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Review

Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus

by
David Gómez-Ríos
*,
Luisa María Gómez-Gaona
and
Howard Ramírez-Malule
School of Chemical Engineering, Universidad del Valle, Cali 760042, Colombia
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(10), 526; https://doi.org/10.3390/fermentation10100526
Submission received: 11 September 2024 / Revised: 15 October 2024 / Accepted: 15 October 2024 / Published: 16 October 2024
(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)
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Abstract

:
Clavulanic acid is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus, widely used in combination with β-lactam antibiotics to combat antimicrobial resistance. This systematic review analyzes the most successful methodologies for clavulanic acid overproduction, focusing on the highest yields reported in bench-scale and bioreactor-scale fermentations. Studies have demonstrated that glycerol is the preferred carbon source for clavulanic acid production over other sources like starch and dextrins. The optimization of feeding strategies, especially in fed-batch operations, has improved glycerol utilization and extended the clavulanic acid production phase. Organic nitrogen sources, particularly soybean protein isolates and amino acid supplements such as L-arginine, L-threonine, and L-glutamate, have been proven effective at increasing CA yields both in batch and fed-batch cultures, especially when balanced with appropriate carbon sources. Strain engineering approaches, including mutagenesis and targeted genetic modifications, have allowed for the obtainment of overproducer S. clavuligerus strains. Specifically, engineering efforts that overexpress key regulatory genes such as ccaR and claR, or that disrupt competing pathways, redirect the metabolic flux towards CA biosynthesis, leading to high clavulanic acid titers. The fed-batch operation at the bioreactor scale emerges as the most feasible alternative for prolonged clavulanic acid production with both wild-type and mutant strains, allowing for the attainment of high titers during cultivations.

1. Introduction

Clavulanic acid (CA) is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus (S. clavuligerus), widely used in combination with β-lactam antibiotics to counteract bacterial resistance. Its role in combating antimicrobial resistance, particularly when paired with antibiotics such as amoxicillin, has made CA an essential compound in the pharmaceutical market. S. clavuligerus is a filamentous Gram-positive bacterium known for its characteristic mycelial morphology and its ability to produce several secondary metabolites apart from CA. Like other members of the Streptomyces genus, the S. clavuligerus life cycle begins with spore germination, followed by the development of vegetative mycelia of branched, multicellular hyphae that grow by tip extension (Figure 1). As the culture matures, the bacterium forms aerial mycelia, which eventually differentiate into chains of spores.
Over the years, extensive research has been conducted to enhance the CA production in S. clavuligerus cultures, with numerous studies focused on improving yields through strain engineering, mutagenesis, and process optimization [1,2,3]. A variety of strategies have been employed, including the modification of culture media—optimizing carbon and nitrogen sources—to enhance the metabolic flux toward CA biosynthesis [3,4]. In addition, CA separation and downstreaming is an expensive and difficult process that would benefit from the high concentrations achieved directly in the bioprocess [3,5].
CA biosynthesis has been elucidated through mechanistic studies, with isotopic carbon labeling and genetic studies revealing the gene clusters encoding biosynthetic enzymes and regulatory factors [1,2,3,6]. CA biosynthesis (Figure 2) is typically divided into the early stages, involving the conversion of N2-(2-carboxyethyl) L-arginine into (3S, 5S)-clavaminic acid, and the late stages, leading to CA and 5S-clavam compounds [7,8]. The initial step in CA biosynthesis consists in the condensation of L-arginine and glyceraldehyde-3-phosphate (GAP), catalyzed by N2-(2-carboxyethyl) L-arginine synthase (CEAS), to form N2-(2-carboxyethyl) L-arginine. This intermediate undergoes further transformation into deoxyguanidinoproclavaminic acid via the β-lactam synthetase (BLS) [9,10]. The clavaminate synthase (CAS), a 2-oxoglutarate-dependent oxygenase, catalyzes three key reactions in CA biosynthesis. First, the hydroxylation of deoxyguanidinoproclavaminic acid produces guanidinoproclavaminic acid [10,11]. Then, proclavaminic acid amidino hydrolase (PAH) yields proclavaminic acid [12] and, again, CAS catalyzes an oxidative cyclization of proclavaminic acid, forming dihydroclavaminic acid, which is desaturated into (3S, 5S)-clavaminic acid [10,13].
From (3S, 5S)-clavaminic acid, the CA biosynthesis diverges into two branches: one leading to CA and another producing various 5S-clavam compounds. A stereochemical inversion of (3S, 5S)-clavaminic acid is required in the late steps, leading to clavulanate-9-aldehyde and, finally, CA. N-glycyl-clavaminic acid is part of this stereochemical inversion from the (3S, 5S) to (3R, 5R) configuration [14,15,16]. Ultimately, clavulanate dehydrogenase (CAD) reduces clavulanate-9-aldehyde to CA [17]. The 5S-clavam metabolites retain the (3S, 5S) stereochemistry of clavaminic acid as a bifurcation point; these metabolites include 2-hydroxymethylclavam, 2-formyloxymethylclavam, clavam-2-carboxylic acid, and alanylclavam [18,19,20,21]. Despite the structural similarities of 5S-clavams to CA, only clavam metabolites with a (3R, 5R) configuration might be effective at inhibiting β-lactamase activity.
The genome of S. clavuligerus comprises a linear chromosome of approximately 6.7 Mb along with four plasmids (pSCL1 to pSCL4), with pSCL4 being the largest at 1.8 Mb. Multi-omics analyses of mutants and wild-type strains have provided insights into CA, cephamycin C, and holomycin biosynthesis and regulation. Interactions between the activator ccaR and regulatory networks (Brp, AreB, AdpA, BldG, RelA) have been studied, showing that ccaR can bind to multiple promoters in the cephamycin and CA clusters. A complete review regarding new molecular genetic information on S. clavuligerus obtained with the aid of omics tools was contributed by Liras and Martin [2].
This systematic review aims to analyze the most successful approaches for CA production, specifically focusing on studies that report the highest yields at both the bench and bioreactor scales. By synthesizing data from these studies, we provide a comprehensive overview of the current best practices in CA production, highlighting the impacts of the carbon and nitrogen source selection, strain improvements through genetic manipulation, and key process parameters on the production efficiency.

2. Materials and Methods

Data were collected through a systematic literature search using the Scopus and PubMed databases, which are recognized for their extensive coverage of scientific citations and abstracts. The search was conducted using specific keywords such as “Streptomyces clavuligerus fermentation”, “Clavulanic acid production”, and “Streptomyces clavuligerus mutants”. To ensure consistency and accuracy, the search was carried out by a single researcher.
To maintain the quality and relevance of the selected studies, only those reporting a maximum CA concentration greater than 250 mg/L were included in the analysis. Furthermore, these studies were required to provide detailed information on the critical fermentation parameters, including the media composition and other key conditions under which the cultures were grown. The review was restricted to experimental studies, including conventional fermentations, extractive fermentations, and immobilization techniques, while excluding simulations and purely theoretical works. The PRISMA algorithm applied for the systematic review is summarized in Figure 3.
Details extracted from each selected study included the strain information, fermentation conditions (such as the media composition, pH, temperature, cultivation time, stirrer speed, and airflow rate, if applicable), fermenter volume, and maximum CA and biomass achieved (see Table 1). For fed-batch cultures, additional details like the feeding media, time of feed, and flow rate were also extracted (see Table 2). The compiled data were then analyzed to compare the maximum concentrations and identify patterns in the fermentation conditions and production methods.

3. Results

The search process initially identified 170 articles (as illustrated in Figure 3). After removing duplicates, 102 articles were retained. Of these, 23 were excluded after a preliminary evaluation of the titles and abstracts. A thorough review of the remaining 55 full-text articles was conducted, resulting in the selection of 42 studies that met the inclusion criteria for qualitative analysis. These studies were categorized into several experimental designs: (a) perturbations related to the shear stress and oxygen levels; (b) use of complex media; (c) supplementation of amino acids in basal media; (d) use of mutant strains; (e) other factors influencing clavulanic acid production; and (f) comparisons between the batch (Table 1) and fed-batch (Table 2) fermentation processes.

