Targeting the cysteine biosynthesis pathway in microorganisms: Mechanism, structure, and drug discovery.

Owing to the global health crisis of resistant pathogenic infections, researchers are emphasizing the importance of novel prevention and control strategies. Existing antimicrobial drugs predominantly target a few pathways, and their widespread use has pervasively increased drug resistance. Therefore, it is imperative to develop new antimicrobial drugs with novel targets and chemical structures. The de novo cysteine biosynthesis pathway, one of the microbial metabolic pathways, plays a crucial role in pathogenicity and drug resistance. This pathway notably differs from that in humans, thereby representing an unexplored target for developing antimicrobial drugs. Herein, we have presented an overview of cysteine biosynthesis pathways and their roles in the pathogenicity of various microorganisms. Additionally, we have investigated the structure and function of enzymes involved in these pathways as well as have discussed drug design strategies and structure-activity relationships of the enzyme inhibitors. This review provides valuable insights for developing novel antimicrobials and offers new avenues to combat drug resistance.

[Cloning and functional analysis of AcFMO from onion during alliine biosynthesis].

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.

Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition.

Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01 Šand with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel ß-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.

Biodesulfurization Induces Reprogramming of Sulfur Metabolism in Rhodococcus qingshengii IGTS8: Proteomics and Untargeted Metabolomics.

Sulfur metabolism in fuel-biodesulfurizing bacteria and the underlying physiological adaptations are not understood, which has impeded the development of a commercially viable bioprocess for fuel desulfurization. To fill these knowledge gaps, we performed comparative proteomics and untargeted metabolomics in cultures of the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 grown on either inorganic sulfate or the diesel-borne organosulfur compound dibenzothiophene as a sole sulfur source. Dibenzothiophene significantly altered the biosynthesis of many sulfur metabolism proteins and metabolites in a growth phase-dependent manner, which enabled us to reconstruct the first experimental model for sulfur metabolism in a fuel-biodesulfurizing bacterium. All key pathways related to assimilatory sulfur metabolism were represented in the sulfur proteome, including uptake of the sulfur sources, sulfur acquisition, and assimilatory sulfate reduction, in addition to biosynthesis of key sulfur-containing metabolites such as S-adenosylmethionine, coenzyme A, biotin, thiamin, molybdenum cofactor, mycothiol, and ergothioneine (low-molecular weight thiols). Fifty-two proteins exhibited significantly different abundance during at least one growth phase. Sixteen proteins were uniquely detected and 47 proteins were significantly more abundant in the dibenzothiophene culture during at least one growth phase. The sulfate-free dibenzothiophene-containing culture reacted to sulfate starvation by restricting sulfur assimilation, enforcing sulfur-sparing, and maintaining redox homeostasis. Biodesulfurization triggered alternative pathways for sulfur assimilation different from those operating in the inorganic sulfate culture. Sulfur metabolism reprogramming and metabolic switches in the dibenzothiophene culture were manifested in limiting sulfite reduction and biosynthesis of cysteine, while boosting the production of methionine via the cobalamin-independent pathway, as well as the biosynthesis of the redox buffers mycothiol and ergothioneine. The omics data underscore the key role of sulfur metabolism in shaping the biodesulfurization phenotype and highlight potential targets for improving the biodesulfurization catalytic activity via metabolic engineering. IMPORTANCE For many decades, research on biodesulfurization of fossil fuels was conducted amid a large gap in knowledge of sulfur metabolism and its regulation in fuel-biodesulfurizing bacteria, which has impeded the development of a commercially viable bioprocess. In addition, lack of understanding of biodesulfurization-associated metabolic and physiological adaptations prohibited the development of efficient biodesulfurizers. Our integrated omics-based findings reveal the assimilatory sulfur metabolism in the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 and show how sulfur metabolism and oxidative stress response were remodeled and orchestrated to shape the biodesulfurization phenotype. Our findings not only explain the frequently encountered low catalytic activity of native fuel-biodesulfurizing bacteria but also uncover unprecedented potential targets in sulfur metabolism that could be exploited via metabolic engineering to boost the biodesulfurization catalytic activity, a prerequisite for commercial application.

