues in controlling selectivity, providing new insights and methodologies for the design of efficient biocatalysts.

In fact, the different positions and configurations of oxygen-containing functional groups on steroids often confer markedly distinct pharmacological activities. For instance, 7α-hydroxy-DHEA may promote weight loss and enhance immune function [128], while 7β-hydroxy-DHEA can act as a neuroprotective agent, anti-inflammatory agent, and immune modulator [7]. Additionally, 14α-hydroxylated steroids serve as starting materials for the synthesis of contraceptives and anti-inflammatory compounds [62], whereas 14β-hydroxy steroids are commonly used in the synthesis of cardiotonic steroid drugs [129].

3.2.1. Hydroxylation of Steroid Nucleus

Common hydroxylation positions on the steroid nucleus include C2, C3, C6, C7, C11, C12, C16, and C17 [125]. The location and number of these hydroxyl groups significantly affect the physicochemical properties and biological functions of steroids [130]. Researchers have developed a series of efficient and highly selective biocatalytic methods targeting steroid hydroxylation reactions at different positions using strategies such as enzyme engineering and metabolic engineering. Song et al. [80] identified and characterized the C14α-hydroxylase (CYP14A) from C. lunatus, screening the best variants I111L-M115K and I111L-V124W, which enabled regioselective C14α-hydroxylation of various high-value steroid substrates and diversified synthesis of C14 functionalized steroids based on this transformation. This chemoenzymatic strategy has also been successfully applied to the efficient synthesis of cardiotonic steroid NPs and their isomers, providing a new compound library for the development of new steroid drugs [131,132]. Gao et al. [32] conducted structure-guided rational design of the CYP154C2 enzyme from Streptomyces avermitilis MA-4680T, discovering that mutants L88F/M191F and M191F/V285L significantly improved the conversion rate of 2α-hydroxylation of testosterone (TES) and androstenedione (ASD), with yields reaching 15–20 mg/L. Yi et al. [33] analyzed and identified five new cytochrome P450 enzymes, and by co-expressing CPR1 and CYP68N1 in engineered cells, they increased the production of 11α-OH-androstenedione (11α-OH-4AD) to 0.845 g/L. Kollerov et al. [133] optimized the cultivation conditions for Backusella lamprospora VKM F-944 to carry out 7α-hydroxylation reactions on pregnenolone and dehydroepiandrosterone. At a substrate concentration of 15 g/L, the absolute yield of 7α-OH-DHEA achieved 94% (w/w). Zhao et al. [134] altered the metabolic pathway of plant sterols by introducing MP450-BM3 into the strain producing 7β-hydroxylated steroids and used a strategy to increase the level of NADPH and the NADPH/NADP+ ratio, synthesizing 7β-hydroxyandrost-4-ene-3,17-dione at a concentration of 164.52 mg/L in one-pot biocatalysis using plant sterols as the substrate.

3.2.2. Hydroxylation of Steroid Side Chains

Side-chain hydroxylation increases the polarity and hydrophilicity of steroids, affecting their positioning in lipid membranes and transmembrane transport. Additionally, it provides sites for further modifications, such as glycosylation and sulfation [135]. Hydroxyl groups on the side chain can occur at C-17, C-20, C-22, C-24, C-25, C-26, or C-27 positions, forming secondary alcohols, tertiary alcohols, or dihydroxy compounds [136]. Hayashi et al. [137] achieved a three-step hydroxylation of vitamin D3 to the biologically active 1α,25-dihydroxyvitamin D₃ and its novel metabolites 1α,25(R),26(OH)₃D₃ and 1α,25(S),26(OH)₃D₃ in recombinant Streptomyces lividans expressing a genetically engineered CYP105A1 enzyme, with a conversion rate of 15.2% for 1α,25-dihydroxyvitamin D₃ and 10% for the novel metabolites. Schmitz et al. [138] identified a new strain, Kutzneria albida, capable of hydroxylating vitamin D₂ and vitamin D₃, and the experimental results showed that the addition of 5% (w/v) 2-hydroxypropyl β-cyclodextrin (2HPβCD) led to yields of 25-OH-D₃ and 25-OH-D₂ reaching 63.7 mg/L and 13.7 mg/L, respectively. Putkaradze et al. [139] successfully converted cholesterol to 24(S)- and 25-hydroxycholesterol using a whole-cell system expressing CYP109E1 in Bacillus megaterium and E. coli both in vitro and in vivo, with yields reaching 44.7 mg/L. Rugor et al. [140] utilized the steroid C25 dehydrogenase (S25DH) under optimized bioreaction conditions to perform region-selective hydroxylation on steroid derivatives, such as cholecalciferol, cholesterol, and other sterol derivatives, successfully synthesizing the biologically active 25-hydroxycholesterol and 25-hydroxycholecalciferol, with titers of 0.8 g/L and 1.4 g/L, respectively. Liu et al. [141] achieved efficient biotransformation from progesterone to 17α-hydroxyprogesterone by expressing a novel CYP17A2 enzyme in engineered Pichia pastoris and optimizing the electron transfer and NADPH regeneration system, with a titer reaching 120.9 mg/L.

