The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and fl...The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and flocculating activity(142.2 U/mL) were simultaneously obtained at the 14th hour when the pH value of the culture medium was maintained at 7.0 during the whole fermentation process. The production of REA-11 kept on a trend of increase till the later phase of fermentation process, which resulted in the ultimate flocculating activity of the culture broth to enhance to nearly 100 U/mL at pH 6.0. A two-stage pH control mode was adopted in REA-11 production in which the pH value of the culture medium was controlled at 7.0 during the first 14 h, then decreased to 6.0 that was maintained until the end of the fermentation process. With the two-stage pH control mode, the maximum flocculating activity reached 178.8 U/mL which was 30% higher than that obtained under the condition of pH 7.0 and the biomass enhanced about 15%. Compared with the fermentation process without pH control, REA-11 production and cell growth via the two-stage pH control mode increased 80% and 25%, respectively.展开更多
Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogena...Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.展开更多
Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling.Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were proposed a...Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling.Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were proposed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose concentrations from 10.0 to 17.5 g·L-1. The initial biomass concentration could shorten the lag time of cell growth,while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of16.22 g·L-1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of2.23 g·L-1was obtained and bioflocculant concentration was enhanced to 176.32 mg·L-1, 18.62% and403.63% higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.展开更多
l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum...l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum is an attractive chassis for l-threonine production.However,the present l-threonine production in C.glutamicum cannot meet the requirement of industrialization due to the relatively low production level of l-threonine and the accumulation of large amounts of by-products(such as l-lysine,l-isoleucine,and glycine).Herein,to enhance the l-threonine biosynthesis in C.glutamicum,releasing the aspartate kinase(LysC)and homoserine dehydrogenase(Hom)from feedback inhibition by l-lysine and l-threonine,respectively,and overexpressing four flux-control genes were performed.Next,to reduce the formation of by-products l-lysine and l-isoleucine without the cause of an auxotrophic phenotype,the feedback regulation of dihydrodipicolinate synthase(DapA)and threonine dehydratase(IlvA)was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by l-lysine and l-isoleucine,respectively.The resulting strain maintained the capability of synthesizing enough amounts of l-lysine and l-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids.To further enhance l-threonine production and reduce the by-product glycine,l-threonine exporter and homoserine kinase were overexpressed.Finally,the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L(17.2%higher)l-threonine with a productivity of 1.20 g/L/h(108.0%higher)in fed-batch fermentation,along with significantly reduced by-product accumulation,representing the record for l-threonine production in C.glutamicum.In this study,we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic l-threonine producing C.glutamicum.The main end by-products including l-lysine,l-isoleucine,and glycine were almost eliminated in fed-batch fermentation of the engineered C.glutamicum strain.This strategy can also be used for engineering producing strains for other amino acids and derivatives.展开更多
Geraniol is a monoterpenoid alcohol with various applications in food,cosmetics,and healthcare.Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosp...Geraniol is a monoterpenoid alcohol with various applications in food,cosmetics,and healthcare.Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosphate pathway.To engineer C.glutamicum to produce geraniol,two different truncated geraniol synthases (GESs) were respectively expressed,and strain LX02 expressing the truncated GESs from Valeriana officinalis (t Vo GES) produced 0.3 mg/L of geraniol.Then,three geranyl diphosphate synthases (GPPSs) were combinatorially co-expressed with t Vo GES to improve geraniol production.The amounts of produced geraniol were all higher than that produced by strain LX02.Strain LX03 co-expressing ERG20 F96W–N127W (ERG20 WW) and t Vo GES produced the highest amount,5.4 mg/L.Subsequently,the co-overexpression of1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) further increased the production to 12.2 mg/L in strain LX03.Lastly,the production of geraniol was increased to 15.2 mg/L via fermentation optimization.To our knowledge,this is the first report on the engineering of C.glutamicum to produce geraniol and thus can serve as a reference for other monoterpenoid production studies.展开更多