Cysteine biosynthesis contributes to ß-methylamino-l-alanine tolerance in Escherichia coli.

In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin ß-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 µg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H2O2 in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

Parallel analysis of global garlic gene expression and alliin content following leaf wounding.

BACKGROUND: Allium sativum (garlic) is an economically important food source and medicinal plant rich in sulfides and other protective substances such as alliin, the precursor of allicin biosynthesis. Cysteine, serine and sulfur is the precursor of alliin biosynthesis. However, little is known about the alliin content under abiotic stress or the mechanism by which it is synthesized. RESULTS: The findings revealed that the content of alliin was lowest in the garlic roots, and highest in the buds. Furthermore, alliin levels decreased in mature leaves following wounding. Transcriptome data generated over time after wounding further revealed significant up-regulation of genes integral to the biosynthetic pathways of cysteine and serine in mature garlic leaves. CONCLUSIONS: The findings suggest that differential expression of cysteine, serine and sulfide-related genes underlies the accumulation of alliin and its precursors in garlic, providing a basis for further analyses of alliin biosynthesis.

Brucella ovis Cysteine Biosynthesis Contributes to Peroxide Stress Survival and Fitness in the Intracellular Niche.

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. The same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform our understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary-phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting cysE (ΔcysE) or both cysK1 and cysK2 (ΔcysK1 ΔcysK2) results in premature entry into stationary phase, reduced culture yield, and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. ΔcysE and ΔcysK1 ΔcysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 h postinfection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.

Cystathionine-gamma-lyase overexpression in T cells enhances antitumor effect independently of cysteine autonomy.

T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long-lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine-gamma-lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8+ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH-expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.

Defect of RNA pyrophosphohydrolase RppH enhances fermentative production of L-cysteine in Escherichia coli.

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Rapid Reaction, Slow Dissociation Aggregation, and Synergetic Multicolor Emission for Imaging the Restriction and Regulation of Biosynthesis of Cys and GSH.

Biosynthesis is a necessary process to maintain life. In recent years, research has fully shown that three kinds of biothiols (Cys, Hcy, GSH) mainly play the role in oxidative stress and maintaining cell homeostasis in cells, and that abnormal concentrations will lead to the occurrence of cardiovascular diseases, cancers, etc. Various fluorescent probes have shown unprecedented advantages in detecting their concentrations and studying their biological functions. As a matter of fact, these three kinds of biothiols are generated in the process of biosynthesis in vivo. It is of great significance to understand their biosynthetic pathways and elucidate their synthetic relationships. In this work, to α,ß-unsaturated ketones conjugated ethylenediamine coumarin and pyrandione was introduced boron fluoride and, through its strong electron deficiency effect, afforded a molecule having near-infrared emission and regulated the rigidity of molecules. At the same time, the conjugated double bond is used to respond to molecular rigidity. The rapid response of the probe to biothiols and the slow dissociation aggregation of the probe itself through the response environment could monitor the absence of biothiols in cells. In addition, based on the difference in sensitivity of response of Cys and GSH to the probe, this work studied the interaction between biosynthetic pathways of Cys and GSH in cells through enzyme inhibition for the first time. The relationship of restriction and regulation of biosynthesis in vivo was revealed.

Fitness of Chassis Cells and Metabolic Pathways for l-Cysteine Overproduction in Escherichia coli.

l-Cysteine is a ubiquitous and unique sulfur-containing amino acid with numerous applications in agricultural and food industries. The efficient production of l-cysteine via microbial fermentation has received a great deal of attention. In this study, the fitness of different Escherichia coli K-12 strains harboring plasmid pLH03 was investigated. The enhancement of the precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of the E. coli BW25113 strain. The expression levels of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on cysteine production. In conjunction, the main degradation pathway genes were also deleted for more efficient production of cysteine. l-Cysteine production was further increased through the manipulation of the sulfur transcription regulator cysB and sulfur supplementation. After process optimization in a 1.5 L bioreactor, LH2A1M0BΔYTS-pLH03 [BW25113 Ptrc2-serA Ptrc1-cysMPtrc-cysBΔyhaMΔtnaAΔsdaA-(pLH03)] accumulated 8.34 g/L cysteine, laying a foundation for application in the cysteine fermentation industry.