3.1. Operational Conditions during Submerging of Cultures of S. clavuligerus

Studies on batch cultures at the bench scale have been more focused on exploring the media effects than the environmental conditions. The usual operating conditions range from 6.5 to 7.0 pH, with 6.8 and 7.0 being more frequent. The culture pH conditions appear to have a significant impact on the CA yield, likely due to degradation processes rather than to the direct inhibition of biosynthesis [63,64]. As noted by Gomez-Ríos et al., CA degradation in culture broths follows a hydrolysis reaction, which is catalyzed by either acidic or basic conditions [65,66]. Experimental evidence indicates that degradation occurs more rapidly in basic media than in acidic environments, suggesting that the reaction rate increases at higher pH levels [63,64]. In general, the degradation process involves the equilibrium-driven opening of the β-lactam ring, initiated by the protonation of the oxygen atom and the subsequent nucleophilic attack of water on the carbonyl group [66]. Once the intermediate structure with an open β-lactam ring is formed, several irreversible pathways can follow, such as nucleophilic attacks by nitrogen atoms on other CA molecules or amino acids, imine formation through decarboxylation, or reactions with other compounds present in the medium [66].
During the cultivation, S. clavuligerus tends to slightly increase the broth pH over 7.0 as the cultivation progresses, but this condition affects the CA accumulation [67]. Therefore, the pH is typically controlled in the range of 6.8–7.0, regardless of the nutritional conditions and scale. Moreover, the pH perturbation during the cultivation by reducing the pH from 6.8 to 6.3 in the production stage might enhance the CA yield, but this kind of perturbation has not been sufficiently studied [67].
The highest CA concentration for a batch operation (1700 mg/L) for a wild-type strain was reported by Saudagar and Singhal using a neutral pH, a temperature of 25 °C, and 200 rpm of agitation, coupled with rich nitrogen sources, such as peptone and yeast extract [44,45]. These conditions were obtained by statistical optimization through the orthogonal design of the experiments [44,45]. Similarly, Costa and Badino and Teodoro et al. achieved notable CA concentrations of 1460–1534 mg/L and 1266.2 mg/L with and without supplementation at a pH of 6.8, a lower cultivation temperature of 20 °C, and a higher stirring rate of 250 rpm [34,47]. Lower temperatures are expected to decrease the hydrolysis rate of CA molecules [65,66,68]. Gouveia et al. preferred a more acidic pH (6.5) but used the same temperature and agitation speed, leading to a maximum concentration of 1140 mg/L [22,23]. Similarly, Salem-Bekhit et al. achieved 1120 mg/L at a neutral pH and 250 rpm [31].
The same range of pH, agitation, and temperature conditions have been used for the cultivation of S. clavuligerus mutant strains. Kim et al. reported a CA concentration of 1750 mg/L with a mutant strain (S. clavuligerus KK) at 400 rpm, pH 7.0, and 28 °C [42]. Qin et al. used a higher pH (7.1) and lower temperature (25 °C) at 250 rpm with the mutant S. clavuligerus strains M3-19 and NEO, whose production reached 4330 and 3260 mg/L, respectively [39].
At the bioreactor scale, the operating conditions for batch fermentations are the same as those used for bench-scale propagation precultures. Thus, the most used conditions are 28 °C and pH 6.8–7.0. The first studies at this scale reported low CA concentrations, the most notable being the study by Belmar-Beiny and Thomas, who reported a maximum CA concentration of 250 mg/L at pH 7.0, 26 °C, and an agitation speed of 1300 rpm [26]. Rosa et al. reported 614 mg/L using 1000 rpm, 28 °C, and pH 6.8 [27]. Ribeiro et al. reached 917.5 mg/L under identical conditions [54]. Cultures conducted under different oxygen transfer and shear rate conditions by manipulating operating variables such as the impeller speed, airflow rate, and gas inlet composition have shown that for the same oxygen transfer rate, higher agitation speeds enhance CA production [54]. At 1000 rpm and 8.13 mmol·L−1·s−1 of aeration, a concentration of 917 mg/L of CA was obtained, 53% higher than at 600 rpm at the same aeration rate [54].
In contrast, Feng et al. used stirring (300 rpm) in a 50 L reactor to obtain 1322 mg/L of CA [55]. A clear relationship between the agitation speed and CA productivity is not possible due to the interaction of multiple factors, such as the nutritional and other environmental conditions. Thus, the highest CA production for both wild-type and mutants has been obtained in a wide agitation range (300–1000 rpm) when cultivated in bioreactors. Ortiz et al. achieved 796 mg/L with a wild-type strain at pH 6.8 and 28 °C with an agitation speed of 800 rpm [43]. Rosa et al. obtained lower concentrations (475–482 mg/L) at the same agitation rate [27], while other authors achieved slightly higher concentrations (454–560 mg/L) with the same conditions [47,48]. Kim et al. reported 3000 mg/L for a mutant strain (S. clavuligerus KK) cultivated in a bioreactor at 500 rpm [42]. Later, using a different mutant strain (S. clavuligerus OL13) and medium composition, a final CA concentration of 1950 mg/L was achieved [46].
Although the emphasis in CA production studies has been on the nutritional regulation of antibiotic secretion, it is known that the hydrodynamic conditions can affect the CA production [60]. Since S. clavuligerus is not particularly shear-sensitive, stirred-tank reactors (STRs) operating under a wide range of agitation conditions are commonly used in CA production, as displayed in Table 1 and Table 2. This kind of bioreactor offers a reliable performance in ensuring efficient mixing and mass transfer. However, the standardization of the conditions is a time-consuming task, since the reactor parameters, such as the geometry, mixing speed, and shear forces, significantly affect the oxygen availability and cellular stress. In the case of S. clavuligerus submerged cultivation, high turbulence enhances the oxygen transfer rate and, consequently, the biomass and antibiotic production [27,54,60,69]. As noticed in Table 1 and Table 2, the aeration rate in bioreactor cultivation is usually set at 0.5–1.0 vvm, since the microorganism is strictly aerobic and the biosynthetic pathway requires molecular oxygen for synthesizing the CA molecule [3,6].
Fed-batch fermentation at the bioreactor scale has been demonstrated to be an effective cultivation technique for enhancing CA titers in submerged cultures of S. clavuligerus, either wild-type or mutant. In this kind of operation, the cultivation conditions are likely the same as those explored in batch operations. Kim et al. achieved 3250 mg/L using the S. clavuligerus KK mutant strain, which was 8% higher than the batch operation [42]. Similarly, Teodoro et al. observed substantial CA yields of up to 1560 mg/L in a bioreactor with a wild-type strain at pH 6.8, 28 °C, and 800 rpm. Gómez-Ríos et al. used a wild-type strain under the same temperature and pH conditions but varied the stirring speed (300–500 rpm) according to the level of dissolved oxygen and the increase in the broth viscosity due to biomass production, yielding 422.7–760 mg/L [60,61]. In this regard, variation in the stirring rates as the biomass increases allows for the maintenance of the reactor’s volumetric oxygen transfer coefficient (kLa) and the operating window of dissolved oxygen (20–80%) that promotes CA biosynthesis [60]. Under the same temperature and pH conditions, at 800 rpm, Bellão et al. reported a CA concentration of 982.1 mg/L [49]. These conditions were previously used by Neto et al. for a final CA concentration of 404 mg/L [59]. In contrast, Chen et al. reported a significantly lower CA production of 270 mg/L at the same temperature, pH 7.0, and 500 rpm, despite utilizing a rich nutrient medium [58]. Li and Townsend worked with an engineered strain, yielding 417 mg/L of CA at the same pH and temperature but at 300 rpm. These studies have shown that CA secretion might be favored by high shear rates caused by agitation speeds over 300 rpm when maintaining sufficient nutrients and oxygen during the cultivation. The intense agitation combined with optimal nutritional conditions promotes a thinner and fragmented mycelial morphology capable of growing, reproducing, and secreting antibiotics in nutrient-rich environments [60,70,71]. The extensive fragmentation leads to a dense and viscous broth that also requires high agitation speeds to ensure the oxygen and nutrient mass transfer [60].

