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
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
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
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
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
Polymyxin B,produced by Paenibacillus polymyxa,is used as the last line of defense clinically.In this study,exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of...Polymyxin B,produced by Paenibacillus polymyxa,is used as the last line of defense clinically.In this study,exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P.polymyxa CJX518-AC(PPAC)from 0.15 g/L and 61.8%to 0.33 g/L and 79.9%,respectively.The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01,which produces high levels of threonine,leucine,and isoleucine,increased polymyxin B1 production to 0.64 g/L.When strains PPAC and C.glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone,polymyxin B1 production was increased to 0.97 g/L.Furthermore,the polymyxin B1 production in the co-culture of strains PPAC and C.glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone.This study provides a new strategy to improve polymyxin B1 production.展开更多
Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may ...Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.展开更多
Heme,an iron-containing porphyrin derivative,holds great promise in fields like medicine,food production and chemicals.Here,we developed an engineered Corynebacterium glutamicum strain for efficient heme production by...Heme,an iron-containing porphyrin derivative,holds great promise in fields like medicine,food production and chemicals.Here,we developed an engineered Corynebacterium glutamicum strain for efficient heme production by combining modular engineering and RBS engineering.The whole heme biosynthetic pathway was methodically divided into 5-ALA synthetic module,uroporphyrinogen III(UPG III)synthetic module and heme synthetic module for further construction and optimization.Three heme synthetic modules were compared and the siroheme-dependent(SHD)pathway was identified to be optimal in C.glutamicum for the first time.To further improve heme production,the expression of genes in UPG III synthetic module and heme synthetic module was coordinated optimized through RBS engineering,respectively.Subsequently,heme oxygenase was knocked out to reduce heme degradation.The engineered strain HS12 showed a maximum iron-containing porphyrin derivatives titer of 1592 mg/L with the extracellular secretion rate of 45.5%in fed-batch fermentation.Our study constructed a C.glutamicum chassis strain for efficient heme accumulation,which was beneficial for the advancement of efficient heme and other porphyrins production.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
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
Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments...Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments.Among the identified proteins encoded by genes within the C.glutamicum genome,nearly 400 are still functionally unknown.Based on previous transcriptome analysis,we found that the hypothetical protein gene cgl2215 was highly upregu-lated in response to phenol,ferulic acid,and vanillin stress.The cgl2215 deletion mutant was shown to be more sensitive than the parental strain to phenolic compounds as well as other environmental factors such as heat,ethanol,and oxidative stresses.Cgl2215 interacts with C.glutamicum mycoloyltransferase A(MytA)and enhances its in vitro esterase activity.Sensitivity assays of theΔmytA andΔcgl2215ΔmytA mutants in response to phenolic stress estab-lished that the role of Cgl2215 in phenolic tolerance was mediated by MytA.Furthermore,transmission electron microscopy(TEM)results showed that cgl2215 and mytA deletion both led to defects in the cell envelope structure of C.glutamicum,especially in the outer layer(OL)and electron-transparent layer(ETL).Collectively,these results indicate that Cgl2215 can enhance MytA activity and affect the cell envelope structure by directly interacting with MytA,thus playing an important role in resisting phenolic and other environmental stresses.展开更多
Trehalose is a disaccharide with many applications in cosmetics,refrigeration,and food.Trehalose synthase is a significant enzyme in trehalose production.Escherichia coli is usually used to express this enzyme heterol...Trehalose is a disaccharide with many applications in cosmetics,refrigeration,and food.Trehalose synthase is a significant enzyme in trehalose production.Escherichia coli is usually used to express this enzyme heterologously.Since E.coli is a pathogenic strain,we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety reasons.Because of its poor permeability,we constructed two recombinant C.glutamicum strains using two anchor proteins,PorH,and short-length NCgl1337,to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from maltose.Studies on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free enzyme.After optimizing the whole-cell transformation,the optimal transformation condition was 35°C,pH 7.0,and OD600 of 30.Under this condition,the conversion rate of 300 g/L maltose reached 69.5%in a 5 L fermentor.The relative conversion rate was still above 75%after repeated five times.展开更多
Corynebacterium glutamicum is a microbial production host established in the industry 60 years ago.It is mainly used for production of feed and food amino acids.As C.glutamicum strain development has been cutting edge...Corynebacterium glutamicum is a microbial production host established in the industry 60 years ago.It is mainly used for production of feed and food amino acids.As C.glutamicum strain development has been cutting edge since its discovery,it has been engineered for production of a plethora of valuable products.This review will focus on recent developments of C.glutamicum strain engineering for biotransformation and fermentation processes towards flavor and fragrance molecules as well as pigments and sweeteners.展开更多
Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals,livestock,food additives,and so on.It is important to develop Corynebacterium glutamicum strains that c...Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals,livestock,food additives,and so on.It is important to develop Corynebacterium glutamicum strains that can efciently produce gamma-aminobutyric acid from glucose.In this study,production of gamma-aminobutyric acid in C.glutamicum CGY700 was improved by construction of CO_(2) anaplerotic reaction and overexpression of citrate synthase.The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck.The expression of ppc and gltA were controlled by promoters P_(tac) and P_(tacM),and the optimal mode of P_(tacM-ppc)-P_(tac)-gltA was determined.Simultaneously,the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted,and glnA2 encoding glutamine synthase was overexpressed in the chromosome.The fnal strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake fask cultivation and 58.33 g/L gamma-aminobutyric acid via FedBatch fermentation with a yield of 0.30 g/g glucose.CGY-PG-304 was constructed by genome editing;therefore,it is stable and not necessary to add any antibiotics and inducer during fermentation.展开更多
4-hydroxyisoleucine(4-HIL)is a potential drug for diabetes and weight control.4-HIL was produced by expressing ido gene in L-isoleucine(Ile)-producing Corynebacterium glutamicum.But L-lysine(Lys)was also accumulated a...4-hydroxyisoleucine(4-HIL)is a potential drug for diabetes and weight control.4-HIL was produced by expressing ido gene in L-isoleucine(Ile)-producing Corynebacterium glutamicum.But L-lysine(Lys)was also accumulated as the main by-product in this recombinant strain SN02.To attenuate Lys synthesis,two genes in Lys synthetic pathway,i.e.,ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02.However,the deletion of ddh increased 4-HIL titer by 28.1%,but did not decrease Lys content;while the deletion of lysE significantly reduced Lys content by 66.7%,but 4-HIL titer also decreased by 19.3%.Therefore,we carried out transcriptome analysis to reveal the global variation in these mutants.Deletion of ddh and lysE(especially lysE)enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway(DapD and DapC)and several enzymes involved in succinyl-CoA accessibility(SucC,SucD and OdhI),suggesting the compensatory synthesis of Lys via succinylase branch.In addition,the transcription of ilvBN in Ile synthesis pathway was improved,while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants.Mere deletion of ddh enhanced the transcription of aceA,ppc and pck,thus promoting oxaloacetate supply and 4-HIL synthesis.Deletion of lysE affected the transcription of some stress-related genes and transporter genes,suggesting that this mutant would be under stress,thus attenuating its 4-HIL synthesis.These findings will be helpful for systematic microbiology and bio-manufacturing of C.glutamicum.展开更多
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 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(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grateful for the financial supports from the National Key R&D Program of China(2018YFA0902200)the National Natural Science Foundation of China(Program:21878224).
文摘Polymyxin B,produced by Paenibacillus polymyxa,is used as the last line of defense clinically.In this study,exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P.polymyxa CJX518-AC(PPAC)from 0.15 g/L and 61.8%to 0.33 g/L and 79.9%,respectively.The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01,which produces high levels of threonine,leucine,and isoleucine,increased polymyxin B1 production to 0.64 g/L.When strains PPAC and C.glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone,polymyxin B1 production was increased to 0.97 g/L.Furthermore,the polymyxin B1 production in the co-culture of strains PPAC and C.glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone.This study provides a new strategy to improve polymyxin B1 production.
基金funded by National Natural Science Foundation of China(no.32272279)the Key R&D project of Qingdao Science and Technology Plan(22-3-3-hygg-29-hy).
文摘Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.
基金This work was financially supported by the National Key Research and Development Program of China,China(2021YFC2100700)the National Natural Science Foundation of China,China(NSFC-22278312).
文摘Heme,an iron-containing porphyrin derivative,holds great promise in fields like medicine,food production and chemicals.Here,we developed an engineered Corynebacterium glutamicum strain for efficient heme production by combining modular engineering and RBS engineering.The whole heme biosynthetic pathway was methodically divided into 5-ALA synthetic module,uroporphyrinogen III(UPG III)synthetic module and heme synthetic module for further construction and optimization.Three heme synthetic modules were compared and the siroheme-dependent(SHD)pathway was identified to be optimal in C.glutamicum for the first time.To further improve heme production,the expression of genes in UPG III synthetic module and heme synthetic module was coordinated optimized through RBS engineering,respectively.Subsequently,heme oxygenase was knocked out to reduce heme degradation.The engineered strain HS12 showed a maximum iron-containing porphyrin derivatives titer of 1592 mg/L with the extracellular secretion rate of 45.5%in fed-batch fermentation.Our study constructed a C.glutamicum chassis strain for efficient heme accumulation,which was beneficial for the advancement of efficient heme and other porphyrins production.
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 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supported by the grant of National Key R&D Program of China(2021YFA0909600 to X.S.)the National Natural Science Foundation of China(31670053 to X.S.,32000022 to H.G.)+1 种基金China Postdoctoral Science Foundation(2019M663829 to H.G.)Northwest A&F University starting research fund(Z1090219039 to H.G.).