Corynebacterium glutamicum is a versatile industrial microorganism for producing various amino acids.However,there have been no reports of well-defined C.glutamicum strains capable of hyperproducing L-tryptophan.This ...Corynebacterium glutamicum is a versatile industrial microorganism for producing various amino acids.However,there have been no reports of well-defined C.glutamicum strains capable of hyperproducing L-tryptophan.This study presents a comprehensive metabolic engineering approach to establish robust C.glutamicum strains for Ltryptophan biosynthesis,including:(1)identification of potential targets by enzyme-constrained genome-scale modeling;(2)enhancement of the L-tryptophan biosynthetic pathway;(3)reconfiguration of central metabolic pathways;(4)identification of metabolic bottlenecks through comparative metabolome analysis;(5)engineering of the transport system,shikimate pathway,and precursor supply;and(6)repression of competing pathways and iterative optimization of key targets.The resulting C.glutamicum strain achieved a remarkable L-tryptophan titer of 50.5 g/L in 48h with a yield of 0.17 g/g glucose in fed-batch fermentation.This study highlights the efficacy of integrating computational modeling with systems metabolic engineering for significantly enhancing the production capabilities of industrial microorganisms.展开更多
L-Homoserine is a valuable intermediate with broad applications in the food,pharmaceutical,and chemical industries.Although Corynebacterium glutamicum has been engineered for the efficient biosynthesis of L-homo-serin...L-Homoserine is a valuable intermediate with broad applications in the food,pharmaceutical,and chemical industries.Although Corynebacterium glutamicum has been engineered for the efficient biosynthesis of L-homo-serine,both production efficiency and glucose conversion remain suboptimal.In this study,an engineered C.glutamicum strain capable of high-yield L-homoserine production from glucose was successfully developed.First,an engineered C.glutamicum strain capable of biosynthesizing L-homoserine using glucose as the sole carbon source was constructed with a yield of 0.38 g/g.To further enhance conversion efficiency,the expression of key genes in the tricarboxylic acid(TCA)cycle was repressed.Among the strategies evaluated,deletion of the aceE gene proved most effective in decoupling glycolysis from the TCA cycle,and acetate supplementation successfully restored cell growth in the decoupled strain.Subsequent metabolic rewiring,including modulation of acetylation efficiency,enhancement of the glyoxylate cycle,and promotion of fumarate-to-L-aspartate con-version,led to substantial L-homoserine accumulation.The engineered strain ultimately achieved an L-homo-serine titer of 17.35 g/L with a yield of 0.56 g/g glucose,representing a 48%increase.Finally,fed-batch fermentation was performed in a 5-L bioreactor using glucose and acetate as mixed carbon sources.The opti-mized strain,ACg23-6,produced 70.54 g/L L-homoserine within 96 h,with a yield of 0.58 g/g glucose and a productivity of 0.73 g/L/h,while consuming 80 g/L acetate.This decoupling strategy provided valuable insights for improving glucose conversion efficiency and acetate utilization in the microbial production of L-aspartatederived compounds.展开更多
L-serine is a versatile,high value-added amino acid,widely used in food,medicine and cosmetics.However,the low titer of L-serine has limited its industrial production.In this study,a cell factory without plasmid for e...L-serine is a versatile,high value-added amino acid,widely used in food,medicine and cosmetics.However,the low titer of L-serine has limited its industrial production.In this study,a cell factory without plasmid for efficient production of L-serine was constructed based on transport engineering.Firstly,the effects of L-serine exporter SerE overexpression and deletion on the cell growth and L-serine titer were investigated in Corynebacterium glutamicum(C.glutamicum)A36,overexpression of serE using a plasmid led to a 15.1%increase in L-serine titer but also caused a 15.1%decrease in cell growth.Subsequently,to increase the export capacity of SerE,we conducted semi-rational design and bioinformatics analysis,combined with alanine mutation and site-specific saturation mutation.The mutant E277K was obtained and exhibited a 53.2%higher export capacity compared to wild-type SerE,resulting in L-serine titer increased by 39.6%.Structural analysis and molecular dynamics simulations were performed to elucidate the mechanism.The results showed that the mutation shortened the hydrogen bond distance between the exporter and L-serine,enhanced complex stability,and reduced the binding energy.Finally,Bayesian optimization was employed to further improve L-serine titer of the mutant strain C-E277K.Under the optimized conditions,47.77 g/L L-serine was achieved in a 5-L bioreactor,representing the highest reported titer for C.glutamicum to date.This study provides a basis for the trans-formation of L-serine export pathway and offers a new strategy for increasing L-serine titer.展开更多