3.2. Carbon Sources Used for High Clavulanic Acid Titers in Submerged Cultures

The selection of the carbon source significantly influences the CA yield in cultivation. Glycerol is widely recognized as the preferred carbon source for CA production given the direct connection of its intermediate GAP, which is the early precursor of all clavams in the clavam pathway [3,6]. Various studies have explored alternative carbon sources apart from glycerol, like starch, sucrose, and acyl glycerides. Nevertheless, comparative studies have shown that glycerol can result in CA titers up to five times higher than those obtained with sucrose or starch as carbon sources [49,72], although substrate inhibition may occur at glycerol concentrations exceeding 50 g/L [72]. The utilization of dextrose or starch tends to promote the secretion of Cephamycin C rather than CA [49,73].
Saudagar and Singhal observed high CA titers when using soy flour (88 g/L) and dextrin (10 g/L) as carbon sources, for a maximum CA yield of 1400 mg/L; however, these authors reported a higher CA yield of 1700 mg/L when using a medium with 15 g/L of glycerol with L-proline and L-glutamic acid as supplements [45]. Costa and Badino used glycerol (15 g/L) in a medium containing soybean protein isolate and tested a pulsating feed of glycerol (100 g/L) for a maximum CA concentration of 1534.3 mg/L [34]. Although glycerol utilization as key precursor of biomass and CA is essential for achieving high titers, this substance is rarely used as the sole carbon source, since secondary sources of carbon and nitrogen are incorporated. Moreover, the maximum CA concentration in glycerol-containing media reaches up to twice the concentration attained in media with starch as the carbon source [49,74].
Maranesi et al. also explored the impact of various oils and glycerol on CA production. When 10 g/L of glycerol was combined with 23 g/L of soybean oil in the medium, the resulting CA concentration was 753 mg/L [24]. Kim et al. found that triolein supplementation might enhance the CA production in wild-type cultures of S. clavuligerus NRRL 3585, achieving a final CA concentration of 989 mg/L [46]. Salem-Bekhit et al. further investigated the use of olive oil as a secondary carbon source added during the cultivation, finding that supplementation resulted in a CA yield of 1120 mg/L in a medium containing starch (10 g/L) and soybean flour. Thus, some acyl glycerides and fatty acids might enhance CA production [31]. Ribeiro et al. confirmed the good results of a glycerol and soybean protein combination via testing at the bioreactor scale, yielding 917.5 mg/L of CA using a medium with glycerol (15 g/L) and soy protein isolate (20 g/L) [54]. Similarly, Feng et al. also demonstrated the efficacy of combining glycerol with other carbon sources by implementing a medium containing glycerol (20 g/L) and dextrin (12.37 g/L), supplemented with soybean meal and triolein, reaching CA titers of 1322 mg/L.
In the case of mutant strains, the media used are likely the same as those identified as favorable for CA production in wild-type strains. Kim et al. used different media at the bench and bioreactor scales [42]. Dextrin (20 g/L) was used as a primary carbon source in the bench setup along with soybean flour, leading to 1750 mg/L of CA [42]. However, in the bioreactor, dextrin was replaced by glycerol (12.6 g/L) and the addition of soybean oil, resulting in a CA concentration of 3000 mg/L [42]. Shin et al. reported 893 mg/L of CA using a mutant strain (ORUN) in a bioreactor setup with glycerol (3%) and soy flour [56]. Li explored the use of a glycolytic-engineered mutant strain, Gap-15-7-30, in a medium containing glycerol (20 g/L) and soybean protein extract [29]. Despite using a substantial concentration of glycerol, the study reported a low CA yield of 282 mg/L, but this was still higher than that of the control experiment [29].
Starch has also been extensively used in mutant strains. Zhihan and Yanping used a mutant strain with a disrupted lat gene cultivated in a medium with soluble starch (15 g/L) and soybean flour, resulting in a low CA concentration of 269.8 mg/L [30]. Cho et al. prepared the S. clavuligerus mutants OL13 and OR, further engineered in the specific clavam regulatory genes cas1, ccaR, and claR. Media containing starch (20 g/L), soy flour, and triolein were employed, yielding notable titers ranging from 4950 to 6010 mg/L [52]. Qin et al. used the mutant strain M3-19 with a medium containing 30 g/L maize starch, soybean powder, and extracts [39]. This combination led to a CA yield of 4330 mg/L at the bioreactor scale. Kizildoğan et al. engineered the industrial strain S. clavuligerus DEPA, obtaining the recombinant S. clavuligerus IDG3, which was able to produce 6690 mg/L, the highest CA titer reported in cultivation at the bench scale, by using a medium with dextrin (20 g/L) and glycerol trioleate (5 g/L) along with soybean flour [50]. In contrast to all other studies, Kim et al. utilized a mutant strain (OL13) in a bioreactor setup with a medium containing 0.5–3% oleic acid as the sole carbon source in the absence of starch, dextrin, or glycerol, and in addition to complex nitrogen sources (N-Z amine type A and yeast extract), leading to CA titers of 1950 mg/L [46].
In fed-batch fermentation processes, the main carbon source in the feeding medium is frequently considered to prolong the growth phase or the stationary phase while supporting secondary metabolite production. Additionally, feeding may incorporate amino acids or complex nitrogen sources to provide precursors toward secondary metabolism in the late phases of cultivation. Across the studies included in this contribution, glycerol is the predominant component in the feed, contributing to higher CA concentrations than in the batch operations, even for mutant strains. Chen et al. used a wild-type strain but incorporated a much higher glycerol concentration (250 g/L) into the feed; however, a modest CA concentration of 270 mg/L was attained [58]. Neto et al. utilized a lower glycerol concentration of 10 g/L in combination with Samprosoy 90NB and malt extract in the feeding medium, which resulted in an enhanced CA yield of 404 mg/L [59]. Domingues et al. used a combination of glycerol and ornithine (40:1 ratio) in the feeding medium, which resulted in a CA yield of 650 mg/L [57]. Similarly, Teodoro et al. fed the culture with glycerol (180 g/L) and ornithine (3.7 g/L), resulting in a high CA yield of 1560 mg/L [47]. In a study by Bellão et al., glycerol at 201 g/L was used in the feeding medium, and a final CA concentration of 982.1 mg/L was attained [49]. Gómez-Rios et al. employed glycerol at 120 g/L in the feeding medium, achieving a CA concentration of 422.7 mg/L [60]. The same authors further optimized the feeding medium with glycerol (27.6 g/L) and monosodium glutamate (25.4 g/L) for a final CA concentration of 760 mg/L [61]. Kim et al. used the mutant S. clavuligerus KK strain in a fed batch with glycerol (66 g/L), obtaining 3250 mg/L of CA [42].

3.3. Amino Acids and Complex Nitrogen Sources for Enhancing Clavulanic Acid Biosynthesis

Glycerol, serving as the C-3 precursor of the CA molecule via GAP, significantly enhances the biosynthesis when the medium is supplemented with amino acids or rich nitrogen sources that supply intermediates convertible to L-arginine as the C-5 precursor of CA [75,76]. In this regard, soy-based nitrogen sources such as soybean flour and soybean protein isolate have been extensively used to enhance CA biosynthesis rates. Soybean flour has mostly been used at a concentration of 20 g/L in both mutant and wild-type strains [24,42,43]. Soybean oil at 23 g/L has also been used as a supplement along with soybean flour with a good performance. Maranesi et al. and Ortiz et al. reached 753 and 696 mg/L of CA at the bench scale using this kind of supplementation [24,43]. Ortiz et al. further scaled up to the bioreactor and a final concentration of 796 mg/L was attained [43]. Shin et al. employed a medium containing a high concentration of soy flour (47 g/L) for the cultivation of the mutant ORUN, which produced 893 mg/L of CA in the bioreactor. Similarly, Feng et al. used a high concentration of soybean meal (39.75 g/L) to reach 1322 mg/L in the bioreactor [55].
These results suggest that soybean derivatives might enhance CA production as an extensive source of amino acids. Among these derivatives, the soybean protein isolates have also been proven to be highly effective at enhancing CA production. Gouveia et al. reported a CA yield of 920 mg/L using 27 g/L of Samprosoy 90NB; when using soybean protein extract (37 g/L), higher CA titers were obtained (1140 mg/L) [22,23]. Costa and Badino achieved the highest bench-scale CA production (1534.3 mg/L) using 25 g/L of soybean protein isolate in combination with glycerol. According to Rodrigues et al., the addition of yeast extract (1 g/L) apart from soybean protein isolate did not further enhance the CA final concentration in the cultivation [40]. Qin et al. cultivated the M3-19 mutant strain using soybean powder (17 g/L) and soybean protein extract (22 g/L) for a CA yield of 4330 g/L at the bioreactor scale [39]. Ribeiro et al. employed soybean protein isolate (20 g/L) as the only complex nitrogen source for a final CA concentration of 917.5 mg/L in the bioreactor.
Amino acid supplementation is another successful strategy for enhancing CA production. Saudagar and Singhal considered the use of free amino acids in the basal medium instead of complex nitrogen sources. The standard medium contained L-proline (22.4 g/L) and L-glutamic acid (16.6 g/L), but threonine was added as a supplement (11.9 g/L), resulting in a CA yield of 1700 mg/L [45]. L-arginine (17.42 g/L) was also tested as a supplement, yielding 1400 mg/L [44]. Similarly, Teodoro et al. employed a feeding supplemented with ornithine (3.7 g/L), while the basal medium contained 20 g/L of soybean protein isolate [47]. This combination yielded a CA concentration of 1560 mg/L in a 10 L bioreactor [47]. Gomez-Rios et al. used monosodium glutamate as a supplement in the basal medium (9.8 g/L) and feeding (25.4 g/L). As in other studies, the use of the amino acid in the feeding favored the accumulation of CA up to 1–8-fold with respect to the non-supplemented feeding, yielding 760 mg/L [60,61].