文摘Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments.Among the identified proteins encoded by genes within the C.glutamicum genome,nearly 400 are still functionally unknown.Based on previous transcriptome analysis,we found that the hypothetical protein gene cgl2215 was highly upregu-lated in response to phenol,ferulic acid,and vanillin stress.The cgl2215 deletion mutant was shown to be more sensitive than the parental strain to phenolic compounds as well as other environmental factors such as heat,ethanol,and oxidative stresses.Cgl2215 interacts with C.glutamicum mycoloyltransferase A(MytA)and enhances its in vitro esterase activity.Sensitivity assays of theΔmytA andΔcgl2215ΔmytA mutants in response to phenolic stress estab-lished that the role of Cgl2215 in phenolic tolerance was mediated by MytA.Furthermore,transmission electron microscopy(TEM)results showed that cgl2215 and mytA deletion both led to defects in the cell envelope structure of C.glutamicum,especially in the outer layer(OL)and electron-transparent layer(ETL).Collectively,these results indicate that Cgl2215 can enhance MytA activity and affect the cell envelope structure by directly interacting with MytA,thus playing an important role in resisting phenolic and other environmental stresses.
基金the National Natural Science Foundation of China(No.32171471)Key Research and Development Project of Shandong Province,China(2019JZZY020605)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Trehalose is a disaccharide with many applications in cosmetics,refrigeration,and food.Trehalose synthase is a significant enzyme in trehalose production.Escherichia coli is usually used to express this enzyme heterologously.Since E.coli is a pathogenic strain,we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety reasons.Because of its poor permeability,we constructed two recombinant C.glutamicum strains using two anchor proteins,PorH,and short-length NCgl1337,to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from maltose.Studies on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free enzyme.After optimizing the whole-cell transformation,the optimal transformation condition was 35°C,pH 7.0,and OD600 of 30.Under this condition,the conversion rate of 300 g/L maltose reached 69.5%in a 5 L fermentor.The relative conversion rate was still above 75%after repeated five times.
基金funding enabled and organized by Projekt DEAL.Support in the framework of the ERA CoBioTech project INDIE(European Union’s Horizon 2020 research and innovation programme under grant agreement No.722361)with national funding is acknowledged by KC(Dutch research council(NWO)grant number 053.80.732)and VFW(Renewable Resources Scheme(FNR)of the Federal Ministry of Food and Agriculture,Germany,grant number 22023517).NAH acknowledges funding by BMBF project KaroTec(grant number:03VP09460).
文摘Corynebacterium glutamicum is a microbial production host established in the industry 60 years ago.It is mainly used for production of feed and food amino acids.As C.glutamicum strain development has been cutting edge since its discovery,it has been engineered for production of a plethora of valuable products.This review will focus on recent developments of C.glutamicum strain engineering for biotransformation and fermentation processes towards flavor and fragrance molecules as well as pigments and sweeteners.
基金Publication costs are funded by the National Key Research and Development Program of China(2021YFC2100900)the Key Technology Project of Inner Mongolia Autonomous Region,China(2019GG302).
文摘Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals,livestock,food additives,and so on.It is important to develop Corynebacterium glutamicum strains that can efciently produce gamma-aminobutyric acid from glucose.In this study,production of gamma-aminobutyric acid in C.glutamicum CGY700 was improved by construction of CO_(2) anaplerotic reaction and overexpression of citrate synthase.The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck.The expression of ppc and gltA were controlled by promoters P_(tac) and P_(tacM),and the optimal mode of P_(tacM-ppc)-P_(tac)-gltA was determined.Simultaneously,the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted,and glnA2 encoding glutamine synthase was overexpressed in the chromosome.The fnal strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake fask cultivation and 58.33 g/L gamma-aminobutyric acid via FedBatch fermentation with a yield of 0.30 g/g glucose.CGY-PG-304 was constructed by genome editing;therefore,it is stable and not necessary to add any antibiotics and inducer during fermentation.
基金funded by the program of State Key Laboratory of Food Science and Technology(SKLF-ZZA-201904)national first-class discipline program of Light Industry Technology and Engineering(LITE2018-10).
文摘4-hydroxyisoleucine(4-HIL)is a potential drug for diabetes and weight control.4-HIL was produced by expressing ido gene in L-isoleucine(Ile)-producing Corynebacterium glutamicum.But L-lysine(Lys)was also accumulated as the main by-product in this recombinant strain SN02.To attenuate Lys synthesis,two genes in Lys synthetic pathway,i.e.,ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02.However,the deletion of ddh increased 4-HIL titer by 28.1%,but did not decrease Lys content;while the deletion of lysE significantly reduced Lys content by 66.7%,but 4-HIL titer also decreased by 19.3%.Therefore,we carried out transcriptome analysis to reveal the global variation in these mutants.Deletion of ddh and lysE(especially lysE)enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway(DapD and DapC)and several enzymes involved in succinyl-CoA accessibility(SucC,SucD and OdhI),suggesting the compensatory synthesis of Lys via succinylase branch.In addition,the transcription of ilvBN in Ile synthesis pathway was improved,while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants.Mere deletion of ddh enhanced the transcription of aceA,ppc and pck,thus promoting oxaloacetate supply and 4-HIL synthesis.Deletion of lysE affected the transcription of some stress-related genes and transporter genes,suggesting that this mutant would be under stress,thus attenuating its 4-HIL synthesis.These findings will be helpful for systematic microbiology and bio-manufacturing of C.glutamicum.