3.3. Hydroxylation of Terpenoids and Their Derivatives

Terpenoids have garnered significant attention due to their broad range of biological activities, including hepatoprotective, anti-inflammatory, antiviral, anticancer, antibacterial, antidiabetic, antioxidant, and antimalarial effects [8,142]. The pharmacological effects of terpenoids are closely associated with the complex and diverse hydroxyl groups on their skeletons [143]. The introduction of hydroxyl groups onto the terpenoid skeleton often leads to significant changes in their biological activities and physicochemical properties, such as improved solubility, reduced toxicity, or enhanced efficacy [144,145]. Many hydroxylated terpenoid derivatives possess important medicinal value. For instance, hydroxylated derivatives of the side chain of paclitaxel are widely used anticancer drugs in clinical settings [146]. Hydroxylated derivatives of oleanolic acid exhibit significant anti-HIV activity [147].

3.3.1. Hydroxylation of Cyclic Terpenoids

Cyclic terpenoids, such as monoterpenes, sesquiterpenes, and diterpenes, can undergo hydroxylation reactions, leading to a series of hydroxylated derivatives with potential application value [148,149,150]. Zhang et al. [151] identified two key enzymes, CYP82D274 and CYP82D263, as the 14-hydroxylases catalyzing the biosynthetic metabolic grid of triptolide. Heterologous expression of these enzymes in Saccharomyces cerevisiae achieved the de novo biosynthesis of 14-hydroxy-dehydroabietic acid, with a yield of 343.87 μg/L. Dai et al. [152] used metabolic engineering strategies to identify a novel cytochrome P450 enzyme, CYP716C49, in hawthorn that catalyzes C-2α hydroxylation and applied it to the recombinant yeast synthesis pathways of maslinic, corosolic, and alphitolic acid, producing 384, 141, and 23 mg/L of the target products, respectively. Görner et al. [64] identified and characterized a novel redox system from Streptomyces afghaniensis that enhances and coordinates the catalytic efficiency of type I hydroxylase cascades. The application of this system in E. coli allowed for the efficient heterologous production of trihydroxylated diterpene cyclooctatin, achieving a yield of 15 mg/L. Mi et al. [153] introduced the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its natural reductase partner, cindoxin, into the solvent-tolerant Pseudomonas putida GS1 strain for heterologous expression, achieving efficient enantioselective hydroxylation of 1,8-cineole, producing 13.3 g/L of (1R)-6β-hydroxy-1,8-cineole. Cannazza et al. [148] studied the recombinant E. coli whole cells carrying CYP153A6 catalyzing different monoterpene derivatives, achieving high-selectivity hydroxylation of various monoterpene derivatives, with yields of (S)-perillyl alcohol and 7-hydroxy-α-terpineol being 3.25 mg/mL and 5.45 mg/mL, respectively. Qin et al. [154] induced hydroxylation reactions in pairs of curcuminoid enantiomers using Aspergillus niger, resulting in the formation of corresponding mirror-image hydroxylated curdiones. Acid-catalyzed rearrangement of these enantiomers led to the production of mirror-image curcumalactones and hydroxylated curcumalactones, offering new insights for drug lead compounds and scaffolds. Wu et al. [155] employed two filamentous fungi, Mortierella ramanniana and Gibberella fujikuroi, to achieve regio- and stereoselective hydroxylation of ingenane diterpenoids, resulting in six novel hydroxylated derivatives. Cytotoxicity assays demonstrated that hydroxylation at the C-13 aliphatic acid ester of 13-oxyingenol dodecanoate significantly reduced its cytotoxic activity. Wang et al. [66] used engineered yeast expressing CYP716E60 from Enkianthus chinensis, which could convert ursane-type and oleanane-type triterpenoids to new structural 6β-hydroxy-α-amyrin and 6β-hydroxy-β-amyrin, showing potential pharmacological value in anti-inflammatory and hepatoprotective activities.

