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
The production of L-alanine was enhanced in Corynebacterium glutamicum ATCC13869 through metabolic engineering of the biosynthesis pathways of L-alanine and fatty acids.Strains ΔfasB, ΔfasBR, ΔfasB Δpks13 and Δfa...The production of L-alanine was enhanced in Corynebacterium glutamicum ATCC13869 through metabolic engineering of the biosynthesis pathways of L-alanine and fatty acids.Strains ΔfasB, ΔfasBR, ΔfasB Δpks13 and ΔfasBR Δpks13 were constructed and exhibited increased L-alanine yields up to 17.29 g/L.Different from ΔfasB mutant constructed from C.glutamicum ATCC13032 in which L-glutamate production accumulated,the muatnt ΔfasB constructed from C.glutamicum ATCC13869 in this study significantly produce L-alanine without L-glutamate accumulation.Transcriptional level analysis revealed that the knockout of fasB upregulated the expression levels of the genes related to L-alanine synthesis but downregulated those associated with fatty acid synthesis,confirming the redirection of metabolic flux from fatty acid synthesis to L-alanine synthesis in these strains.L-alanine productions were further enhanced in strains ΔfasB and ΔfasBR through the combinatorial expression of heterologous genes Bacillus subtilis alaD encoding alanine dehydrogenase and Escherichia coli alaE encoding alanine export protein,and the yields reached 55.21 g/L and 54.95 g/L,respectively.Finally,69.9 g/L L-alanine was obtained in ΔfasB/pJYW-5-alaDE after 60 h of fermentation by supplementing glucose.Our data indicate that disrupting the fatty acid biosynthesis could redirect metabolic flux towards L-alanine biosynthesis.These results provide a new strategy for increasing the production of L-alanine in C.glutamicum.展开更多
This study described that a low-producing mutagenic strain was transformed to a l-lysine high-producing recombinant strain by optimizing the l-lysine metabolic pathway of Corynebacterium glutamicum.The nucleotide sequ...This study described that a low-producing mutagenic strain was transformed to a l-lysine high-producing recombinant strain by optimizing the l-lysine metabolic pathway of Corynebacterium glutamicum.The nucleotide sequence results revealed that the lysC of mutant strain CgK37 mutated at 279th codon.Based on this site,site-directed saturation mutation was performed to screen for the mutant with better effect in relieving aspartate kinase feedback inhibition.Then,the supply of oxaloacetate and nicotinamide adenine dinucleotide phosphate was increased via knockout and overexpression of related genes.In order to solve the problem of low utilization efficiency of culture medium,fructokinase gene gmuE was heterologous expressed in CgK37,which could directly use intracellular fructose to improve the growth rate.In addition,the synthesis of partial by-products was weakened at the gene transcription level to avoid carbon excessively flowing into the branch metabolism.Finally,a large-scale fermentation experiment was conducted in 5 L jar fermenter.The l-lysine yield of CgK37-11 was 196.58±1.68 g/L,which was 83.24%original higher than CgK37,and the productivity reached 2.46 g/L/h.展开更多
Corynebacterium glutamicum is widely used in the production of amino acids.C.glutamicum possesses seven sigma factors,among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic...Corynebacterium glutamicum is widely used in the production of amino acids.C.glutamicum possesses seven sigma factors,among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic acid(MA)and its derivatives,the unique cell envelope of C.glutamicum.To understand the influence of MA synthesis on amino acid production and membrane phenotype of C.glutamicum,the expression of sigD gene and some mycolyltransferase genes,i.e.,cmt1,cop1 and cmt2,were regulated by several growth-regulated promoters in this study.Except for 2 mutant strains of P_(cg3096)-sigD and P_(cg1633)-cop1,the growth and 4-hydroxyisoleucine(4-HIL)titer of most modified strains did not change significantly.But the 4-HIL titer of P_(odhI)-sigD strain increased by 20.73%(142.45±3.69 mM)compared to that of control strain(117.99±0.34 mM).After it was cultivated in bioreactor,4-HIL titer reached 372.56 mM.This may be caused by the increase of MA content,and 17%decrease of cell hydrophobicity and 12%increase of membrane permeability were observed at the exponential phase.In conclusion,we proved that rearrangements in regulation of sigD expression contributed to the improved fermentation performance of C.glutamicum and promoted 4-HIL production.展开更多