3.4. Mutant and Engineered S. clavuligerus Strains for Clavulanic Acid Overproduction

In an effort to enhance the CA productivity of S. clavuligerus, random mutagenesis and strain engineering have been explored. Nevertheless, very high titers are not usual, even with mutant strains. Kim et al. tested the random mutation on wild-type S. clavuligerus ATCC 27064 by using methylnitronitrosoguanidine (NTG) [42]. Although the genome of the resultant S. clavuligerus KK was not studied by the authors, the strain was able to grow in the same media used for the wild-type strain and it was able to synthesize CA. The bacterium was immobilized in polyurethane pellets and applied at the bench and bioreactor scales in fed-batch operation [42]. The bioreactor cultivation produced 3250 mg/L, representing a 220% increase in productivity compared to the fed-batch cultivation of free wild-type cells, which produced 1500 mg/L [42].
Li and Townsend explored the enhancement of CA biosynthesis by targeting the glycolytic pathway to address the limited availability of GAP as a C-3 precursor of the CA molecule [29]. The vectors pSET152/1–95 and pGAP2Am were prepared to disrupt one of two genes, gap1 or gap2, to increase the carbon flux toward the clavam pathway. The disruption of the gap1 gene increased the CA production more than 2-fold with respect to the wild-type strain [29]. The gap2 mutant did not show any change in CA production, indicating that the enzyme encoded by gap2 might be involved in the metabolism of carbon sources other than glycerol [29].
S. clavuligerus NRRL 3585 was engineered using a pKC1139-lat vector to disrupt the lat gene encoding the Lysine-ε-aminotransferase responsible for the incorporation of L-Lysine in cephamycin C biosynthesis. Four mutants (M-1 to M-4) were constructed and no significant differences in the biomass or morphology were observed between the lat gene-disrupted mutants and the wild-type strains. However, it was found that mutations affecting cephamycin C production may improve the CA secretion rate. All the lat-gene-disrupted mutants duplicated, at least, the CA concentration of the wild-type strain and showed no significant cephamycin C production. Among the four mutants, the M-1 strain reached 2.6-fold the concentration of the wild-type strain in shake flask experiments [30].
Kim et al. selected mutants resistant to high concentrations of oleic acid. S. clavuligerus NRRL 3585 was cultivated using different vegetable oils; triolein was found to be the most effective for CA production [46]. However, the free fatty acids generated from oil hydrolysis in the culture broth may negatively impact growth and CA production. To overcome this issue, the wild-type strain was subjected to stepwise selection in the presence of increasing concentrations of oleic acid [46]. The screened strains were then treated with NTG and plated on agar containing oleic acid at concentrations higher than the minimum inhibitory concentration [46]. The mutant S. clavuligerus OL13 exhibited a minimum inhibitory concentration to oleic acid of 2.1 g/L, significantly higher than the 0.4 g/L observed in the parent strain [46]. This resistance led to improved cell growth and a maximum CA production of 1950 mg/L in the batch bioreactor, which is approximately 2-fold more than that observed in the parent strain [46].
Guo et al. explored the role of the glycerol utilization operon (gyl) in CA production [37]. The gyl operon supplies GAP and is regulated by the GylR protein in response to glycerol. An additional copy of the regulatory gene ccaR was expressed under the control of the gyl promoter (Pgyl) and integrated into S. clavuligerus NRRL 3585 [37]. The resulting transformants carrying the Pgyl-controlled ccaR exhibited a 3.19-fold increase in CA production in glycerol-enriched batch cultures compared to the control strain expressing ccaR under its native promoter PccaR [37]. This increase in CA production was associated with significantly upregulated transcription levels of ccaR, ceas2, and claR, highlighting the effectiveness of linking CA biosynthesis with glycerol metabolism to enhance production [37].
S. clavuligerus ATCC 27064 was treated with UV radiation and subjected to screening for further cultivation in a 5 L bioreactor in batch mode with glycerol and a complex nitrogen source [51]. The mutant strain 70 produced approximately 1.6-fold more CA than the wild strain at the bioreactor scale [51]. This mutant exhibited a higher CA productivity (29.5 mg/h/L), despite showing a 43% lower specific growth rate.
Qin et al. engineered the transcriptional regulator claR by fusing the promoterless kanamycin resistance gene neo downstream of the claR, resulting in the strain S. clavuligerus F613-1 NEO [39]. This claR-neo fusion strain was then subjected to physical and chemical mutageneses, followed by screening under high concentrations of kanamycin to isolate high-yield CA producers [39]. After three rounds of mutagenesis and selection, the strain M3-19 was identified as a high producer, achieving CA titers of 4330 mg/L in shake flasks, representing a 33% increase over the titer of 3260 mg/L observed in the parent strain S. clavuligerus F613-1, an industrial producer of CA [39]. Transcriptional analysis revealed that claR-neo was overexpressed more than 30-fold in the mutant strain M3-19 compared to the original strain NEO, leading to the upregulation of biosynthetic genes involved in the late stages of CA synthesis [39].
Cho et al. performed a genome analysis of the S. clavuligerus OL13 strain and another UV-prepared mutant denominated OR, revealing a mutation in the cas1 gene [52]. The overexpression of the intact cas1 gene in the OR strain led to a significant enhancement in the CA production, with a 25% increase, yielding 4.95 g/L of CA [52]. Further, the overexpression of the ccaR and claR regulators in the OR strain boosted the CA yield by about 43% up to 5.66 g/L. However, the co-expression of the intact cas1 gene with ccaR-claR did not result in an additional improvement in the CA production [52]. The cultivation at the bioreactor scale with the OR strain expressing both wild-type cas1 and ccaR-claR reached 5.52 and 6.01 g/L, respectively [52].
Shin et al. observed that the S. clavuligerus OR strain consumed less glycerol compared to the wild-type strain [56]. To address this issue, the glycerol utilization operon (glp), including the gylR-glpF1-glpK1-glpD1 genes, was overexpressed relative to the parent OR strain [56]. The OR strain was engineered with the kanamycin resistance gene neo downstream of the glp operon through homologous recombination between the glp operon and a neo gene insertion plasmid, pKD04 [56]. The promoterless neo gene was fused downstream of the glpD1 gene, which encodes glycerol-3-phosphate dehydrogenase [56]. To enhance the co-transcription of the glp operon with the neo gene, a two-step mutagenesis process involving UV irradiation and NTG was applied [56]. Following the mutagenesis and screening, the mutant strain with the highest CA titer was selected and designated as S. clavuligerus ORUN [56]. The resulting ORUN strain exhibited a 31.3% increase in CA production compared to the original OR strain, reaching 5.210 g/L in the flask culture and 6110 g/L in the bioreactor [56].
Likewise, the industrial CA strain S. clavuligerus DEPA was further engineered to enhance CA production by introducing single or multiple copies of ccaR, claR regulators, and cas2 genes under the control of various promoters [50]. Introducing an additional copy of ccaR under its native promoter, PermE, resulted in 7.6-fold and 2.3-fold increases in the CA production in the recombinant strains AK9 and ID3, respectively [50]. Additionally, integrating an extra copy of cas2 under PermE into the S. clavuligerus DEPA genome enhanced the CA production by 3.8-fold in the recombinant strain GV61 [50]. Notably, the multicopy expression of ccaR driven by PglpF led to a substantial 25.9-fold increase in CA titers in the recombinant strain IDG3 [50]. The resulting recombinant strain, S. clavuligerus IDG3, produced the highest CA concentration reported in the literature so far (6690 mg/L) [50]. Despite harboring a recombinant multicopy plasmid, IDG3 demonstrated its potential as an effective strain for industrial CA fermentation, even after seven subcultures, maintaining stable expression up to 93% in nonselective media [50].