Liquid-liquid phase separation(LLPS)-driven membraneless organelles(MLOs)have been employed to enhance metabolic efficiency in various microbial cell factories.However,their application in the industrial bacterium Cor...Liquid-liquid phase separation(LLPS)-driven membraneless organelles(MLOs)have been employed to enhance metabolic efficiency in various microbial cell factories.However,their application in the industrial bacterium Corynebacterium glutamicum has not been explored.Here,we report the formation of liquid protein condensates in C.glutamicum using the RGG domain of Caenorhabditis elegans LAF-1.We optimized conditions for condensate formation,including the pre-induction period,inducer concentration,and cultivation temperature.Using the indigoidine biosynthesis pathway as a model,we demonstrated that LLPS-mediated MLOs enhanced indigoidine production.Furthermore,we applied these MLOs to modulate the toxicity of antimicrobial peptides(AMPs)to host cells,facilitating the expression of AMPs,including melittin and lactoferricin B.These findings provide insights into MLOs engineering in C.glutamicum and suggest broader applications of LLPS-mediated systems in industrial biotechnology.展开更多
5-Aminolevulinic acid(5-ALA),a versatile precursor for tetrapyrrole derivatives(such as heme,chlorophyll,and cobalamin),drives advancing microbial cell factories to meet growing biomedical and industrial demands.Howev...5-Aminolevulinic acid(5-ALA),a versatile precursor for tetrapyrrole derivatives(such as heme,chlorophyll,and cobalamin),drives advancing microbial cell factories to meet growing biomedical and industrial demands.However,there remain two challenges that limit yield and scalability:the limitations of conventional plasmid-based gene expression systems and the lack of fine regulation of succinyl-CoA.In this study,to address these limitations,we integrated multiple copies of hemAC132A of the heterologous C4 pathway on the genome.For fine regulating the supply of succinyl-CoA,the genes related to the tricarboxylic acid cycle(TCA cycle)oxidation branch pathway were combinatorially screened.The optimal combination of icd and lpd was confirmed by ribosome binding site(RBS)engineering,which was integrated on the genome with optimized expression in-tensity.Succinyl-CoA supply was further increased by genome integration and expression optimization of key CoA biosynthetic gene coaA,pantothenic acid synthesis-related gene panB-panC,andβ-alanine synthesis-related gene panD.The optimized genomically stable chassis achieved a high 5-ALA production of 6.38±0.16 g/L,which was 8.63-fold higher than the single hemAC132A copy strain A1(0.74±0.07 g/L).From these findings,a stable and high-yield 5-ALA synthetic strain was successfully constructed,providing a new strategy for pro-duction of biochemicals derived from succinyl-CoA in C.glutamicum.展开更多
Exporter protein systems play a crucial role in the efficient production of valuable chemicals.However,the lack of active exporters significantly limits the application of industrial bio-based production,making the id...Exporter protein systems play a crucial role in the efficient production of valuable chemicals.However,the lack of active exporters significantly limits the application of industrial bio-based production,making the identification and utilization of novel exporters highly important.In this study,we discovered a novel L-Homoserine exporter,Cg0701,in Corynebacterium glutamicum through homology analysis.First,tolerance assays revealed that the cg0701 overexpression strain(CgH-2)exhibited a 10.45%increase in cell growth compared to the control when cultivated with 30 g/L-Homoserine.Additionally,export assays demonstrated that the L-Homoserine export capacity of CgH-2 increased by approximately 30%.Furthermore,genomic overexpression of cg0701 in an L-Homoserine-producing chassis also enhanced both tolerance and export activity.As a result,the recombinant strain CgH-11 produced 10.79 g/L-Homoserine in shake flask cultures and 48.72 g/L in a 5 L fermenter,representing improvements of 19.89%and 24.44%,respectively.In summary,our results indicate that Cg0701 is a novel L-Homoserine exporter in C.glutamicum,enriching our understanding of amino acid export systems and providing a valuable target for the construction of L-Homoserine microbial cell factories.展开更多