L-Methionine is widely used in food,agricultural and pharmaceutical industries.In this study,the L-methionine production in Corynebacterium glutamicum ATCC13032 was promoted by eliminating the feedback inhibition of k...L-Methionine is widely used in food,agricultural and pharmaceutical industries.In this study,the L-methionine production in Corynebacterium glutamicum ATCC13032 was promoted by eliminating the feedback inhibition of key rate-limiting enzymes,blocking L-threonine biosynthesis,and strengthening the downstream pathway of L-homoserine.ATCC13032 does not accumulate L-threonine,we found that overexpressing the genes lysC and hom^(G378S) could accumulate 0.6 g/L L-threonine.Deleting the genes thrB,McbR,and metD in ATCC13032 could accumulate 0.49 g/L L-methionine.Next,enhancing oxaloacetate supply,overexpressing brnFE,and deleting Ncgl2640 that involved in the repression of sulphuric metabolism could accumulate 0.92 g/L L-methionine.Further overexpressing the genes related to L-homoserine downstream pathway,the resulting strain ZBW011/pEC-metYX could produce 1.82 g/L L-methionine.Finally,the gene pyk2 was deleted and the final strain ZBW014/pEC-metYX produced 7.06 g/L L-methionine in a 2.4-L fermenter.The strategies presented in this study would be useful to engineer C.glutamicum for industrial L-methionine production.展开更多
L-serine is utilized in various applications across the pharmaceutical and food industry.Corynebacterium glutamicum,a non-pathogenic strain,is extensively used in amino acid production.However,the current titer and pr...L-serine is utilized in various applications across the pharmaceutical and food industry.Corynebacterium glutamicum,a non-pathogenic strain,is extensively used in amino acid production.However,the current titer and productivity of L-serine through direct fermentation are insufficient to meet the demands of industrial production.This shortfall arises from the strain’s inadequacy sucrose utilization,which affects both L-serine productivity and sucrose consumption rate.To solve the problem,this research conducted three strategies to increase the carbon flow from sucrose to L-serine.Initially,ALE was performed using a stress of 300 g/L sucrose based on A36-pDSer,and a biosensor-assisted high-throughput screening platform was utilized to identify the mutants with higher intracellular L-serine concentration.The strain A36-mut achieved 39.0 g/L L-serine titer,marking a notable 25.3%improvement over the parent strain A36(31.1 g/L).Subsequently,the overexpression of the L-serine exporter serE along with its transcription factor serR in strain A36-mut led to an improvement in the L-serine production,reaching 44.8 g/L.Finally,by optimizing the fed-batch fermentation process,the L-serine titer and productivity were improved to 53.7 g/L and 0.50 g/L/h,respectively.This research presented the highest L-serine titer from sucrose in C.glutamicum to date,offering the possibility for the industrialization production of L-serine by fermentation.展开更多
4-Hydroxyisoleucine(4-HIL)holds potential value in the treatment of diabetes.It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine(Ile)producing Corynebacterium glutamicum stra...4-Hydroxyisoleucine(4-HIL)holds potential value in the treatment of diabetes.It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine(Ile)producing Corynebacterium glutamicum strains.But the stable expression of ido on plasmids relies on the usage of antibiotics.To make the harboring of ido independent of plasmid,this study developed a chromosome-engineered strain for synthesizing 4-HIL directly from glucose.First,the ido-cat-ido expressing cassette was inserted into the chromosome of C.glutamicum,and the copy number of ido was increased through chemically inducible chromosome evolution(CIChE).After successive rounds of CIChE by increasing chloramphenicol concentration,7 copies of ido were integrated in the chromosome of C.glutamicum SE04,and the 4-HIL production reached 20.3±4.99 g/L,3.5-fold higher than the initial strain SC12 harboring two-copies of ido.To cease further homologous recombination,recA was deleted in CIChE strains,but cell growth and 4-HIL production were damaged.Notably,the stability of chromosomally inserted genes in the evolved strain SE04 was confirmed.Ultimately,the evolved C.glutamicum SE04 strain produced 30.3 g/L of 4-HIL in a 2-L bioreactor.This study established a plasmid-free strain of C.glutamicum for 4-HIL production,offering new insights into utilizing multi-copy integration methods for producing other valuable biochemical substances in C.glutamicum.展开更多
Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system ...Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system of the strain.By building a signal peptide library,we screened several Sce-type signal peptides and analyzed the relationship between their constitutive properties and secretory efficiency.To further meet the safety requirements in industrial applications,fifteen constitutive promoters were screened,and protein expression was optimized by promoter tandem strategy.In the WYJ1,WYJ2,WYJ3,and WYJ4 engineering strains,we confirmed that the modification of cell permeability favored protein secretion.The engineering strains WYJ2P35SP35 and WYJ4P35SP35 were scaled up for culture,and their extracellular enzyme activities and proteins reached 26.42 U/mL and 19.65 mg/L,and 23.97 U/mL and 13.84 mg/L,respectively.This secretory expression system increases the potential of industrial applications of Corynebacterium glutamicum and lays the foundation for applications.展开更多
2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but ...2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose.In this study,C.glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose.The gene futC from Helicobacter pylori encodingα-1,2-fucosyltransferase was codon optimized and mutated at four amino acids(F40S/Q150H/C151R/Q239S).The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C.glutamicum,resulting in CW002.CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane.Therefore,the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002,resulting in CW006.CW006 did synthesize 2′-fucosyllactose as expected.It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed,suggesting that the expression levels of the four genes in CW006 might have to well balanced.C.glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.展开更多
β-Alanine is the only naturally occurring β-type amino acid,with various applications in the pharmaceutical,food,and chemical industries.Given the growing market demand,the study of β-alanine production is importan...β-Alanine is the only naturally occurring β-type amino acid,with various applications in the pharmaceutical,food,and chemical industries.Given the growing market demand,the study of β-alanine production is important.This study utilized a modified lysine-producing strain as a chassis cell line to further promote β-alanine synthesis through metabolic engineering.In order to reduce the consumption of oxaloacetate,the gene pck was deleted.A promoter mutation library was constructed to screen the original promoter of the stronger promoter replacement gene pyc to enhance the oxaloacetate synthesis pathway and further increase the intracellular supply of oxaloacetate.Next,the gene poxB was deleted,and pyruvate accumulation further promoted β-alanine synthesis.Then,the aspartate kinase-coding gene lysC was weakened by predicting the RBS sequence,thus reducing the synthesis of lysine by-products and improving β-alanine synthesis.Ultimately,the carbon flux in the β-alanine biosynthetic pathways was increased by overexpressing aspartate-α-decarboxylase,aspartate ammonia-lyase,and aspartate aminotransferase using the strong promoter Ptrc.The resulting strain QBA9 was cultured in a 5-L fermenter by fed-batch to produce 70.8 g/L of β-alanine with a productivity of 0.98 g/L/h.These modification strategies demonstrate the potential for efficient β-alanine production by the lysine-producing strain and provide an innovative idea for the developing β-alanine-producing strains.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
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 Research and Development Program of China(2021YFC2100900).
文摘The production of L-alanine was enhanced in Corynebacterium glutamicum ATCC13869 through metabolic engineering of the biosynthesis pathways of L-alanine and fatty acids.Strains ΔfasB, ΔfasBR, ΔfasB Δpks13 and ΔfasBR Δpks13 were constructed and exhibited increased L-alanine yields up to 17.29 g/L.Different from ΔfasB mutant constructed from C.glutamicum ATCC13032 in which L-glutamate production accumulated,the muatnt ΔfasB constructed from C.glutamicum ATCC13869 in this study significantly produce L-alanine without L-glutamate accumulation.Transcriptional level analysis revealed that the knockout of fasB upregulated the expression levels of the genes related to L-alanine synthesis but downregulated those associated with fatty acid synthesis,confirming the redirection of metabolic flux from fatty acid synthesis to L-alanine synthesis in these strains.L-alanine productions were further enhanced in strains ΔfasB and ΔfasBR through the combinatorial expression of heterologous genes Bacillus subtilis alaD encoding alanine dehydrogenase and Escherichia coli alaE encoding alanine export protein,and the yields reached 55.21 g/L and 54.95 g/L,respectively.Finally,69.9 g/L L-alanine was obtained in ΔfasB/pJYW-5-alaDE after 60 h of fermentation by supplementing glucose.Our data indicate that disrupting the fatty acid biosynthesis could redirect metabolic flux towards L-alanine biosynthesis.These results provide a new strategy for increasing the production of L-alanine in C.glutamicum.
基金funded by the“Important Amino Acid Industrial Strain System Transformation and Industrial Demonstration”project[Grant Numbers 2021YFC2100900].