Cystathionine ß-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii.

Cystathionine ß-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T. gondii CGL gene encodes a functional enzyme. Herein, we cloned and biochemically characterized cDNA encoding CBS from T. gondii (TgCBS), which represents a first example of protozoan CBS that does not bind heme but possesses two C-terminal CBS domains. We demonstrated that TgCBS can use both serine and O-acetylserine to produce cystathionine, converting these substrates to an aminoacrylate intermediate as part of a PLP-catalyzed ß-replacement reaction. Besides a role in cysteine biosynthesis, TgCBS can also efficiently produce hydrogen sulfide, preferentially via condensation of cysteine and homocysteine. Unlike the human counterpart and similar to CBS enzymes from lower organisms, the TgCBS activity is not stimulated by S-adenosylmethionine. This study establishes the presence of an intact functional reverse transsulfuration pathway in T. gondii and demonstrates the crucial role of TgCBS in biogenesis of H2S.

Cellular Source of Cysteinyl Leukotrienes Following Chlorine Exposure.

Exposure of mice to high concentrations of chlorine leads to the synthesis of cysteinyl leukotrienes (cysLTs). CysLTs contribute to chlorine-induced airway hyperresponsiveness. The aim of the current study was to determine the cellular source of the cysLTs. To achieve this aim, we exposed mice to 100 ppm of chlorine for 5 minutes. Intranasal instillation of clodronate in liposomes and of diphtheria toxin in CD11c-DTR mice was used to deplete macrophages. CCR2-/- mice were used to assess the contribution of recruited macrophages. Eosinophils and neutrophils were depleted with specific antibodies. Platelet-neutrophil aggregation was prevented with an antibody against P-selectin. The potential roles of phagocytosis of neutrophils by macrophages and of transcellular metabolism between epithelial cells and neutrophils were explored in coculture systems. We found that depletion of neutrophils was the only intervention that inhibited the synthesis of cysLTs at 24 hours after chlorine exposure. Although macrophages did synthesize cysLTs in response to phagocytosis of neutrophils, depletion of macrophages did not reduce the increment in cysLTs triggered by chlorine exposure. However, coculture of airway epithelial cells with neutrophils resulted in a significant increase in the synthesis of cysLTs, dependent on the expression of 5-lipoxygenase by neutrophils. We conclude that cysLT synthesis following chlorine exposure may be dependent on transcellular metabolism by neutrophil-epithelial interactions.

Enhancement of Sulfur Conversion Rate in the Production of l-Cysteine by Engineered Escherichia coli.

Cysteine is a commercially important sulfur-containing amino acid widely used as a supplement in the agricultural and food industries. It is extremely desirable to achieve a high sulfur conversion rate in the fermentation-based cysteine production. Here, the metabolic engineering of Escherichia coli was performed to enhance the sulfur conversion rate in cysteine biosynthesis. Accordingly, the reduction of sulfur loss by the regulator decR and its yhaOM operons were deleted. serACB was integrated into chromosome with constitutive promoter to coordinately increase sulfur utilization. The sulfur assimilation pathways and sulfur transcriptional regulator cysB were overexpressed to regulate sulfur metabolism and enhance sulfur conversion significantly. After the process optimization in fed-batch fermentation, LH16 [SLH02 ΔyhaM Ptrc1-serACB-cysM-nrdH-(pLH03, pTrc99a-cysB)] produced 7.5 g/L of cysteine with a sulfur conversion rate of 90.11%. These results indicate that cysteine production by LH16 is a valuable process in the agricultural and food industries.

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ß-Lactam Production.

To produce high levels of ß-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for ß-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production.IMPORTANCEPenicillium rubens is an important industrial producer of ß-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

X-ray crystallographic structure of BshB, the zinc-dependent deacetylase involved in bacillithiol biosynthesis.

Many gram-positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three-enzyme pathway that includes the action of the zinc-dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X-ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.

Discovery and Characterization of the Antimetabolite Action of Thioacetamide-Linked 1,2,3-Triazoles as Disruptors of Cysteine Biosynthesis in Gram-Negative Bacteria.

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N-(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.

Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum.

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.

Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a.

Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16 bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.