Biotin concentration affects anaplerotic reactions functioning in glutamic acid production in Corynebacterium glutamicum.

The study investigates the effect of biotin concentration on the role of anaplerotic reactions catalysed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) in glutamic acid production by Corynebacterium glutamicum. C. glutamicum requires biotin for its growth, and its glutamic acid production can be induced by the addition of Tween 40 or penicillin or by biotin limitation. The biotin enzyme PC and the non-biotin enzyme PEPC catalyse two anaplerotic reactions to supply oxaloacetic acid to the TCA cycle in C. glutamicum. Therefore, they are crucial for glutamic acid production in this bacterium. In this study, we investigated the contribution of each anaplerotic reaction to Tween 40- and penicillin-induced glutamic acid production using disruptants of PEPC and PC. In the presence of 20 µg l-1 biotin, which is sufficient for growth, the PEPC-catalysed anaplerotic reaction mainly contributed to Tween 40- and penicillin-induced glutamic acid production. However, when increasing biotin concentration 10-fold (i.e. 200 µg l-1), both PC- and PEPC-catalysed reactions could function in glutamic acid production. Western blotting revealed that the amount of biotin-bound PC was reduced by the addition of Tween 40 and penicillin in the presence of 20 µg l-1. However, these induction treatments did not change the amount of biotin-bound PC in the presence of 200 µg l-1 biotin. These results indicate that both anaplerotic reactions are functional during glutamic acid production in C. glutamicum and that biotin concentration mainly affects which anaplerotic reactions function during glutamic acid production.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes.

BACKGROUND: With a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs. RESULTS: In this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL-1) and cutinase titre (660 mg L-1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L-1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%. CONCLUSION: Extracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Sustainable production of the drug precursor tyramine by engineered Corynebacterium glutamicum.

Tyramine has attracted considerable interest due to recent findings that it is an excellent starting material for the production of high-performance thermoplastics and hydrogels. Furthermore, tyramine is a precursor of a diversity of pharmaceutically relevant compounds, contributing to its growing importance. Given the limitations of chemical synthesis, including lack of selectivity and laborious processes with harsh conditions, the biosynthesis of tyramine by decarboxylation of L-tyrosine represents a promising sustainable alternative. In this study, the de novo production of tyramine from simple nitrogen and sustainable carbon sources was successfully established by metabolic engineering of the L-tyrosine overproducing Corynebacterium glutamicum strain AROM3. A phylogenetic analysis of aromatic-L-amino acid decarboxylases (AADCs) revealed potential candidate enzymes for the decarboxylation of tyramine. The heterologous overexpression of the respective AADC genes resulted in successful tyramine production, with the highest tyramine titer of 1.9 g L-1 obtained for AROM3 overexpressing the tyrosine decarboxylase gene of Levilactobacillus brevis. Further metabolic engineering of this tyramine-producing strain enabled tyramine production from the alternative carbon sources ribose and xylose. Additionally, up-scaling of tyramine production from xylose to a 1.5 L bioreactor batch fermentation was demonstrated to be stable, highlighting the potential for sustainable tyramine production. KEY POINTS: • Phylogenetic analysis revealed candidate l-tyrosine decarboxylases • C. glutamicum was engineered for de novo production of tyramine • Tyramine production from alternative carbon substrates was enabled.

Metabolic engineering of Corynebacterium glutamicum for the production of pyrone and pyridine dicarboxylic acids.