文摘This study described that a low-producing mutagenic strain was transformed to a l-lysine high-producing recombinant strain by optimizing the l-lysine metabolic pathway of Corynebacterium glutamicum.The nucleotide sequence results revealed that the lysC of mutant strain CgK37 mutated at 279th codon.Based on this site,site-directed saturation mutation was performed to screen for the mutant with better effect in relieving aspartate kinase feedback inhibition.Then,the supply of oxaloacetate and nicotinamide adenine dinucleotide phosphate was increased via knockout and overexpression of related genes.In order to solve the problem of low utilization efficiency of culture medium,fructokinase gene gmuE was heterologous expressed in CgK37,which could directly use intracellular fructose to improve the growth rate.In addition,the synthesis of partial by-products was weakened at the gene transcription level to avoid carbon excessively flowing into the branch metabolism.Finally,a large-scale fermentation experiment was conducted in 5 L jar fermenter.The l-lysine yield of CgK37-11 was 196.58±1.68 g/L,which was 83.24%original higher than CgK37,and the productivity reached 2.46 g/L/h.
基金supported by the program of State Key Laboratory of Food Science and Technology(SKLF-ZZA-201904).
文摘Corynebacterium glutamicum is widely used in the production of amino acids.C.glutamicum possesses seven sigma factors,among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic acid(MA)and its derivatives,the unique cell envelope of C.glutamicum.To understand the influence of MA synthesis on amino acid production and membrane phenotype of C.glutamicum,the expression of sigD gene and some mycolyltransferase genes,i.e.,cmt1,cop1 and cmt2,were regulated by several growth-regulated promoters in this study.Except for 2 mutant strains of P_(cg3096)-sigD and P_(cg1633)-cop1,the growth and 4-hydroxyisoleucine(4-HIL)titer of most modified strains did not change significantly.But the 4-HIL titer of P_(odhI)-sigD strain increased by 20.73%(142.45±3.69 mM)compared to that of control strain(117.99±0.34 mM).After it was cultivated in bioreactor,4-HIL titer reached 372.56 mM.This may be caused by the increase of MA content,and 17%decrease of cell hydrophobicity and 12%increase of membrane permeability were observed at the exponential phase.In conclusion,we proved that rearrangements in regulation of sigD expression contributed to the improved fermentation performance of C.glutamicum and promoted 4-HIL production.
基金supported by the National Key Research and Development Program of China(2021YFC2100900).
文摘L-Methionine is widely used in food,agricultural and pharmaceutical industries.In this study,the L-methionine production in Corynebacterium glutamicum ATCC13032 was promoted by eliminating the feedback inhibition of key rate-limiting enzymes,blocking L-threonine biosynthesis,and strengthening the downstream pathway of L-homoserine.ATCC13032 does not accumulate L-threonine,we found that overexpressing the genes lysC and hom^(G378S) could accumulate 0.6 g/L L-threonine.Deleting the genes thrB,McbR,and metD in ATCC13032 could accumulate 0.49 g/L L-methionine.Next,enhancing oxaloacetate supply,overexpressing brnFE,and deleting Ncgl2640 that involved in the repression of sulphuric metabolism could accumulate 0.92 g/L L-methionine.Further overexpressing the genes related to L-homoserine downstream pathway,the resulting strain ZBW011/pEC-metYX could produce 1.82 g/L L-methionine.Finally,the gene pyk2 was deleted and the final strain ZBW014/pEC-metYX produced 7.06 g/L L-methionine in a 2.4-L fermenter.The strategies presented in this study would be useful to engineer C.glutamicum for industrial L-methionine production.
基金financially supported by the National Natural Science Foundation of China(32171470).
文摘L-serine is utilized in various applications across the pharmaceutical and food industry.Corynebacterium glutamicum,a non-pathogenic strain,is extensively used in amino acid production.However,the current titer and productivity of L-serine through direct fermentation are insufficient to meet the demands of industrial production.This shortfall arises from the strain’s inadequacy sucrose utilization,which affects both L-serine productivity and sucrose consumption rate.To solve the problem,this research conducted three strategies to increase the carbon flow from sucrose to L-serine.Initially,ALE was performed using a stress of 300 g/L sucrose based on A36-pDSer,and a biosensor-assisted high-throughput screening platform was utilized to identify the mutants with higher intracellular L-serine concentration.The strain A36-mut achieved 39.0 g/L L-serine titer,marking a notable 25.3%improvement over the parent strain A36(31.1 g/L).Subsequently,the overexpression of the L-serine exporter serE along with its transcription factor serR in strain A36-mut led to an improvement in the L-serine production,reaching 44.8 g/L.Finally,by optimizing the fed-batch fermentation process,the L-serine titer and productivity were improved to 53.7 g/L and 0.50 g/L/h,respectively.This research presented the highest L-serine titer from sucrose in C.glutamicum to date,offering the possibility for the industrialization production of L-serine by fermentation.