3.3.2. Hydroxylation of Acyclic Terpenoids

Acyclic terpenoids, formed by the head-to-tail linkage of a certain number of isoprene units, also serve as important substrates for hydroxylation metabolism [156,157]. Nankai et al. [158] utilized the plant pathogenic fungus Glomerella cingulata as a biocatalyst to perform biotransformation on two saturated acyclic monoterpene compounds, tetrahydrogeraniol and tetrahydrolavandulol. The results indicated that the fungus could regioselectively oxidize the isopropyl group of the acyclic terpenoid compounds in a 2 L fermentation medium, producing 260 mg of hydroxycitronellol and 290 mg of 5-hydroxytetrahydrolavandulol, respectively. Davies et al. [159] overcame the bottleneck of the heterologous expression pathway by combinatorially assessing variants of geraniol-8-hydroxylase and cytochrome P450 reductase as well as changes in copy number, successfully enhancing the synthetic efficiency of 8-hydroxygeraniol. Under optimized conditions, the titer of 8-hydroxygeraniol reached 72.52 mg/L/OD₆₀₀. Sintupachee et al. [160] reconstructed the heterologous expression system of plant-derived CYP76F45 and CsCPR I enzymes in E. coli, demonstrating that these enzymes could synergistically catalyze the conversion of geraniol to 8-hydroxygeraniol, participating in the biosynthetic process of monoterpene indole alkaloids.

3.4. Hydroxylation of Lipids and Their Derivatives

Lipids play important roles in the life activities of living organisms, including a variety of functions such as energy storage, signaling, regulation of body temperature, maintenance of acid–base homeostasis, immune response, substance transport, and cell recognition [161,162,163]. Fatty acids are components of many complex lipid molecules and rarely occur as free molecules in nature [164,165]. Currently, hydroxylation of fatty acids can be achieved by chemical synthesis and enzyme catalysis [166,167]. For example, fatty acid desaturases, epoxidases, and epoxide hydrolases catalyzed the synthesis of 9,10-dihydroxyhexadecanoic acid from hexadecanoic acid [67] and the synthesis of chiral hydroxyl fatty acid derivatives (R)-9-hydroxystearic acid and (+)-2-hydroxy-6-(10′-hydroxypentadec-8′(E)-enyl)benzoic acid [67].

