Systems Microbiology and Biomanufacturing
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2025
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IF 2.9
Li Zihan,Zhao Guihong,Zhang Dezhi,Tang Yaqun,Liu Geer,Wang Xiaoyuan
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 encoding α-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.
研究领域:
微生物学
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生物工程
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分子生物学
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食品科学
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2018
·Vol. 102
·期 14
·6039-6046
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IF 3.34
Tigu Fitsum,Zhang Junli,Liu Guoxia,Cai Zhen,Li Yin
d -Pantothenic acid (vitamin B 5 ) has wide applications in the feed, food, chemical, and pharmaceutical industries. Its biological production routes which employ pantothenate synthetase (PS) as the key enzyme are attractive since they avoid the tedious and time-consuming optical resolution process. However, little data is available on the activity and kinetics of this enzyme, hampering the rational selection of an efficient enzyme for the biological production of d -pantothenic acid. In this study, six phylogenetically distant PS-encoding genes, from Escherichia coli , Corynebacterium glutamicum , Bacillus subtilis , Bacillus thuringiensis , Bacillus cereus , and Enterobacter cloacae , were expressed in E. coli . The PS from C. glutamicum exhibited a specific activity of 205.1 U/mg and a turnover number of 127.6 s -1 , which to our best knowledge are the highest values ever reported. The addition of substrates ( d -pantoic acid and β-alanine) to the E. coli strain harboring this enzyme during the early log phase of fermentation resulted in the production of 97.1 g/L of d -pantothenic acid within 32 h, corresponding to a conversion yield of 99.1% and a productivity of 3.0 g/L/h. To the best of our knowledge, this is the highest productivity reported to date.
研究领域:
生物化学
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微生物学
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工业生物技术
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2025
·
IF 3.8
Ying-Ying Xu,Bin Zhang,Jie Bao
This study investigated Corynebacterium glutamicum for GABA production using corncob residue, a cheap carbon source, as carbohydrate feedstock instead of grains. Current fermentative production of γ-aminobutyric acid (GABA) mainly relies on starch and sugar feedstocks. Therefore, the use of cheap carbon sources can effectively reduce the production costs of GABA. This study validated the feasibility of an available non-food lignocellulose feedstock, corncob residue, for GABA fermentation. An engineered C. glutamicum was constructed by secretory expression of glutamate decarboxylase gadBmut and knock-out the GABA biodegradation gene. The fermentation parameters in shake flasks and bioreactors were optimized including the sugar concentration, nitrogen levels, cofactor levels and aeration rate, and fermentation mode. The fed-batch fermentation of the concentrated corncob residue hydrolysate achieved a record high GABA production of 93.15 g/L from lignocellulosic feedstock with a yield of 0.43 g/g glucose. The results provided technical support for the industrial production of GABA from low-cost lignocellulosic carbohydrates.
研究领域:
生物技术
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微生物学
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DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2021
·
IF 3.307
Jingbai Wen,Jie Bao
Fermentative production of γ-aminobutyric acid by the glutamate overproducing Corynebacterium glutamicum from cheap sugar feedstock is generally regarded as one of the most promising methods to reduce the production cost. However, the intracellularly expressed glutamate decarboxylase in C. glutamicum often showed feeble catalysis activity to convert glutamate into γ-aminobutyric acid. Here we tried to secretory express glutamate decarboxylase to achieve efficient extracellular decarboxylation of glutamate, thus improving the γ-aminobutyric acid production by C. glutamicum . We first tested glutamate decarboxylases from different sources, and the mutated glutamate decarboxylase GadBmut from E. coli with better catalytic performance was selected. Then, a signal peptide of the SEC translocation pathway directed the successful secretion of glutamate decarboxylase in C. glutamicum . The extracellular catalysis by secreted glutamate decarboxylase increased the γ-aminobutyric acid generation by three-fold, compared with intracellular catalysis. Enhancing glutamate decarboxylase expression and decreasing γ-aminobutyric acid degradation further increased γ-aminobutyric acid production by 39 %. The fed-batch fermentation of the engineered C. glutamicum strain reached the record high titer (77.6 ± 0.0 g /L), overall yield (0.44 ± 0.00 g/g glucose), and productivity (1.21 ± 0.00 g/L/h). This study demonstrated a unique design of extracellular catalysis for efficient γ-aminobutyric acid production by C. glutamicum .