4. Discussion

The uptake of C-3 and C-5 sources impacts the growth rate of S. clavuligerus, as these are the primary nutrients utilized during cultivation. An overview of the connections of these precursors with CA biosynthesis is displayed in Figure 4. Metabolic engineering has focused mainly on increasing the carbon fluxes toward the clavam pathway by increasing the fluxes of glyceraldehyde 3-phosphate from glycolysis and L-arginine either from TCA or urea cycle intermediates. Other approaches are intended to increase the biosynthesis rate directly in the clavam pathway by engineering the regulation factors related to biosynthetic enzymes.
Our results show that S. clavuligerus is mostly cultivated using glycerol as the main carbon source. This is partly due to its inefficient glucose transport that limits the utilization of other carbohydrates. When comparing different carbon sources, glycerol has been shown to yield CA titers up to five times higher than those obtained using starch and derivatives (see Table 1 and Table 2). Moreover, it has been reported that the use of dextrose or starch tends to favor other secondary metabolites, like cephamycin C, affecting the CA accumulation [49,73,77]. The preferential use of glycerol over glucose, due to its direct conversion to the glycolysis intermediate GAP, directly aligns with the metabolic demands of the clavam biosynthetic pathway through N2-(2-Carboxyethyl)arginine, which is the first intermediate in the pathway, as shown in Figure 3 [6,47,57].
Metabolic engineering in connection with experimental observations has provided insights into the influence of glycerol uptake in CA production. In silico fluxomics suggests that the flux through glyceraldehyde-3-phosphate dehydrogenase (GAPD) is heavily influenced by the extracellular glycerol concentration and, consequently, by the glycerol uptake [61,62]. In all scenarios, particularly in batch processes, glycerol depletion results in a significant reduction in glycolytic flux, a trend that must be reflected in the activity of pyruvate dehydrogenase (PDH) [61,75]. Therefore, rational strategies for feeding in fed-batch cultivations have improved CA titers only by maintaining longer extracellular glycerol availability [34,47,61].
Beyond the optimization of the operating variables with the aid of experimental approaches and metabolic modeling tools, strategies for improving glycerol utilization during CA production include rational strain engineering. The introduction of an extra copy of the ccaR gene into the gyl operon make their expressions responsive to glycerol presence, causing an early transcription of CA biosynthetic genes while glycerol is available, synchronizing the C-3 flux with CA biosynthesis, favoring its accumulation [37]. Similarly, co-transcriptional strategies involving the glp operon and the kanamycin resistance neo gene have further confirmed the essential role of glycerol uptake and utilization in enhancing CA biosynthetic fluxes [56]. The improvement in glycerol utilization during CA production has probably been the most successful strategy for improving CA titers so far, yielding up to 3.6-fold the highest CA titers reported for wild-type S. clavuligerus.
Medema et al. studied the genetic changes in an industrial strain, S. clavuligerus DS48802, which was obtained by several random mutation cycles [78]. In this strain, genes involved in glycerol uptake and metabolism are upregulated more than two-fold, evidencing the increased capacity of glycerol utilization and, hence, the GAP fluxes as a crucial CA precursor. At the same time, aconitase and citrate synthases from the TCA cycle are downregulated, suggesting reduced fluxes from glycolysis, favoring CA biosynthesis instead [78]. This is commensurate with the observations of Li and Townsend when deleting the gap1, blocking the conversion of GAP into 1,3-bisphosphoglycerate, decreasing the flux toward the TCA cycle, and increasing the GAP availability for further condensation with arginine [29]. The deletion of pyk genes has been suggested via dynamic flux balance analysis as a promising modification that would contribute to increasing the GAP fluxes toward CA production, showing an improved balance between glycerol utilization, biomass production, and CA secretion [79]. The modification of pyk decreases pyruvate synthesis from phosphoenolpyruvate, causing the carbon flux to be rerouted toward the pentose–phosphate pathway and CA biosynthesis [79]. To compensate for the pyruvate demand from pyruvate dehydrogenase, the malic enzyme’s activity drives the demand for L-malate, inverting the flux through malate dehydrogenase, thereby maintaining the TCA cycle fluxes. In this sense, the downregulation of pyk genes encoding one isoform of pyruvate kinase was experimentally observed in the DS48802 strain, highlighting the potential contribution to increase the GAP flux during CA production [78].
As observed in Table 1 and Table 2, either in batch or fed-batch operations, the addition of protein isolates, protein hydrosilates, or free amino acids has a positive effect on the net CA secretion in S. clavuligerus cultures. However, the complex interactions between the amino acid uptake, TCA, and urea cycles along with CA biosynthesis are not completely understood. Furthermore, it is difficult to trace the effect of specific amino acid profiles when complex nitrogen sources are used. In contrast, experimental approaches focused on the effect of individual amino acids have contributed to understanding the nutritional regulation of CA biosynthesis. From these experimental studies, it is clear that several amino acids influence the CA production in S. clavuligerus, such as, in descending order of impact, L-glutamic acid, L-ornithine, L-threonine, L-arginine, and L-proline [28,40,45,57,61].
The utilization of L-arginine and L-ornithine as supplements contribute directly to CA biosynthesis, since N2-(2-Carboxyethyl)arginine recruits L-arginine directly from the urea cycle [28,45,57]. Simultaneously, L-ornithine uptake decreases L-arginine degradation, increasing the availability of this C-5 precursor for CA biosynthesis [75]. Furthermore, in the mutant strain Gap15-7-30, supplementation with L-arginine contributes to overcoming the limitation caused by the lack of sufficient C-5 precursors under the abundant pool of GAP generated by the gap1 disruption [29]. Under these conditions, N2-(2-Carboxyethyl)arginine synthase (CEAS) is expected to be saturated with both precursors, GAP, and L-arginine, with a net increase along the clavam pathway [29].
Transcriptomics and RT-qPCR have suggested that rich organic nitrogen sources enhance CA biosynthesis by upregulating the expression of the clavam gene clusters, including ceas1, ceas2, bls1, bls2, cas2, pah2, pah1, gcaS, cad, and claR [41]. Additionally, transcriptomic and metabolomic data have revealed the link between L-arginine synthesis and CA production. In this regard, rich organic nitrogen influences the expression of genes involved in arginine metabolism (argJ, argB, argC, argD, argR, argG, and argH) [41]. Genes involved in the conversion of glutamine into arginine are also upregulated, supporting the hypothesis that supplementation with glutamic acid, arginine, or glutamine favors CA production [41].
Supplementation with C-5 precursors along the glutamate/aspartate degradation pathway boosts CA secretion more effectively, potentially due to the role of 2-oxoglutarate in both TCA cycle regulation and CA biosynthesis. Increasing the 2-oxoglutarate availability, whether through glutamate or its precursors, amplifies the fluxes through the urea cycle toward arginine and CA synthesis. In this sense, L-glutamate has been referenced by in silico dynamic fluxomics as one of the best supplements for enhancing CA production, which has also been experimentally confirmed [61,62].
L-threonine also has a remarkable effect on CA production, despite not being directly involved in L-arginine synthesis, the urea cycle, or CA biosynthesis [44,45]. A flux balance analysis suggests that the metabolism of L-threonine can also yield glutamate via isoleucine through transamination, although this is a more complex pathway that produces several by-products [75]. Moreover, L-threonine contributes to the pool of Acetyl-CoA, which is extensively required for the amino acids’ metabolism. L-proline degradation also produces L-glutamate via L-proline and glutamate-γ-semialdehyde dehydrogenases, enhancing fluxes toward L-aspartate and L-arginine in the urea cycle and, consequently, CA biosynthesis [75]. The high availability of L-proline and L-glutamate also affects reactions involving 2-oxoglutarate, thereby stimulating the flux of pyruvate dehydrogenase, which regulates the carbon path to the TCA cycle downstream [62]. Therefore, it is expected that a complex amino acid profile provides influxes of several C-5 precursors, enhancing the carbon flux in the urea cycle and its connected pathways.
The genome of the industrial strain DS48802 showed that preserving a significant acetyl-CoA pool through the incomplete downregulation of the TCA cycle would increase C-5 precursors like L-ornithine and L-glutamate [78]. More important is the capacity of the strain to sustain 2-oxoglutarate production, which is directly linked to L-glutamate degradation. In addition, three molecules of 2-oxoglutarate are consumed per CA molecule synthesized, primarily during the oxidative steps catalyzed by clavaminate synthase, which yields succinate [62,78].
Although they are synthesized in different secondary metabolic pathways, cephamycin C and CA seem to compete for precursors, especially nitrogen from amino acid metabolism [30,77]. It has been shown that the addition of L-lysine and maltose increases CA production, despite L-lysine being a precursor for cephamycin C, while the addition of high concentrations of diaminopropane and L-lysine enhances cephamycin C but decreases CA production [77]. The disruption of the lat gene in S. clavuligerus led to an important increase in CA production as a consequence of the impossibility of incorporating L-α-aminoadipate, L-cysteine, and L-valine into the biosynthesis of cephamycin C, and some of these precursors were likely redirected toward the glycolysis, urea, and TCA cycles, favoring the flux along the clavam pathway [30]. Both pathways, clavams, and cephamycin C biosynthesis are regulated by the same gene, ccaR, suggesting that the elimination of the capacity to synthesize metabolites in the cephamycin C pathway would exert a regulatory influence on the expression of CA biosynthetic genes, thereby impacting CA production [30].
The importance of preserving adequate nutritional conditions for achieving high CA titers is evident in experiments involving fed-batch operations. As observed in Table 2, fed-batch operations have consistently been shown to enhance the CA production for both wild-type and mutant strains of S. clavuligerus. When compared with the batch processes, the fed-batch process supports longer biomass production and an extended CA production phase [28,49,57,61,62]. In fed-batch operations, the glycerol fed in excess supports the steady production of biomass at a rate approximately equal to the dilution until the end of the cultivation [60]. Although adding the primary carbon source to the feed has been shown to improve CA accumulation, the addition of amino acids to the feed seems to increase the CA biosynthesis rate more.
Dynamic flux analysis has been used as a tool to optimize CA production by rationally modifying the operational parameters, such as the feed composition and the timing of the fed-batch initiation. These adjustments resulted in more efficient carbon utilization and extended CA biosynthesis, demonstrating that relatively simple process modifications can significantly increase the CA production in S. clavuligerus [61,62]. Feed media with glycerol, L-threonine, and L-arginine have been reported to maintain C-3 fluxes into CA biosynthesis, especially by promoting pyruvate production from the degradation of L-threonine to glycine and L-serine [29,45]. In silico studies have suggested that serine is further metabolized into pyruvate, contributing to the C-3 precursor availability in the absence of glycerol [62]. Fed-batch operations might contribute to maintaining stable fluxes through CA biosynthetic pathways beyond 50 h of cultivation, extending the productive phase [61,62]. Such stability contrasts with the rapid decline in fluxes that occurs in batch cultures once the substrates deplete [61,62]. A balanced feeding rate and adequate carbon/supplement concentrations based on the substrate uptake prevent substrate accumulation that could lead to overflow metabolism, negatively impacting the flux through CA biosynthesis [61,79]. In this regard, the feed medium formulation should provide adequate amounts of glycerol as a C-3 precursor and C-5 precursor, such as L-glutamate or L-proline, along with controlled concentrations of ammonia and phosphate at narrow levels to ensure the nutrient limitation triggering CA synthesis while supporting central metabolism activity [62,75].
Phosphate is essential for biomass synthesis in S. clavuligerus, but its uptake must be controlled due to its repressive effect on CA biosynthesis [45,80]. Saudagar and Singhal demonstrated that concentrations as low as 200 mM inhibit CA production drastically, while the optimal phosphate concentrations for CA production must be around 10 mM. Phosphate limitation slightly reduces growth rates without completely stopping them, suggesting the existence of metabolic mechanisms that help to regulate phosphate compensation and energy utilization through antibiotic biosynthesis [81,82]. In silico metabolic studies suggest that phosphate limitation increases reaction fluxes in several glutamate-dependent pathways related to phosphate (Pi) and pyrophosphate (PPi), which are connected to CA biosynthesis, that release Pi and PPi molecules as by-products [69]. In the Streptomyces genus, the phosphate limitation triggers adaptive responses that induce the strong activation of oxidative and amino acid metabolism, thus generating ATP and favoring antibiotic synthesis as a way to adjust the ATP levels under low-phosphate conditions [69,82,83]. In this regard, a flux balance analysis using a metabolic model of S. clavuligerus suggested that CA biosynthesis occurs alongside reduced ATP generation given the negative shadow prices for the ADP/ATP and NADP+/NADPH ratios [76].
Apart from nutritional conditions and genetic factors, aeration and agitation also significantly impact CA yields. In stirred-tank bioreactors, higher oxygen mass transfer is achieved by controlling the aeration rate and, especially, the stirring velocities. Both factors, the aeration and shear stress, seem to favor CA biosynthesis, as the higher the agitation velocity, the higher the CA accumulation. Moreover, a strong correlation between the mycelial macromorphology of S. clavuligerus and CA production has been observed [54,60]. Increased agitation speeds boost production but higher oxygen flows do not always have the same effect [48,54]. Ribeiro et al. concluded that, comparatively, the shear rate exhibited the greatest impact on CA production: a 145% increase in the oxygen transfer rate resulted in a 29% increase in CA productivity, while a 134% increase in the shear rate led to enhanced CA productivity up to 53% [54]. It is worth mentioning that CA biosynthesis requires molecular oxygen in the reaction steps catalyzed by the clavaminate synthase. Indeed, experimental studies indicate that high shear stress enhances growth rates and CA production, while low-shear stress conditions lead to larger and thicker mycelial structures, potentially reducing productivity due to changes in the nutrient and oxygen transport efficiency [60]. Intense agitation leads to the formation of shorter and thinner viable hyphal fragments capable of growing under sufficient nutrient availability. This morphology favors mass transfer, enhancing the S. clavuligerus growth rate and CA secretion [60].

