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
Systems metabolic engineering is a multidisciplinary area that integrates systems biology,synthetic biology and evolutionary engineering.It is an efficient approach for strain improvement and process optimization,and ...Systems metabolic engineering is a multidisciplinary area that integrates systems biology,synthetic biology and evolutionary engineering.It is an efficient approach for strain improvement and process optimization,and has been successfully applied in the microbial production of various chemicals including amino acids.In this review,systems metabolic engineering strategies including pathwayfocused approaches,systems biology-based approaches,evolutionary approaches and their applications in two major amino acid producing microorganisms:Corynebacterium glutamicum and Escherichia coli,are summarized.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
Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated...Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glu- tamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.展开更多
Rational microbial chassis design and engineering for improving production of amino acids have attracted a considerable attention.l-glutamate,l-lysine,l-threonine and l-tryptophan are the main amino acids demanded in ...Rational microbial chassis design and engineering for improving production of amino acids have attracted a considerable attention.l-glutamate,l-lysine,l-threonine and l-tryptophan are the main amino acids demanded in the food industry.Systems metabolic engineering and synthetic biology engineering generally are believed as the comprehensive engineering approaches to obtain rationally designed strains and construct high-performance platforms for amino acids.The strate-gies focus on microbial chassis characterization optimization,precise metabolic engineering such as promoter engineer-ing,modular pathway engineering,transporter engineering,and dynamic switch systems application,and global genome streamline engineering to reduce cell burden.In this review,we summarized the efficient engineering strategies to optimize Corynebacterium glutamicum and Escherichia coli cell factories for improving the production of l-glutamate,l-lysine,l-threonine,and l-tryptophan.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
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
研究不同树脂对L-瓜氨酸的吸附能力,发现D001树脂对L-瓜氨酸的吸附效果最好。采用静态吸附法研究L-瓜氨酸在D001型阳离子交换树脂上的热力学和动力学特性,考察不同温度、pH和溶液初始浓度对离子交换过程的影响。结果表明:L-瓜氨酸在D00...研究不同树脂对L-瓜氨酸的吸附能力,发现D001树脂对L-瓜氨酸的吸附效果最好。采用静态吸附法研究L-瓜氨酸在D001型阳离子交换树脂上的热力学和动力学特性,考察不同温度、pH和溶液初始浓度对离子交换过程的影响。结果表明:L-瓜氨酸在D001型阳离子交换树脂上的吸附等温线符合Freundlich和Langmuir等温吸附方程,其中,Langmuir吸附方程能更好地描述该过程。吸附过程焓变ΔH=-45.01 k J/mol(<0),说明该吸附过程放热。树脂对L-瓜氨酸的吸附过程速度控制步骤为颗粒扩散。随着温度升高,树脂的最大平衡吸附量减小;当pH=6时,树脂达到最大吸附量135.5 mg/g;L-瓜氨酸溶液初始质量浓度为8 g/L时,扩散系数达到最大,为8.53×10-3,吸附速率最快。展开更多
During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating en- zymes, which are phosphotransacetylase (PT...During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating en- zymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an over- expressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glu- cose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the pta-ack operon.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
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
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
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
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 research was supported by National High Technology Research and Development Program of China(2015AA021003)National Natural Science Foundation of China(31470211)+1 种基金Natural Science Foundation of Tianjin(17JCQNJC09500)Foundation(No.2016IM104)of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology(Tianjin Universityof Science&Technology).
文摘Systems metabolic engineering is a multidisciplinary area that integrates systems biology,synthetic biology and evolutionary engineering.It is an efficient approach for strain improvement and process optimization,and has been successfully applied in the microbial production of various chemicals including amino acids.In this review,systems metabolic engineering strategies including pathwayfocused approaches,systems biology-based approaches,evolutionary approaches and their applications in two major amino acid producing microorganisms:Corynebacterium glutamicum and Escherichia coli,are summarized.
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This work was supported by the Chinese Academy of Sciences(KSCX2-SW-1I3)National Natural Science Foundation of China(Grant No.30230010).
文摘Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacte- ria has been well studied, this branch is less understood in Gram+ bacteria. In this study, Cory- nebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glu- tamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.
基金supported by the National Natural Science Foundation of China(32100023)the Provincial Natural Science Foundation of Jiangsu Province(BK20210466)+2 种基金the National Natural Science Foundation of China(32000020)the Provincial Natural Science Foundation of Jiangsu Province(BK20200615)and the Youth Fund for Basic Research Program of Jiangnan University(JUSRP122009).
文摘Rational microbial chassis design and engineering for improving production of amino acids have attracted a considerable attention.l-glutamate,l-lysine,l-threonine and l-tryptophan are the main amino acids demanded in the food industry.Systems metabolic engineering and synthetic biology engineering generally are believed as the comprehensive engineering approaches to obtain rationally designed strains and construct high-performance platforms for amino acids.The strate-gies focus on microbial chassis characterization optimization,precise metabolic engineering such as promoter engineer-ing,modular pathway engineering,transporter engineering,and dynamic switch systems application,and global genome streamline engineering to reduce cell burden.In this review,we summarized the efficient engineering strategies to optimize Corynebacterium glutamicum and Escherichia coli cell factories for improving the production of l-glutamate,l-lysine,l-threonine,and l-tryptophan.
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.
文摘研究不同树脂对L-瓜氨酸的吸附能力,发现D001树脂对L-瓜氨酸的吸附效果最好。采用静态吸附法研究L-瓜氨酸在D001型阳离子交换树脂上的热力学和动力学特性,考察不同温度、pH和溶液初始浓度对离子交换过程的影响。结果表明:L-瓜氨酸在D001型阳离子交换树脂上的吸附等温线符合Freundlich和Langmuir等温吸附方程,其中,Langmuir吸附方程能更好地描述该过程。吸附过程焓变ΔH=-45.01 k J/mol(<0),说明该吸附过程放热。树脂对L-瓜氨酸的吸附过程速度控制步骤为颗粒扩散。随着温度升高,树脂的最大平衡吸附量减小;当pH=6时,树脂达到最大吸附量135.5 mg/g;L-瓜氨酸溶液初始质量浓度为8 g/L时,扩散系数达到最大,为8.53×10-3,吸附速率最快。
文摘During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating en- zymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an over- expressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glu- cose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the pta-ack operon.
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 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 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.
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.