Environmental concerns from plastic waste are driving interest in alternative monomers from bio-based sources. Pseudoaromatic dicarboxylic acids are promising alternatives with chemical structures similar to widely used petroleum-based aromatic dicarboxylic acids. However, their use in polyester synthesis has been limited due to production challenges. Here, we report the fermentative production of five pseudoaromatic dicarboxylic acids, including 2-pyrone-4,6-dicarboxylic acid (PDC) and pyridine dicarboxylic acids (PDCAs: 2,3-, 2,4-, 2,5-, and 2,6-PDCA), from glucose using five engineered Corynebacterium glutamicum strains. A platform C. glutamicum chassis strain was constructed by modulating the expression of nine genes involved in the synthesis and degradation pathways of precursor protocatechuate (PCA) and the glucose-uptake system. Comparative transcriptome analysis of the engineered strain against wild-type C. glutamicum identified iolE (NCgl0160) as a target for PDC production. Optimized fed-batch fermentation conditions enabled the final engineered strain to produce 76.17 ± 1.24 g/L of PDC. Using this platform strain, we constructed 2,3-, 2,4-, and 2,5-PDCA-producing strains by modulating the expression of key enzymes. Additionally, we demonstrated a previously uncharacterized pathway for 2,3-PDCA biosynthesis. The engineered strains produced 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA through fed-batch fermentation. To complete the portfolio, we introduced the 2,6-PDCA biosynthetic pathway to an L-aspartate pathway-enhanced C. glutamicum strain, producing 15.01 ± 0.03 g/L of 2,6-PDCA in fed-batch fermentation. The metabolic engineering strategies developed here will be useful for the production of pseudoaromatic chemicals.

Thermo-adaptive evolution of Corynebacterium glutamicum reveals the regulatory functions of fasR and hrcA in heat tolerance.

BACKGROUND: High-temperature fermentation technology is promising in improving fermentation speed and product quality, and thereby widely used in various fields such as food, pharmaceuticals, and biofuels. However, extreme temperature conditions can disrupt cell membrane structures and interfere with the functionality of biological macromolecules (e.g. proteins and RNA), exerting detrimental effects on cellular viability and fermentation capability. RESULTS: Herein, a microbial thermotolerance improvement strategy was developed based on adaptive laboratory evolution (ALE) for efficient high-temperature fermentation. Employing this strategy, we have successfully obtained Corynebacterium glutamicum strains with superior resistance to high temperatures. Specifically, the genome analysis indicated that the evolved strains harbored 13 missense genetic mutations and 3 same-sense genetic mutations compared to the non-evolved parent strain. Besides, reverse transcription quantitative PCR analysis (RT qPCR) of the hrcA-L119P mutant demonstrated that both groEL genes were upregulated under 42 °C, which enabled the construction of robust strains with improved heat tolerance. Furthermore, a significant increase in FAS-IA and FAS-IB expression of the fasR-L102F strain was proved to play a key role in protecting cells against heat stress. CONCLUSIONS: This work systematically reveals the thermotolerance mechanisms of Corynebacterium glutamicum and opens a new avenue for revolutionizing the design of cell factories to boost fermentation efficiency.

Biosensor-based growth-coupling as an evolutionary strategy to improve heme export in Corynebacterium glutamicum.

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of PhrtB, creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

Fine-Regulating the Carbon Flux of l-Isoleucine Producing Corynebacterium glutamicum WM001 for Efficient l-Threonine Production.

l-Threonine, an essential amino acid, is widely used in various industries, with an annually growing demand. However, the present Corynebacterium glutamicum strains are difficult to achieve industrialization of l-threonine due to low yield and purity. In this study, we engineered an l-isoleucine-producing C. glutamicum WM001 to efficiently produce l-threonine by finely regulating the carbon flux. First, the threonine dehydratase in WM001 was mutated to lower the level of l-isoleucine production, then the homoserine dehydrogenase and aspartate kinase were mutated to release the feedback inhibition of l-threonine, and the resulting strain TWZ006 produced 14.2 g/L l-threonine. Subsequently, aspartate ammonia-lyase and aspartate transaminase were overexpressed to accumulate the precursor l-aspartate. Next, phosphoenolpyruvate carboxylase, pyruvate carboxylase and pyruvate kinase were overexpressed, and phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase were inactivated to fine-regulate the carbon flux among oxaloacetate, pyruvate and phosphoenolpyruvate. The resulting strain TWZ017 produced 21.5 g/L l-threonine. Finally, dihydrodipicolinate synthase was mutated with strong allosteric inhibition from l-lysine to significantly decrease byproducts accumulation, l-threonine export was optimized, and the final engineered strain TWZ024/pXTuf-thrE produced 78.3 g/L of l-threonine with the yield of 0.33 g/g glucose and the productivity of 0.82 g/L/h in a 7 L bioreactor. To the best of our knowledge, this represents the highest l-threonine production in C. glutamicum, providing possibilities for industrial-scale production.