5. Concluding Remarks

This systematic review highlights the substantial advancements made in enhancing clavulanic acid production. Multidisciplinary approaches integrating nutrient optimization, systems biology, strain engineering, and process control have contributed to understanding the complex relationships between nutritional regulation and antibiotic biosynthesis in S. clavuligerus. Glycerol is the preferred carbon source due to its direct involvement in the clavulanic acid biosynthetic pathway by providing GAP, the C-3 precursor in CA biosynthesis. Glycerol enables higher CA titers compared to alternative carbon sources like starch and dextrins. Optimized feeding strategies in fed-batch operations have further improved carbon utilization during CA production.
Organic nitrogen sources, in the form of both complex substrates like soybean protein isolates and free amino acids such as L-arginine and L-glutamate, play a critical role in enhancing CA biosynthetic fluxes. These nitrogen supplements provide the C-5 precursors required for CA biosynthesis through the TCA and urea cycles. The successful application of these nitrogen sources in batch and fed-batch operations supports the importance of balancing the nutrient uptake during S. clavuligerus cultivation. Thus, optimized fed-batch operations emerge as an effective strategy at the bioreactor scale for sustaining the biomass and antibiotic secretion by carefully regulating the nutrient supply.
Genetic perturbation has been the most effective tool for enhancing the CA biosynthetic capacity of S. clavuligerus. Techniques like UV radiation and chemical mutagenesis have generated stable mutants for CA overproduction. Additionally, rational strain engineering focused on overexpressing key regulatory genes, such as ccaR and claR, under glycerol-inducible promoters have demonstrated significant enhancement in the flux through CA biosynthesis. The targeted disruption of competing pathways, like cephamycin C biosynthesis, redirects metabolic fluxes toward the clavam pathway and ultimately CA. These strategies have not only significantly improved CA production but have also helped to stabilize industrial strains, reducing strain degeneration and the need for frequent strain screening.

6. Future Perspectives

The integration of systems biology approaches with process optimization would also contribute to achieving even higher CA titers in industrial settings. Data from omics technologies combined with dynamic metabolic flux analysis will enable the development of more predictive models for strain improvement and process optimization. The use of machine learning and artificial intelligence approaches to analyze large datasets from fermentation processes could further accelerate the identification of the optimal operational parameters during bioprocess development.
Additionally, the continued refinement of genetic engineering techniques, particularly through the application of genome editing tools such as CRISPR-Cas9, could be employed to precisely manipulate regulatory and biosynthetic genes within S. clavuligerus. For instance, targeting bottlenecks in the biosynthesis pathway or enhancing the expression of regulatory genes could unlock further improvements in CA yields. Additionally, the overexpression of transport proteins responsible for the precursors’ uptake could further improve nutrient assimilation, thereby driving higher productivity. The construction of synthetic regulatory circuits and biosynthetic pathways, potentially integrating non-native enzymes or regulatory proteins, could offer more refined control over clavam biosynthesis, leading to S. clavuligerus strains capable of more efficient precursor utilization and reduced by-product formation.