L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engine...L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.展开更多
Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated...Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glu- tamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.展开更多
L-glutamate family amino acids(GFAAs),consisting of L-glutamate,L-arginine,L-citrulline,L-ornithine,L-proline,L-hydroxyproline,γ-aminobutyric acid,and 5-aminolevulinic acid,are widely applied in the food,pharmaceutic...L-glutamate family amino acids(GFAAs),consisting of L-glutamate,L-arginine,L-citrulline,L-ornithine,L-proline,L-hydroxyproline,γ-aminobutyric acid,and 5-aminolevulinic acid,are widely applied in the food,pharmaceutical,cosmetic,and animal feed industries,accounting for billions of dollars of market activity.These GFAAs have many functions,including being protein constituents,maintaining the urea cycle,and providing precursors for the biosynthesis of pharmaceuticals.Currently,the production of GFAAs mainly depends on microbial fermentation using Corynebacterium glutamicum(including its related subspecies Corynebacterium crenatum),which is substantially engineered through multistep metabolic engineering strategies.This review systematically summarizes recent advances in the metabolic pathways,regulatory mechanisms,and metabolic engineering strategies for GFAA accumulation in C.glutamicum and C.crenatum,which provides insights into the recent progress in L-glutamate-derived chemical production.展开更多
Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tigh...Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.展开更多
Glucosamine(GlcN)and its acetylated derivative N-acetylglucosamine(GlcNAc)are widely used in the pharmaceutical industries.Here,we attempted to achieve efficient production of GlcNAc via genomic engineering of Coryneb...Glucosamine(GlcN)and its acetylated derivative N-acetylglucosamine(GlcNAc)are widely used in the pharmaceutical industries.Here,we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum.Specifically,we ligated the GNA1 gene,which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate,into the plasmid pJYW4 and then transformed this recombinant vector into the C.glutamicum ATCC 13032,ATCC 13869,ATCC 14067,and S9114 strains,and we assessed the GlcNAc titers at 0.5 g/L,1.2 g/L,0.8 g/L,and 3.1 g/L from each strain,respectively.This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C.glutamicum strains.Therefore,we performed whole genome sequencing of the S9114 strain,which has not been previously published,and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested.Next,nagA(encoding GlcNAc-6-phosphate deacetylase)and gamA(encoding GlcN-6-phosphate deaminase)were deleted in C.glutamicum S9114 to block the catabolism of intracellular GlcNAc,leading to a 54.8%increase in GlcNAc production(from 3.1 to 4.8 g/L)when grown in a shaker flask.In addition,lactate synthesis was blocked by knockout of ldh(encoding lactate dehydrogenase);thus,further increasing the GlcNAc titer to 5.4 g/L.Finally,we added a key gene of the GlcN synthetic pathway,glmS,from different sources into the expression vector pJYW-4-ceN,and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L.This is the first report concerning the metabolic engineering of C.glutamicum,and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.展开更多
基金Supported by the Innovative Project for Young Scientific Scholars of Fujian Province(No.2 0 0 2 J0 4 4 )
文摘The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and flocculating activity(142.2 U/mL) were simultaneously obtained at the 14th hour when the pH value of the culture medium was maintained at 7.0 during the whole fermentation process. The production of REA-11 kept on a trend of increase till the later phase of fermentation process, which resulted in the ultimate flocculating activity of the culture broth to enhance to nearly 100 U/mL at pH 6.0. A two-stage pH control mode was adopted in REA-11 production in which the pH value of the culture medium was controlled at 7.0 during the first 14 h, then decreased to 6.0 that was maintained until the end of the fermentation process. With the two-stage pH control mode, the maximum flocculating activity reached 178.8 U/mL which was 30% higher than that obtained under the condition of pH 7.0 and the biomass enhanced about 15%. Compared with the fermentation process without pH control, REA-11 production and cell growth via the two-stage pH control mode increased 80% and 25%, respectively.