Microbial synthesis of sedoheptulose from glucose by metabolically engineered Corynebacterium glutamicum.

BACKGROUND: Seven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production. RESULTS: In this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production. CONCLUSION: We achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.

[Efficient biosynthesis of guanidoacetic acid by a recombinant strain of Bacillus subtilis].

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.

[Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum].

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Design of fully synthetic signal peptide library and its use for enhanced secretory production of recombinant proteins in Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging. RESULT: We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for ⍺-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results. CONCLUSIONS: By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.

Enhanced molecular stability of ApxII antigen during secretion in Corynebacterium glutamicum by rational design.

ApxII is a vaccine antigen used to protect against porcine contagious pleuropneumonia, which is a significant threat to the pig industry. Here, we aimed to improve the proteolytic degradation stability of ApxII during its secretion by establishing a complete screening process of stable variants through bioinformatics and site-directed mutagenesis. We employed a combination of semi-rational and rational design strategies to create 34 single-point variants of ApxII. Among them, R114E and T115D variants exhibited better stability without compromising antigen activity. Furthermore, we constructed a multi-site variant, R114E/T115D, which demonstrated the best stability, activity, and yield. Protein stability and molecular dynamic analysis indicated that the greater solubility and lower structural expansion coefficient might explain the increased stability of R114E/T115D. Additionally, site T115 was identified as a key point of truncated ApxII stability. The R114E/T115D variant, with its proven stability and intact antigenic activity, holds promising prospects for industrial-scale applications in the prevention of porcine contagious pleuropneumonia.

Mannosidase-inhibiting iminosugar production by recombinant Corynebacterium glutamicum harboring the 1-deoxynojirimycin biosynthetic gene cluster.

The iminosugar class of carbohydrate-active enzyme inhibitors has therapeutic applications in metabolic syndrome conditions, viral infections and cancer. Compared to chemical synthesis, microbial iminosugar production has benefits of cost, sustainability and optimization. In this study, the 1-deoxynojirimycin (DNJ) biosynthetic gene cluster from Bacillus velezensis MBLB0692, and its individual genes, were cloned into Corynebacterium glutamicum (Cg). Characterizations of the encoded aminotransferase GabT1, phosphatase Yktc1, and dehydrogenase GutB1, were performed with purified enzymes and whole cell biocatalysts bearing individual and clustered (TYB) genes. GabT1 showed a variable pattern in its half-reaction with a slow turnover. GutB1 was an alkaline dehydrogenase with a broad substrate specificity and no divalent ion dependency while the zinc-dependent phosphatase Yktc1 had substrate specificity that was both pH- and ion-dependent. The CgYktc1 and CgGutB1 whole cells were viable biocatalysts with wider ranges of substrates than their enzyme counterparts. The CgTYB cells produced mannosidase-inhibiting iminosugars corresponding to mannojirimycin dehydrate (162 m/z) and deoxymannojirimycin (164 m/z). Mannosidase inhibitors have been found to be effective in treating orphan diseases, cancer and viral infections, and their biosynthesis by recombinant C. glutamicum can be optimized for industrial production and novel drug development.

Branched-chain amino acids: physico-chemical properties, industrial synthesis and role in signaling, metabolism and energy production.