Author Contributions

Conceptualization, D.G.-R. and H.R.-M.; methodology, D.G.-R. and H.R.-M.; validation, D.G.-R. and H.R.-M.; formal analysis, L.M.G.-G. and D.G.-R.; investigation, L.M.G.-G. and D.G.-R.; resources, D.G.-R. and H.R.-M.; data curation, L.M.G.-G.; writing—original draft preparation, L.M.G.-G. and D.G.-R.; writing—review and editing, D.G.-R. and H.R.-M.; visualization, L.M.G.-G.; supervision, D.G.-R.; project administration, D.G.-R.; funding acquisition, D.G.-R. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge the financial support of Universidad de Valle, grant CI 21258.

Data Availability Statement

All data generated and analyzed are included in this research article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 10 00526 g001
Figure 1. Typical vegetative mycelium of S. clavuligerus in submerged cultivation: (a) shake flask; (b) bioreactor.
Figure 1. Typical vegetative mycelium of S. clavuligerus in submerged cultivation: (a) shake flask; (b) bioreactor.
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Figure 2. CA biosynthesis in S. clavuligerus. Red, blue, and green carbons correspond to atoms coming from L-arginine, glyceraldehyde 3-phosphate, and glycine, respectively.
Figure 2. CA biosynthesis in S. clavuligerus. Red, blue, and green carbons correspond to atoms coming from L-arginine, glyceraldehyde 3-phosphate, and glycine, respectively.
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Figure 3. PRISMA algorithm for article selection.
Figure 3. PRISMA algorithm for article selection.
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Figure 4. Connections of C-3 and C-5 precursors with clavulanic acid biosynthesis.
Figure 4. Connections of C-3 and C-5 precursors with clavulanic acid biosynthesis.
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Table 1. Studies on batch cultivation of S. clavuligerus for CA production.
Table 1. Studies on batch cultivation of S. clavuligerus for CA production.
Ref.TypeStrainScaleMedium
(g/L)		Volume
(mL)		Time
(h)		Aeration
(vvm)		pHTemperature
(°C)		Agitation
(rpm)		Supplement
(g/L)		CA
(mg/L)		Biomass
(g/L)
[22]WildNRRL 3585BenchGlycerol (15); bacteriological peptone (10); K2HPO4 (0.8)50096-6.528250Samprosoy 90NB (27)9204.8
Soybean meal (80)4724.8
[23]WildNRRL 3585BenchGlycerol (15); K2HPO4 (0.8)50072-6.528250Corn steep liquor (36.5); protein extract of soybean (27)400-
Yeast extract (12.8); protein extract of soybean (27)725-
Bacteriological peptone (10); protein extract of soybean (27)900-
Protein extract of soybean (37)1140-
[24]WildATCC 27064BenchSoluble starch (10); soybean flour (20); soybean oil (23); K2HPO4 (1.2)500100-7.028250-458-
Soybean flour (20); soybean oil (28); K2HPO4 (1.2)500120-7.028250-478-
Glycerol (10); soybean flour (20); soybean oil (23); K2HPO4 (1.2)500120-7.028250Soybean oil (16)420-
Soybean oil (23)722-
Soybean oil (30)753-
Corn oil (23)680-
Sunflower oil (23)660-
[25]WildATCC 27064BenchGlycerol (18.75); soybean meal powder (42); KH2PO4 (0.125); MgSO4·7H2O (0.25); FeSO4·7H2O (0.3); ornithine (1.32)300---28250-526-
[26]WildATCC 20764BioreactorGlycerol (20); malt extract (10); bacteriological peptone (10)50001200.57.0261300-2505.4
[27]WildATCC 27064BioreactorGlycerol (15); Samprosoy 90NB (10); malt extract (10); yeast extract (1); K2HPO4 (2.5)4000800.56.828600-254-
800-475-
1000-614-
800-482-
[28]WildATCC 27064BioreactorGlycerol (15); malt extract (10); yeast extract (1); K2HPO4 (2.5); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)4000640.56.828800Samprosoy 90NB (20)376
[29]MutantGap-15-7-30-Glycerol (20); soybean protein extract (5.5); K2HPO4 (0.8)-216-7.028300-2823.0 1
[30]MutantpKC1139-lat gene disruptedBenchSoybean flour (15); soluble starch (4.7); K2HPO4 (0.1); FeSO4·7H2O (0.2)25096-6.828220-269.86.0
[31]WildATCC 27064BenchStarch (10); soybean flour (20); oil (23); phosphate (1.2); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)500140-7.028250Olive oil1120-
[32]WildDSM 738BenchMalt extract (0.3); glycerol (0.05); soy flour (0.05); FeSO4 (0.00001); and MnSO4 (0.00001)25072--28220-343.87-
[33]WildMTCC 1142BenchGlycerol (15); sucrose (20); L-arginine (1.7); L-glutamic acid (16.8); CaCl2 (0.4); FeCl3·6H2O (0.1); K2HPO4 (2); NaCl (5); MnCl2·4H2O (0.1); ZnCl2 (0.05); MgSO4·7H2O (1)250120-7.025200-1129-
[34]WildATCC 27064BenchGlycerol (15); soybean protein isolate (25); K2HPO4 (0.8); MgSO4·7H2O (0.75)500250-6.820250-1266.2-
-631.6 2-
Glycerol (100)1534.3-
Glycerol (200)1495.1-
Glycerol (300)1465.4-
Glycerol (400)1460.0-
[35]Mutantlat::scarBench--120--28--790.686
[36]WildATCC 27064BenchSoybean flour (20);
glycerol (10); K2HPO4 (1.2); soy oil (23); MnCl2.4H2O (0.001); FeSO47H2O (0.001); ZnSO47H2O (0.001)		500120-7.225250-815.88.8
[37]MutantpSGRBenchSoybean flour (20); dextrin (10); glycerol (5); KH2PO4 (0.6); MOPS (8)50072-7.128250-838.7-
[38]Mutant120A3BenchSoluble starch (25); glycerol (10 mL); soybean
meal (30); KH2PO4 (0.1); FeSO47H2O (0.1)		250144-7.028200-2755.6
[39]MutantM3-19BenchSoybean powder (1.7); soybean protein extract (2.2); maize starch (3); KCl (0.15); MgCl2·6H2O (0.1); MgSO4·7H2O (0.2); CaCl2·2H2O (0.04) 3100120-7.125250-4330-
F613-1Bench3260-
[40]WildATCC 27064BenchGlycerol (15); yeast extract (1); soybean protein isolate (20); K2HPO4 (0.5); MgSO4·7H2O (0.4); FeSO4·7H2O (0.4); MnCl2·4H2O (0.001); ZnSO4·7H2O (0.001)25072–84-6.827250-437-
[41]MutantF613-1BenchPeptone from soybean (0.08); dextrin (0.01); glycerol trioleate (0.008); KCl (0.015); MgCl2·6H2O (0.01); K2HPO4 (0.02); CaCl2·2H2O (0.004); FeCl2·6H2O (0.0008); ZnCl2 (0.0001); NaCl (0.0018); MOPS (0.08)250144-7.125200-39018.7
(wet weight)
[42]MutantKKBenchSoybean flour (10); dextrin (20); soybean oil (2 4)25019217.028400–500-1750-
BioreactorSoybean flour (15); glycerol (12.6); KH2PO4 (1); MgSO4 (4); soybean oil (2 5)250019217.028400–500-3000-
[43]WildATCC 27064BenchGlycerol (10); soybean flour (20); soybean oil (23); K2HPO4 (1.2); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)500120–160-6.828250-698-
Bioreactor40001000.56.828800-796-
[44]WildMTCC 1142BioreactorSoybean oil (4); soybean flour (88); yeast extract (15); dextrin (10); K2HPO4 (2)-96-7.025200L-arginine (17.42)1400-
7.525200-495-
[45]WildMTCC 1142BioreactorGlycerol (15); L-proline (22.4);
L-glutamic acid (16.6); sucrose (20); K2HPO4 (2); CaCl2 (0.4); FeCl3·6H2O (0.1); NaCl (5); MnCl2·4H2O (0.1); ZnCl2 (0.05); MgSO4·7H2O (1)		-96-7.0--L-threonine (11.9)1700-
K2HPO4 (1.742)87824.0