Hydroxylated fatty acids (HFAs) are a class of long-chain fatty acids that contain one or more hydroxyl groups in their chemical structure and occur as components of triacylglycerols, waxes, cerebrosides, and other essential lipids in living organisms [168]. HFAs have special properties, such as higher reactivity, stability, and viscosity compared to non-hydroxylated fatty acids, and these properties have attracted great interest [68,166]. They are widely used as starting materials for the synthesis of nylon, resins, waxes, plastics, biopolymers, and lubricants as well as additives for coatings and paints [166,169]. Cao et al. [170] transformed E. coli using metabolic engineering to co-express acetyl-CoA carboxylase (ACCase) and leadless acyl-CoA thioesterase (TesA), knockdown endogenous acyl-CoA synthetase (FadD), and introducing fatty acid hydroxylase (CYP102A1) from B. megaterium, achieving efficient direct synthesis of HFAs from glucose in a yield of 548 mg/L. This yield was significantly higher than the amount of HFAs produced by the team’s previously engineered E. coli strain (117.0 mg/L) [171], suggesting that the optimization of metabolic engineering strategies is important for improving the yield of HFAs. Liu et al. [68] genetically engineered S. cerevisiae to disrupt genes related to fatty acid metabolism while introducing the cytochrome P450 monooxygenase CYP52M1, achieving efficient biosynthesis of long-chain ω-hydroxy fatty acids from glucose, with a titer as high as 347 mg/L. Zong et al. [172] performed targeted saturation mutagenesis of BAMF2522, a self-sufficient CYP102 enzyme from Bacillus amyloliquefaciens DSM 7, and successfully enhanced the enzyme’s regioselectivity for the internal hydroxylation of medium- and long-chain fatty acids, with the products containing a total ω-7, ω-8, and ω-9 hydroxy fatty acids up to 84% of the total content. Gomez de Santos et al. [173] used directed evolution to genetically engineer long UPO from Cyclocybe (Agrocybe) aegerita (AaeUPO) and efficiently expressed the Fett mutant in P. pastoris to synthesize 1.4 g of (ω-1)-hydroxytetradecanoic acid in a continuous-feed reactor, achieving a high degree of regioselectivity at the ω-1 position of fatty acid hydroxylation. Lee et al. [174] used structure-directed engineering techniques to mutate the double-oxygenating 15R-lipoxygenase (15R-LOX) into the single-oxygenating 15R-LOX, resulting in the quantitative conversion of C20 and C22 polyunsaturated fatty acids into 15R-hydroxyeicosatetraenoic acid, 15R-hydroxyeicosapentaenoic acid, and 17R-hydroxydocosahexaenoic acid in recombinant E. coli expressing the monooxygenase system. In addition, the research team utilized E. coli expressing 15S-lipoxygenase in a bioreactor to efficiently convert oil rich in docosahexaenoic acid into the anti-inflammatory lipid mediator Resolvin D5, which possesses hydroxyl groups at the C7 and C17 positions; the process achieved a yield of over 1.0 g/L, with a recovery rate exceeding 87% (w/w) [175].

3.5. Hydroxylation of Phenylpropanoids and Their Derivatives

Phenylpropanoids are a class of plant specialized metabolites synthesized from the amino acid phenylalanine through a series of enzymatic reactions. The core structure of phenylpropanoids consists of an aromatic ring (designated as C6) and a three-carbon side chain (designated as C3), which is typically a propionic acid or propanoid moiety. Many phenylpropanoids also possess phenolic hydroxyl groups attached to the aromatic ring [70,176]. Due to their antimicrobial, antioxidant, anti-inflammatory, antidiabetic, and anticancer activities, phenylpropanoids have been widely used in the fields of food, pharmaceuticals, cosmetics, textiles, and biofuels [176,177]. The introduction of hydroxyl groups can significantly enhance the solubility, stability, structural diversity, and biological activity of these secondary metabolites [70]. Various hydroxylated phenylpropanoid compounds have been found to possess potential biological and pharmacological properties. For example, picetannol, a 3′-hydroxylated derivative of resveratrol, has the ability to inhibit biofilm formation, reduce bacterial motility, and decrease the synthesis of extracellular enzymes [70,178].