基金Supported by the National Natural Science Foundation of China (30970089,20876181,20831006)the Natural Science Foundation of Guangdong Province (9351027501000003)
文摘Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.
基金Supported by the National Natural Science Foundation of China(21206143,51378444)the program for New Century Excellent Talents of Education Ministry of China(ncet-13-0501)
文摘Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling.Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were proposed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose concentrations from 10.0 to 17.5 g·L-1. The initial biomass concentration could shorten the lag time of cell growth,while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of16.22 g·L-1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of2.23 g·L-1was obtained and bioflocculant concentration was enhanced to 176.32 mg·L-1, 18.62% and403.63% higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.
基金funded by the National Key Research and Development Program of China(2021YFC2100900)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-CXRC-058)+2 种基金the National Natural Science Foundation of China(32270101)the Key R&D Program of Shandong Province(2021CXGC010602)the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)。
文摘l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation.The GRAS(generally regarded as safe)industrial workhorse Corynebacterium glutamicum is an attractive chassis for l-threonine production.However,the present l-threonine production in C.glutamicum cannot meet the requirement of industrialization due to the relatively low production level of l-threonine and the accumulation of large amounts of by-products(such as l-lysine,l-isoleucine,and glycine).Herein,to enhance the l-threonine biosynthesis in C.glutamicum,releasing the aspartate kinase(LysC)and homoserine dehydrogenase(Hom)from feedback inhibition by l-lysine and l-threonine,respectively,and overexpressing four flux-control genes were performed.Next,to reduce the formation of by-products l-lysine and l-isoleucine without the cause of an auxotrophic phenotype,the feedback regulation of dihydrodipicolinate synthase(DapA)and threonine dehydratase(IlvA)was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by l-lysine and l-isoleucine,respectively.The resulting strain maintained the capability of synthesizing enough amounts of l-lysine and l-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids.To further enhance l-threonine production and reduce the by-product glycine,l-threonine exporter and homoserine kinase were overexpressed.Finally,the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L(17.2%higher)l-threonine with a productivity of 1.20 g/L/h(108.0%higher)in fed-batch fermentation,along with significantly reduced by-product accumulation,representing the record for l-threonine production in C.glutamicum.In this study,we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic l-threonine producing C.glutamicum.The main end by-products including l-lysine,l-isoleucine,and glycine were almost eliminated in fed-batch fermentation of the engineered C.glutamicum strain.This strategy can also be used for engineering producing strains for other amino acids and derivatives.
基金supported by the National Natural Science Foundation of China (No. 21878220)。
文摘Geraniol is a monoterpenoid alcohol with various applications in food,cosmetics,and healthcare.Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosphate pathway.To engineer C.glutamicum to produce geraniol,two different truncated geraniol synthases (GESs) were respectively expressed,and strain LX02 expressing the truncated GESs from Valeriana officinalis (t Vo GES) produced 0.3 mg/L of geraniol.Then,three geranyl diphosphate synthases (GPPSs) were combinatorially co-expressed with t Vo GES to improve geraniol production.The amounts of produced geraniol were all higher than that produced by strain LX02.Strain LX03 co-expressing ERG20 F96W–N127W (ERG20 WW) and t Vo GES produced the highest amount,5.4 mg/L.Subsequently,the co-overexpression of1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) further increased the production to 12.2 mg/L in strain LX03.Lastly,the production of geraniol was increased to 15.2 mg/L via fermentation optimization.To our knowledge,this is the first report on the engineering of C.glutamicum to produce geraniol and thus can serve as a reference for other monoterpenoid production studies.