[46]WildNRRL 3585BioreactorStarch (10); soybean flour (20); oil (23); phosphate (1.2); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)7000140-7.028220Triolein (22)989-
MutantOL13BioreactorN-Z amine type A (2); yeast extract (1); beef extract (1); oleic acid (0.5–3)7000140-8.028250Triolein 19502.0
Olive oil500-
[47]WildATCC 27064BioreactorGlycerol (15); soybean protein isolate (20); K2HPO4 (0.8); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001); salt solution (1); soybean oil (1)5000660.56.828800Ornithine (0.66)560-
[48]WildATCC 27064BioreactorGlycerol (15); soybean protein isolated (10); malt extract (10); yeast extract (1); K2HPO4 (2.5); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)6000603.06.830--454-
4000600.56.830800-402-
[49]WildDSM 41826BioreactorGlycerol (10); soybean meal (11); L-lysine (18.3); yeast extract (0.5); K2HPO4 (1.75); MgSO4·7H2O (0.75); CaCl2·2H2O (0.2); NaCl (2.0); FeSO4·7H2O (0.005); MnCl2·4H2O (0.005); ZnSO4·7H2O (0.005); sodium thiosulfate (1)5000800.56.828800Glycerol (15)348.5-
[50]MutantIDG3BioreactorSoybean flour (20); dextrin (10); KH2PO4 (0.6); GTO (5); MOPS (10.5); oligo element solution (CaCl2) (10); MgCl2.6H2O (10); FeCl3 (3); ZnCl2 (0.5); MnSO4 H2O (0.5); NaCl (10) (10 mL)----23.5-Glycerol trioleate (0.8 mL/40 mL)6647-
[51]MutantMutant 70BioreactorGlycerol (15);
malt extract (10); yeast extract (1); soytone (15);
arginine (2.62); KH2PO4 (0.63); MgSO47H2O (0.75);
salt solution (1 mL); MnCl2.4H2O (0.001);
FeSO47H2O (0.001); ZnSO47H2O (0.001);
MOPS buffer (21) 		5000480.56.828800-500-
[52]MutantOR/pCCAR-CLARBioreactorStarch (10); soybean meal (20); triolein (23); phosphate (1.2); trace elements; antifoam (1 mL/L)7000-Over 0.37.0-300–700-6010-
[53]WildDM738BioreactorYeast extract (4); malt extract (10); dextrose (4)1800480.257.0301000Olive pomace oil (0.6% v/v)32536.0
[54]WildATCC 27064BioreactorGlycerol (15); soybean
protein isolate (20); MOPS (21); MgSO47H2O (0.75);
K2HPO4 (0.80); and salt solutions (1)		5100720.56.8281000-917.538.4
[55]WildATCC 27064BioreactorGlycerol (20); dextrin (12.37); soybean meal (39.75); KH2PO4 (1.2); triolein (26.98 mL); trace element solution (2 mL)50,000620.287.028300-1322-
[56]MutantORUNBioreactorGlycerol (0.3); soy flour (0.47); MOPS (0.105); NaH2PO4 (0.016); NaCl (0.001); MgSO4·7H2O (0.001); FeCl3·6H2O (0.0004); MnSO4·H2O (0.00005); CuCl2·2H2O (0.00005); ZnCl2 (0.00005)7000136--28220Glycerol (10)893-
1 Reported cell wet weight (mg). 2 This run was subject to a temperature reduction with Ti = 25 °C. 3 Media compositions are reported in %. 4 mL per liter. 5 mL per liter.
Table 2. Studies on fed-batch cultivation of S. clavuligerus for CA production.
Table 2. Studies on fed-batch cultivation of S. clavuligerus for CA production.
Ref.TypeStrainScaleMedium
(g/L)		Feed
(g/L)		Volume (mL)Feed Time
(h)		Flow
(mL/h)		Time
(h)		Aeration
(vvm)		pHTemperature
(°C)		Agitation
(rpm)		CA
(mg/L)		Biomass
(g/L)
[29]MutantGap 15-7-30BenchGlycerol (20); protein extract from soybean (5.5); K2HPO4 (0.8); MOPS (21)Arginine (2.35)-60-216-7.02830041725.0 1
[57]WildATCC 27064BenchGlycerol (15); soybean protein isolate (15.5); yeast extract (1); K2HPO4 (0.8); MgSO4·7H2O (0.75); MnCl2·4H2O (1); FeSO4·7H2O (1); ZnSO4·7H2O (1)Glycerol:ornithine (40:1) 250048-144-6.8282503908.0
BioreactorGlycerol:ornithine (40:1) 2500048-14416.828-6507.2
[42]MutantKKBioreactorSoybean flour (15); glycerol (12.6); KH2PO4 (1); MgSO4 (4); soybean oil (2 3)Glycerol (66 4)250048-19217.028400–5003250-
[58]WildATCC 27064BioreactorGlycerol (20); soy meal extract (1000) 5; peptone (10); KH2PO4 (1); MgSO4·7H2O (1)Glycerol (250)5000601012017.02850027010.6
[59]WildATCC 27064BioreactorGlycerol (15); Samprosoy 90NB (10); malt extract (10); yeast extract (1); K2HPO4 (2.5); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)Glycerol (10); Samprosoy 90NB (10); malt extract (10); yeast extract (1); K2HPO4 (2.5); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001)30002480960.56.8288004049.9
[45]WildMTCC 1142BioreactorGlycerol (15); L-proline (22.4); L-glutamic acid (16.6); sucrose (20); K2HPO4 (2); CaCl2 (0.4); FeCl3·6H2O (0.1); NaCl (5); MnCl2·4H2O (0.1); ZnCl2 (0.05); MgSO4·7H2O (1)Threonine (595.6)-60-130-7.0--186313.0
[47]WildATCC 27064BioreactorGlycerol (15); soybean protein isolate (20); K2HPO4 (0.8); MgSO4·7H2O (0.75); MnCl2·4H2O (0.001); FeSO4·7H2O (0.001); ZnSO4·7H2O (0.001); salt solution (1); soybean oil (1)Glycerol (180); ornithine (3.7)500024101200.56.8288001506-
10,00024201200.56.8288001560-
[49]WildDSM 41826BioreactorGlycerol (10); soybean meal (11); L-lysine (18.3); yeast extract (0.5); K2HPO4 (1.75); MgSO4·7H2O (0.75); CaCl2·2H2O (0.2); NaCl (2.0); FeSO4·7H2O (0.005); MnCl2·4H2O (0.005); ZnSO4·7H2O (0.005); sodium thiosulfate (1)Glycerol (201)5000245-0.56.828800982.1-
[60]WildDSM 41826BioreactorGlycerol (9.3); K2HPO4 (0.8); (NH4)2SO4 (1.26); monosodium glutamate (9.8); FeSO4·7H2O (0.18); MgSO4·7H2O (0.72)Glycerol (120); K2HPO4 (2); (NH4)2SO4 (8)15,00037351570.66.828300–500422.712.3
[61]WildDSM 41826BioreactorGlycerol (0.8);
(NH4)2SO4 (1.26); monosodium glutamate (9.8); FeSO47H2O (0.18);
MgSO47H2O (0.72); MOPS (10.5); trace element solution (1.44 mL)		Glycerol (120);
K2HPO4 (2); (NH4)2SO4 (8)		15,00037351100.5–0.76.828300–500467.213.8
[62]WildDSM 41826BioreactorGlycerol (15); L-proline (22.4); L-glutamic acid (16.6); sucrose (20); K2HPO4 (2); CaCl2 (0.4); FeCl3·6H2O (0.1); NaCl (5); MnCl2·4H2O (0.1); ZnCl2 (0.05); MgSO4·7H2O (1)Arginine (21.07); threonine (119.12); K2HPO4 (2); (NH4)2SO4 (8)5000180.02 L−11200.66.828300–6003293.321.2
1 Reported cell wet weight (mg). 2 Molar ratio. 3 mL per liter. 4 mL. 5 Media composition was reported in mL/L.
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MDPI and ACS Style

Gómez-Ríos, D.; Gómez-Gaona, L.M.; Ramírez-Malule, H. Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus. Fermentation 2024, 10, 526. https://doi.org/10.3390/fermentation10100526

AMA Style

Gómez-Ríos D, Gómez-Gaona LM, Ramírez-Malule H. Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus. Fermentation. 2024; 10(10):526. https://doi.org/10.3390/fermentation10100526

Chicago/Turabian Style

Gómez-Ríos, David, Luisa María Gómez-Gaona, and Howard Ramírez-Malule. 2024. "Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus" Fermentation 10, no. 10: 526. https://doi.org/10.3390/fermentation10100526

APA Style

Gómez-Ríos, D., Gómez-Gaona, L. M., & Ramírez-Malule, H. (2024). Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus. Fermentation, 10(10), 526. https://doi.org/10.3390/fermentation10100526

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