Branched-chain amino acids (BCAAs)-leucine (Leu), isoleucine (Ile), and valine (Val)-are essential nutrients with significant roles in protein synthesis, metabolic regulation, and energy production. This review paper offers a detailed examination of the physico-chemical properties of BCAAs, their industrial synthesis, and their critical functions in various biological processes. The unique isomerism of BCAAs is presented, focusing on analytical challenges in their separation and quantification as well as their solubility characteristics, which are crucial for formulation and purification applications. The industrial synthesis of BCAAs, particularly using bacterial strains like Corynebacterium glutamicum, is explored, alongside methods such as genetic engineering aimed at enhancing production, detailing the enzymatic processes and specific precursors. The dietary uptake, distribution, and catabolism of BCAAs are reviewed as fundamental components of their physiological functions. Ultimately, their multifaceted impact on signaling pathways, immune function, and disease progression is discussed, providing insights into their profound influence on muscle protein synthesis and metabolic health. This comprehensive analysis serves as a resource for understanding both the basic and complex roles of BCAAs in biological systems and their industrial application.

Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria.

BACKGROUND: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. RESULTS: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. CONCLUSION: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation. ONE-SENTENCE SUMMARY: Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

Riboflavin overproduction from diverse feedstocks with engineeredCorynebacterium glutamicum.

Riboflavin overproduction byCorynebacterium glutamicumwas achieved by screening synthetic operons, enabling fine-tuned expression of the riboflavin biosynthetic genesribGCAH.The synthetic operons were designed by means of predicted translational initiation rates of each open reading frame, with the best-performing selection enabling riboflavin overproduction without negatively affecting cell growth. Overexpression of the fructose-1,6-bisphosphatase (fbp) and 5-phosphoribosyl 1-pyrophosphate aminotransferase (purF) encoding genes was then done to redirect the metabolic flux towards the riboflavin precursors. The resulting strain produced 8.3 g l-1of riboflavin in glucose-based fed-batch fermentations, which is the highest reported riboflavin titer withC. glutamicum. Further genetic engineering enabled both xylose and mannitol utilization byC. glutamicum, and we demonstrated riboflavin overproduction with the xylose-rich feedstocks rice husk hydrolysate and spent sulfite liquor, and the mannitol-rich feedstock brown seaweed hydrolysate. Remarkably, rice husk hydrolysate provided 30% higher riboflavin yields compared to glucose in the bioreactors.

Regulation of oxidative stress response and antioxidant modification in Corynebacterium glutamicum.

As an efficient and safe industrial bacterium, Corynebacterium glutamicum has extensive application in amino acid production. However, it often faces oxidative stress induced by reactive oxygen species (ROS), leading to diminished production efficiency. To enhance the robustness of C. glutamicum, numerous studies have focused on elucidating its regulatory mechanisms under various stress conditions such as heat, acid, and sulfur stress. However, a comprehensive review of its defense mechanisms against oxidative stress is needed. This review offers an in-depth overview of the mechanisms C. glutamicum employs to manage oxidative stress. It covers both enzymatic and non-enzymatic systems, including antioxidant enzymes, regulatory protein families, sigma factors involved in transcription, and physiological redox reduction pathways. This review provides insights for advancing research on the antioxidant mechanisms of C. glutamicum and sheds light on its potential applications in industrial production.

Metabolic Engineering of Corynebacterium glutamicum for the High-Level Production of l-Valine under Aerobic Conditions.

l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a Corynebacterium glutamicum strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway. Subsequently, we weakened anaplerotic pathways, and attenuated the tricarboxylic acid cycle via start codon substitution in icd, encoding isocitrate dehydrogenase. Finally, to balance bacterial growth and l-valine production, an l-valine biosensor-dependent genetic circuit was established to dynamically repress citrate synthase expression. The engineered strain Val19 produced 103 g/L of l-valine with a high yield of 0.35 g/g glucose and a productivity of 2.67 g/L/h. This represents the highest reported l-valine production in C. glutamicum via direct fermentation and exhibits potential for its industrial-scale production, leveraging the advantages of C. glutamicum over other microbes.

AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.