文摘4-Hydroxyisoleucine(4-HIL)holds potential value in the treatment of diabetes.It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine(Ile)producing Corynebacterium glutamicum strains.But the stable expression of ido on plasmids relies on the usage of antibiotics.To make the harboring of ido independent of plasmid,this study developed a chromosome-engineered strain for synthesizing 4-HIL directly from glucose.First,the ido-cat-ido expressing cassette was inserted into the chromosome of C.glutamicum,and the copy number of ido was increased through chemically inducible chromosome evolution(CIChE).After successive rounds of CIChE by increasing chloramphenicol concentration,7 copies of ido were integrated in the chromosome of C.glutamicum SE04,and the 4-HIL production reached 20.3±4.99 g/L,3.5-fold higher than the initial strain SC12 harboring two-copies of ido.To cease further homologous recombination,recA was deleted in CIChE strains,but cell growth and 4-HIL production were damaged.Notably,the stability of chromosomally inserted genes in the evolved strain SE04 was confirmed.Ultimately,the evolved C.glutamicum SE04 strain produced 30.3 g/L of 4-HIL in a 2-L bioreactor.This study established a plasmid-free strain of C.glutamicum for 4-HIL production,offering new insights into utilizing multi-copy integration methods for producing other valuable biochemical substances in C.glutamicum.
基金supported by the National Key Research and Development Program of China(No.2021YFC2100900)the National Natural Science Foundation of China(No.32171471)+1 种基金the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX24_2586)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Corynebacterium glutamicum is a safe strain with great potential for industrial applications,but more research is needed on secretory expression systems.Here,we constructed a non-inducible secretory expression system of the strain.By building a signal peptide library,we screened several Sce-type signal peptides and analyzed the relationship between their constitutive properties and secretory efficiency.To further meet the safety requirements in industrial applications,fifteen constitutive promoters were screened,and protein expression was optimized by promoter tandem strategy.In the WYJ1,WYJ2,WYJ3,and WYJ4 engineering strains,we confirmed that the modification of cell permeability favored protein secretion.The engineering strains WYJ2P35SP35 and WYJ4P35SP35 were scaled up for culture,and their extracellular enzyme activities and proteins reached 26.42 U/mL and 19.65 mg/L,and 23.97 U/mL and 13.84 mg/L,respectively.This secretory expression system increases the potential of industrial applications of Corynebacterium glutamicum and lays the foundation for applications.
文摘2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth.Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose.In this study,C.glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose.The gene futC from Helicobacter pylori encodingα-1,2-fucosyltransferase was codon optimized and mutated at four amino acids(F40S/Q150H/C151R/Q239S).The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C.glutamicum,resulting in CW002.CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane.Therefore,the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002,resulting in CW006.CW006 did synthesize 2′-fucosyllactose as expected.It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed,suggesting that the expression levels of the four genes in CW006 might have to well balanced.C.glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.
基金funded by the“Important Amino Acid Industrial Strain System Transformation and Industrial Demonstration”project[grant numbers 2021YFC2100900].
文摘β-Alanine is the only naturally occurring β-type amino acid,with various applications in the pharmaceutical,food,and chemical industries.Given the growing market demand,the study of β-alanine production is important.This study utilized a modified lysine-producing strain as a chassis cell line to further promote β-alanine synthesis through metabolic engineering.In order to reduce the consumption of oxaloacetate,the gene pck was deleted.A promoter mutation library was constructed to screen the original promoter of the stronger promoter replacement gene pyc to enhance the oxaloacetate synthesis pathway and further increase the intracellular supply of oxaloacetate.Next,the gene poxB was deleted,and pyruvate accumulation further promoted β-alanine synthesis.Then,the aspartate kinase-coding gene lysC was weakened by predicting the RBS sequence,thus reducing the synthesis of lysine by-products and improving β-alanine synthesis.Ultimately,the carbon flux in the β-alanine biosynthetic pathways was increased by overexpressing aspartate-α-decarboxylase,aspartate ammonia-lyase,and aspartate aminotransferase using the strong promoter Ptrc.The resulting strain QBA9 was cultured in a 5-L fermenter by fed-batch to produce 70.8 g/L of β-alanine with a productivity of 0.98 g/L/h.These modification strategies demonstrate the potential for efficient β-alanine production by the lysine-producing strain and provide an innovative idea for the developing β-alanine-producing strains.
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.