基金supported by the National Key R&D Program of China(No.2021YFC2100900)the National Natural Science Foundation of China(Grant Nos.21938004,22078172)Tsinghua University Initiative Scientific Research Program(No.20223080016).
文摘Corynebacterium glutamicum is a versatile industrial microorganism for producing various amino acids.However,there have been no reports of well-defined C.glutamicum strains capable of hyperproducing L-tryptophan.This study presents a comprehensive metabolic engineering approach to establish robust C.glutamicum strains for Ltryptophan biosynthesis,including:(1)identification of potential targets by enzyme-constrained genome-scale modeling;(2)enhancement of the L-tryptophan biosynthetic pathway;(3)reconfiguration of central metabolic pathways;(4)identification of metabolic bottlenecks through comparative metabolome analysis;(5)engineering of the transport system,shikimate pathway,and precursor supply;and(6)repression of competing pathways and iterative optimization of key targets.The resulting C.glutamicum strain achieved a remarkable L-tryptophan titer of 50.5 g/L in 48h with a yield of 0.17 g/g glucose in fed-batch fermentation.This study highlights the efficacy of integrating computational modeling with systems metabolic engineering for significantly enhancing the production capabilities of industrial microorganisms.
基金supported by the National Natural Science Foundation of China(22108098)the Natural Science Foundation of Guangxi Province(2023JJA130304).
文摘L-Homoserine is a valuable intermediate with broad applications in the food,pharmaceutical,and chemical industries.Although Corynebacterium glutamicum has been engineered for the efficient biosynthesis of L-homo-serine,both production efficiency and glucose conversion remain suboptimal.In this study,an engineered C.glutamicum strain capable of high-yield L-homoserine production from glucose was successfully developed.First,an engineered C.glutamicum strain capable of biosynthesizing L-homoserine using glucose as the sole carbon source was constructed with a yield of 0.38 g/g.To further enhance conversion efficiency,the expression of key genes in the tricarboxylic acid(TCA)cycle was repressed.Among the strategies evaluated,deletion of the aceE gene proved most effective in decoupling glycolysis from the TCA cycle,and acetate supplementation successfully restored cell growth in the decoupled strain.Subsequent metabolic rewiring,including modulation of acetylation efficiency,enhancement of the glyoxylate cycle,and promotion of fumarate-to-L-aspartate con-version,led to substantial L-homoserine accumulation.The engineered strain ultimately achieved an L-homo-serine titer of 17.35 g/L with a yield of 0.56 g/g glucose,representing a 48%increase.Finally,fed-batch fermentation was performed in a 5-L bioreactor using glucose and acetate as mixed carbon sources.The opti-mized strain,ACg23-6,produced 70.54 g/L L-homoserine within 96 h,with a yield of 0.58 g/g glucose and a productivity of 0.73 g/L/h,while consuming 80 g/L acetate.This decoupling strategy provided valuable insights for improving glucose conversion efficiency and acetate utilization in the microbial production of L-aspartatederived compounds.
基金supported by the National Natural Science Foundation of China(32171470).
文摘L-serine is a versatile,high value-added amino acid,widely used in food,medicine and cosmetics.However,the low titer of L-serine has limited its industrial production.In this study,a cell factory without plasmid for efficient production of L-serine was constructed based on transport engineering.Firstly,the effects of L-serine exporter SerE overexpression and deletion on the cell growth and L-serine titer were investigated in Corynebacterium glutamicum(C.glutamicum)A36,overexpression of serE using a plasmid led to a 15.1%increase in L-serine titer but also caused a 15.1%decrease in cell growth.Subsequently,to increase the export capacity of SerE,we conducted semi-rational design and bioinformatics analysis,combined with alanine mutation and site-specific saturation mutation.The mutant E277K was obtained and exhibited a 53.2%higher export capacity compared to wild-type SerE,resulting in L-serine titer increased by 39.6%.Structural analysis and molecular dynamics simulations were performed to elucidate the mechanism.The results showed that the mutation shortened the hydrogen bond distance between the exporter and L-serine,enhanced complex stability,and reduced the binding energy.Finally,Bayesian optimization was employed to further improve L-serine titer of the mutant strain C-E277K.Under the optimized conditions,47.77 g/L L-serine was achieved in a 5-L bioreactor,representing the highest reported titer for C.glutamicum to date.This study provides a basis for the trans-formation of L-serine export pathway and offers a new strategy for increasing L-serine titer.