研究领域:
微生物学
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生物技术
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2025
·
IF 3.8
Ying-Ying Xu,Bin Zhang,Jie Bao
Industrial production of cadaverine primarily relies on whole-cell catalysis of lysine using engineered Escherichia coli as host cells. The growing interest in cadaverine as a monomer of biobased polyamides requires inexpensive, non-food carbohydrate feedstock for its bioproduction, rather than using food-derived sugars as feedstock. This study used corncob residue as the starting carbohydrate feedstock followed by enzymatic hydrolysis to obtain the sugars. Since E. coli was not tolerant to even minor inhibitor residues in corncob residue hydrolysate, the robust Corynebacterium glutamicum was used as the host bacterium followed by the metabolic modifications of secretory expression of lysine decarboxylase via Ncgl1289 and cgR_0949 , and cadaverine degradation pathway knockout. The final whole-cell catalysis of C. glutamicum recombinant using corncob hydrolysate as carbohydrate feedstock achieved a record-high 78.19 g/L of cadaverine with a conversion yield of 91%.
研究领域:
工业微生物学
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生物工程
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代谢工程
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产品关联
DOI
高影响因子
ELECTROPHORESIS
·
2019
·Vol. 40
·期 20
·2665-2671
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IF 2.754
Wei Wang,Ruiguang Bai,Huimin Zhang,Xiaoyu Cai
Corynebacterium glutamicum ( C. glutamicum ) is a well-known workhorse for the industrial production of amino acids. Different carbon, nitrogen, and sulfur source may force the bacterium to produce specific metabolites. In this work, a method of high-speed MEKC with LIF detection was developed to rapidly analyze the amino acid metabolites released by C. glutamicum , which is fed with different culture mediums. Corynebacterium glutamicum was cultured in microbial fuel cells to monitor its metabolism process and collect its metabolites. In the CE system, a microliter-scale sample reservoir was designed and applied to perform tiny volume sample injection. With the assistance of microwave, the derivatization time for amino acids with FITC was greatly shortened to 6 min. Under the optimized condition, the eight candidate amino acids of metabolites could be rapidly separated within 2 min. The whole analysis process for real samples, from sampling to determination, could be shortened to less than 10 min. The results showed that C. glutamicum could produce additional l -lysine and l -valine as the metabolites when fed with glucose and l -methionine, respectively. The method proved that culture mediums used to feed C. glutamicum had great effect on the bacterium's metabolites.
研究领域:
微生物学
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生物化学
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分析化学
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产品关联
DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
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2024
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IF 4.4
Hui-Zhong Sun,Si-Yu Wei,Qiu-Man Xu,Wei Shang,Qing Li,Jing-Sheng Cheng,Ying-Jin Yuan
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.
研究领域:
微生物学
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生物工程
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药学
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DOI
PubMed
高影响因子
Systems Microbiology and Biomanufacturing
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2024
·
IF 0
Huang Yu,Li Hedan,Zhao Guihong,Hu Xiaoqing,Wang Xiaoyuan
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 Δ fasB , Δ fasBR , Δ fasB Δ pks13 and Δ fasBR Δ pks13 were constructed and exhibited increased L-alanine yields up to 17.29 g/L. Different from Δ fasB mutant constructed from C. glutamicum ATCC13032 in which L-glutamate production accumulated, the muatnt Δ fasB 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 Δ fasB and Δ fasBR 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 Δ fasB /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 . Graphical Abstract
研究领域:
微生物学
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生物工程
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分子生物学
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DOI
ACS Catalysis
·
2025
·
IF 13.1
Mengkai Hu,Teng Bao,Zhen Qin,Qiang Wang,Hengwei Zhang,Yujue Wang,Jiajia You,Zhenglian Xue,Rongzhen Zhang,Shang-Tian Yang,Xian Zhang,Zhiming Rao
Surface-displayed whole-cell biocatalysis offers significant advantages over traditional intracellular enzyme-based methods but faces challenges in complex multienzyme processes and low efficiency. We present Scaffold-Mediated Assembly Regulation Tool (SMART), a programmable platform for dynamic control of multienzyme biocatalysts on Corynebacterium glutamicum surfaces. SMART integrates orthogonal cross-linked scaffolds with the twin Catcher/Tag pairs (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag), controllable molecular ratios, and high loading capacity, offering user-defined functionality for enzyme assembly. We optimized SMART through computational modeling and experimental validation and created bifunctional scaffolds and long tandem repeats for optimal enzyme loading. The versatility of SMART was demonstrated by codisplaying in a two enzyme sequential cascade with tunable stoichiometry, significantly improving the conversion of maltodextrin to trehalose. Furthermore, we integrated a Bxb1 recombinase-based state machine (RSM) genetic circuit, allowing temporal regulation of enzyme and scaffold expression within a single cell. Finally, the system’s applicability was showcased in isomaltulose production from low-cost sucrose. SMART design represents a significant advancement in surface-displayed biocatalysis, offering a programmable platform for complex multienzyme reactions with potential applications in various industrially relevant biocatalysts.