As an important member of the phenylpropanoid family, flavonoids are widely present in fruits, vegetables, herbs, and other plants [179]. Flavonoids possess activities such as anti-obesity, anticancer, anti-inflammatory, and antidiabetic effects [180], showing promising applications in the treatment of neurological and cardiovascular diseases, Parkinson’s disease, and Alzheimer’s disease [181]. The hydroxylation of flavonoids can significantly enhance their solubility and antioxidant activity [70,182]. The expression of plant P450-related enzymes (hydroxylases and cytochrome P450 reductases) is crucial for the production of hydroxylated flavonoids [183]. Lv et al. [182] used Y. lipolytica as a host to optimize the biosynthetic pathway of flavonoids and their hydroxylated derivatives by adjusting the gene copy numbers of the key enzymes chalcone synthase and cytochrome P450 reductase, producing 252.4 mg/L of naringenin, 134.2 mg/L of eriodictyol, and 110.5 mg/L of taxifolin, respectively. Hu et al. [184] achieved regioselective hydroxylation of various flavonoid compounds using whole-cell P450 sca-2 as a biocatalyst, optimizing electron transfer partners, sequence-directed enzyme engineering, and biotransformation conditions, preparing eriodictyol, dihydroquercetin, quercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone with yields of 77.3, 66.3, 5.7, 31.8, and 75.1 mg/L, respectively. Wang et al. [185] constructed multiple 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) expression vectors and optimized fermentation conditions, successfully synthesizing B-ring hydroxylated flavonoids, producing 46.84 ± 2.85 mg/L of eriodictyol, confirming the catalytic activity of HpaBC on dihydrokaempferol and kaempferol. Li et al. [186] co-expressed multiple hydroxylases in S. cerevisiae and used metabolic engineering strategies, such as multi-copy gene integration and optimization of promoter strength, to improve the biosynthetic efficiency of dihydromyricetin (DHM), achieving direct synthesis of DHM from glucose, with a titer of 246.4 mg/L. Jones et al. [72] used the native hydroxylase complex HpaBC in E. coli to hydroxylate naringenin and p-coumaric acid; under optimized culture conditions, the titer of eriodictyol from naringenin reached 62.7 mg/L, and the titer of caffeic acid from p-coumaric acid was 3.5 g/L. Jeong et al. [187] used recombinant B. megaterium DY804 strain tyrosinase as a biocatalyst, under optimized reaction conditions, catalyzing the conversion of naringenin to eriodictyol and dihydrotricetin, two hydroxylated products. Sordon et al. [9] utilized the Rhodotorula glutinis to regioselectively ortho-hydroxylate various flavonoids, including hesperetin and chrysin, converting them into hydroxylated derivatives with enhanced antioxidant activities. Specifically, the yield of 8-hydroxyhesperetin from hesperetin was 17.0%, and the yield of norwogonin from chrysin was 30.8%.

4. Conclusions

Hydroxylation of NPs has made significant progress in the fields of synthetic biology and biocatalysis. Through the study of enzymatic and chemical hydroxylation reactions of representative NPs, such as amino acids, flavonoids, terpenoids, and steroids, the catalytic effects of various hydroxylases have been revealed, laying the foundation for the development of efficient and highly selective biocatalysts. Utilizing genetic engineering and metabolic engineering strategies, researchers have constructed artificial biosynthetic pathways for a series of hydroxylated natural product derivatives, achieving green biomanufacturing of hydroxylated NPs. In addition, the catalytic efficiency and regioselectivity of natural hydroxylases have been improved through directed evolution and other means, expanding the synthesis of non-natural hydroxylated products. Although the hydroxylation of NPs has achieved gratifying results in the field of synthetic biology, it still faces many challenges. For example, the catalytic efficiency and selectivity of many natural hydroxylases need to be further improved, and the product yield and stability of heterologous expression systems also need to be optimized. To break through these bottlenecks and accelerate the application of natural product hydroxylation in the fields of drug development, functional foods, and biomaterials, it is necessary to integrate interdisciplinary technologies, such as synthetic biology, enzyme engineering, and metabolic engineering, for collaborative research. In addition, the development of new types of biocatalysts, such as artificial metalloenzymes, and the development of high-throughput screening and directed evolution technologies will help discover and create hydroxylases with excellent performance to catalyze a more diverse range of hydroxylation reactions. This can not only greatly enrich the structural diversity of NPs but also is expected to achieve precise chemical modification and customized synthesis of NPs, meeting the growing demands in the fields of drug development, agricultural production, and industrial applications. In summary, the study of natural product hydroxylation not only has important theoretical significance but also shows broad application prospects in the fields of synthetic biology and biocatalysis and is worth further in-depth exploration.