基金supported by the National Natural Science Foundation of China(12375352)China+2 种基金the Anhui Provincial Natural Science Foundation(2308085MC76),ChinaChina Postdoctoral Science Foundation(2025M770911),Chinathe Major Projects of Science and Technology of Anhui Province(202103a06020003),China.
文摘Liquid-liquid phase separation(LLPS)-driven membraneless organelles(MLOs)have been employed to enhance metabolic efficiency in various microbial cell factories.However,their application in the industrial bacterium Corynebacterium glutamicum has not been explored.Here,we report the formation of liquid protein condensates in C.glutamicum using the RGG domain of Caenorhabditis elegans LAF-1.We optimized conditions for condensate formation,including the pre-induction period,inducer concentration,and cultivation temperature.Using the indigoidine biosynthesis pathway as a model,we demonstrated that LLPS-mediated MLOs enhanced indigoidine production.Furthermore,we applied these MLOs to modulate the toxicity of antimicrobial peptides(AMPs)to host cells,facilitating the expression of AMPs,including melittin and lactoferricin B.These findings provide insights into MLOs engineering in C.glutamicum and suggest broader applications of LLPS-mediated systems in industrial biotechnology.
基金supported by the National Natural Science Foundation of China(NSFC-22278312)the Central Guidance for Local Science and Technology Development Projects(2024FRD05057)key research and development program of Ningxia Hui Autonomous Region(2024BEE02005).
文摘5-Aminolevulinic acid(5-ALA),a versatile precursor for tetrapyrrole derivatives(such as heme,chlorophyll,and cobalamin),drives advancing microbial cell factories to meet growing biomedical and industrial demands.However,there remain two challenges that limit yield and scalability:the limitations of conventional plasmid-based gene expression systems and the lack of fine regulation of succinyl-CoA.In this study,to address these limitations,we integrated multiple copies of hemAC132A of the heterologous C4 pathway on the genome.For fine regulating the supply of succinyl-CoA,the genes related to the tricarboxylic acid cycle(TCA cycle)oxidation branch pathway were combinatorially screened.The optimal combination of icd and lpd was confirmed by ribosome binding site(RBS)engineering,which was integrated on the genome with optimized expression in-tensity.Succinyl-CoA supply was further increased by genome integration and expression optimization of key CoA biosynthetic gene coaA,pantothenic acid synthesis-related gene panB-panC,andβ-alanine synthesis-related gene panD.The optimized genomically stable chassis achieved a high 5-ALA production of 6.38±0.16 g/L,which was 8.63-fold higher than the single hemAC132A copy strain A1(0.74±0.07 g/L).From these findings,a stable and high-yield 5-ALA synthetic strain was successfully constructed,providing a new strategy for pro-duction of biochemicals derived from succinyl-CoA in C.glutamicum.
文摘Exporter protein systems play a crucial role in the efficient production of valuable chemicals.However,the lack of active exporters significantly limits the application of industrial bio-based production,making the identification and utilization of novel exporters highly important.In this study,we discovered a novel L-Homoserine exporter,Cg0701,in Corynebacterium glutamicum through homology analysis.First,tolerance assays revealed that the cg0701 overexpression strain(CgH-2)exhibited a 10.45%increase in cell growth compared to the control when cultivated with 30 g/L-Homoserine.Additionally,export assays demonstrated that the L-Homoserine export capacity of CgH-2 increased by approximately 30%.Furthermore,genomic overexpression of cg0701 in an L-Homoserine-producing chassis also enhanced both tolerance and export activity.As a result,the recombinant strain CgH-11 produced 10.79 g/L-Homoserine in shake flask cultures and 48.72 g/L in a 5 L fermenter,representing improvements of 19.89%and 24.44%,respectively.In summary,our results indicate that Cg0701 is a novel L-Homoserine exporter in C.glutamicum,enriching our understanding of amino acid export systems and providing a valuable target for the construction of L-Homoserine microbial cell factories.