研究领域:
合成生物学
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生物化学
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代谢工程
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DOI
高影响因子
BIOCHEMICAL GENETICS
·
2019
·Vol. 57
·期 3
·443-454
·
IF 1.931
Zhang Yu,Zhang Xiaohuan,Xiao Shiyuan,Qi Wei,Xu Jingliang,Yuan Zhenhong,Wang Zhongming
3-Methyl-1-butanol (3MB) is a promising biofuel that can be produced from 2-ketoisocaproate via the common l -leucine biosynthesis pathway. Corynebacterium glutamicum was chosen as a host bacterium because of its strong resistance to isobutanol. In the current study, several strategies were designed to overproduce 3MB in C. glutamicum through a non-fermentation pathway. The engineered C. glutamicum mutant was obtained by silencing the pyruvate dehydrogenase gene complex ( aceE ) and deleting the lactic dehydrogenase gene ( ldh ), followed by mutagenesis with diethyl sulfate (DES) and selection with Fmoc-3-4-thiazolyl- l -alanine (FTA). The mutant could produce 659 mg/L of 3MB after 12 h of incubation. To facilitate carbon flux to 3MB biosynthesis, the engineered recombinant was also constructed without branched-chain acid aminotransferase ( ilvE ) activity by deleting the ilvE gene. This recombinant could produce 697 mg/L of 3MB after 12 h of incubation.
研究领域:
微生物学
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生物技术
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代谢工程
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产品关联
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Wang Qing,Zhang Jie,Zhao Zhe,Li Yichen,You Jiajia,Wang Yi,Li Xiangfei,Xu Meijuan,Rao Zhiming
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the ‘dock’ of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
研究领域:
微生物学
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遗传学
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合成生物学
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产品关联
DOI
PubMed
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2023
·Vol. 39
·期 6
·1-13
·
IF 4.1
Zhu Di,Wu Caiyun,Niu Chenxin,Li Hong,Ge Fanglan,Li Wei
Corynebacterium glutamicum porphobilinogen synthase (PBGS) is a metal enzyme with a hybrid active site metal binding sequence. In this study, the porphobilinogen synthase gene of C. glutamicum was cloned and heterogeneously expressed in Escherichia coli. C. glutamicum PBGS was purified, and its enzymatic characteristics were analyzed. The results showed that C. glutamicum PBGS is a Zn 2+ -dependent enzyme, and Mg 2+ has allosteric regulation. The allosteric Mg 2+ plays a vital role in forming the quaternary structure of C. glutamicum PBGS. Based on the ab initio predictive structure modeling of the enzyme and the molecular docking model of 5-aminolevulinic acid (5-ALA), 11 sites were selected for site-directed mutagenesis. When the hybrid active site metal binding site of C. glutamicum PBGS is converted into a cysteine-rich motif (Zn 2+ -dependent) or an aspartic acid-rich motif (Mg 2+ /K + -dependent), the enzyme activity is basically lost. Four residues, D128, C130, D132, and C140, in the metal binding site, were the binding sites of Zn 2+ and the active center of the enzyme. The band migration, from the native PAGE, of five variants with mutations in the center of enzyme activity was the same as that of the variant enzymes as purified, individually adding two metal ion chelating agents. Their Zn 2+ active center structures were abnormal, and the quaternary structure equilibrium was altered. The destroyed active center affects the construction of its quaternary structure. The quaternary structural balance between octamer and hexamer through dimers was regulated by the allosteric regulation of C. glutamicum PBGS. The enzyme activity was also affected by the change of the active site lid structure and (α β) 8 -barrel structure caused by mutation. Structural changes in the variants were analyzed to understand C. glutamicum PBGS better.
研究领域:
生物化学
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分子生物学
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酶学
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微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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