基金supported by grants from Ministry of Science and Technology of China (Grant Nos.2008ZX09401-05 and 2010ZX09401-403)the National Natural Science Foundation of China (Grant No. 31100074)Chinese Academy of Sciences (Grant No. XBXA-2011-009)
文摘L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.
基金This work was supported by the Chinese Academy of Sciences(KSCX2-SW-1I3)National Natural Science Foundation of China(Grant No.30230010).
文摘Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glu- tamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.
基金This work was supported by National Natural Science Foundation of China[No.32000057]Open Funding Project of the State Key Laboratory of Biocatalysis and Enzyme Engineering(SKLBEE2019015)+1 种基金Jiangxi Provincial Natural Science Foundation(No.20202BAB213023)Jiangxi Province Postgraduate Innovation Special Fund Project[No.YC2020-S258].
文摘L-glutamate family amino acids(GFAAs),consisting of L-glutamate,L-arginine,L-citrulline,L-ornithine,L-proline,L-hydroxyproline,γ-aminobutyric acid,and 5-aminolevulinic acid,are widely applied in the food,pharmaceutical,cosmetic,and animal feed industries,accounting for billions of dollars of market activity.These GFAAs have many functions,including being protein constituents,maintaining the urea cycle,and providing precursors for the biosynthesis of pharmaceuticals.Currently,the production of GFAAs mainly depends on microbial fermentation using Corynebacterium glutamicum(including its related subspecies Corynebacterium crenatum),which is substantially engineered through multistep metabolic engineering strategies.This review systematically summarizes recent advances in the metabolic pathways,regulatory mechanisms,and metabolic engineering strategies for GFAA accumulation in C.glutamicum and C.crenatum,which provides insights into the recent progress in L-glutamate-derived chemical production.
基金This work received funding from the National Natural Science Foundation of China(No.21878124,22078128,and 21938004)the Fundamental Research Funds for the Central Universities(No.JUSRP221032)+1 种基金the 111 Project(No.111-2-06)the national first-class discipline program of Light Industry Technology and Engineering(LITE2018-24).
文摘Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.
基金This work was financially supported by the National Natural Science Foundation of China(31622001,31671845,31600068)the Natural Science Foundation of Jiangsu Province(BK20160176)the 111 Project(111-2-06).
文摘Glucosamine(GlcN)and its acetylated derivative N-acetylglucosamine(GlcNAc)are widely used in the pharmaceutical industries.Here,we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum.Specifically,we ligated the GNA1 gene,which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate,into the plasmid pJYW4 and then transformed this recombinant vector into the C.glutamicum ATCC 13032,ATCC 13869,ATCC 14067,and S9114 strains,and we assessed the GlcNAc titers at 0.5 g/L,1.2 g/L,0.8 g/L,and 3.1 g/L from each strain,respectively.This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C.glutamicum strains.Therefore,we performed whole genome sequencing of the S9114 strain,which has not been previously published,and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested.Next,nagA(encoding GlcNAc-6-phosphate deacetylase)and gamA(encoding GlcN-6-phosphate deaminase)were deleted in C.glutamicum S9114 to block the catabolism of intracellular GlcNAc,leading to a 54.8%increase in GlcNAc production(from 3.1 to 4.8 g/L)when grown in a shaker flask.In addition,lactate synthesis was blocked by knockout of ldh(encoding lactate dehydrogenase);thus,further increasing the GlcNAc titer to 5.4 g/L.Finally,we added a key gene of the GlcN synthetic pathway,glmS,from different sources into the expression vector pJYW-4-ceN,and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L.This is the first report concerning the metabolic engineering of C.glutamicum,and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.
