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公开(公告)号：US12049661B2
公开(公告)日：2024-07-30
申请号：US17479018
申请日：2021-09-20
申请人： Jiangnan University
发明人： Liming Liu , Guipeng Hu , Xiulai Chen , Danlei Ma , Jia Liu , Qiuling Luo
CPC分类号： C12P7/46 , C12N1/20 , C12N9/0006 , C12N9/001 , C12N9/0093 , C12N9/1205 , C12N9/88 , C12N9/93 , C12N15/70 , C12Y101/01001 , C12Y101/01028 , C12Y101/0104 , C12Y103/05004 , C12Y117/01 , C12Y207/01029 , C12Y402/01002 , C12Y602/01001
摘要： The disclosure discloses construction and application of an engineered strain of E. coli for producing malic acid by fixing CO2, and belongs to the field of fermentation. The engineered strain is obtained by performing genetic engineering transformation on Escherichia coli MG1655; the genetic engineering transformation includes knocking out a fumarate reductase gene, a fumarase gene, a lactate dehydrogenase gene and an alcohol dehydrogenase gene and freely overexpressing a formate dehydrogenase, an acetyl coenzyme A synthetase, an acylated acetaldehyde dehydrogenase, a formaldehyde lyase, a dihydroxyacetone kinase, a malic enzyme and a phosphite oxidoreductase to obtain a strain GH0407. The strain is used for producing malic acid by fermentation, anaerobic fermentation is performed for 72 hours with CO2 and glucose as a co-substrate, the production of malic acid reaches 39 g/L, the yield is 1.53 mol/mol, and accumulation of malic acid in the original strain is not achieved.
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公开(公告)号：US11976267B2
公开(公告)日：2024-05-07
申请号：US17256906
申请日：2020-11-12
申请人： JIANGNAN UNIVERSITY
发明人： Liming Liu , Wenxiu Tang , Chen Shen , Qiuling Luo , Xiulai Chen , Jia Liu , Cong Gao , Wei Song
CPC分类号： C12N1/205 , C12N9/0004 , C12N9/1025 , C12N9/88 , C12N15/52 , C12P1/04 , C12R2001/19
摘要： The invention provides a recombinant Escherichia coli strain for producing succinic acid and a construction method thereof. The by-product encoding genes in the E. coli strain FMME-N-2 are knocked out to obtain the E. coli strain FMME-N-5 (ΔfocA-pflB-ΔldhA-Δpta-ackA); and the phosphoenolpyruvate carboxykinase pck derived from Actinobacillus succinogenes and the phosphite dehydrogenase ptxD derived from Pseudomonas stutzeri were overexpressed. The constructed plasmid pTrcHisA-pck-ptxD was introduced into the expression host E. coli FMME-N-5 (ΔfocA-pflB-ΔldhA-Δpta-ackA), and the cells were screened in a plate containing ampicillin, to obtain an engineered strain E. coli FMME-N-5 (ΔfocA-pflB-ΔldhA-Δpta-ackA)-pck-ptxD that can efficiently produce succinic acid. After fermentation by a two-stage fermentation strategy, the production of succinic acid reaches 137 g/L, the yield of succinic acid is up to 1 g/g glucose, and the space time yield is 1.43 g/L/h, while no by-products of lactic acid and formic acid are accumulated, and the acetic acid content is 1-2 g/L.
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公开(公告)号：US11781118B2
公开(公告)日：2023-10-10
申请号：US17351318
申请日：2021-06-18
申请人： Jiangnan University
发明人： Jing Wu , Shanshan Pei , Jia Liu , Wei Song , Xiulai Chen , Qiuling Luo
CPC分类号： C12N9/0022 , C12P7/40 , C12Y104/03002
摘要： The disclosure discloses preparation of an L-amino acid deaminase mutant and application thereof, belonging to the technical field of gene engineering. Through pmirLAAD protein modification, analysis of a flexible loop structure around a binding site of the pmirLAAD product, and design of the best mutant, the modification method of the disclosure overcomes the defect that the catalytic efficiency of the previous wild-type enzyme is reduced due to product inhibition, and is tested by experiments. Compared with the control, the catalytic efficiency (1.61 mM−1·min−1) and the product inhibition constant (5.4 mM) of the finally obtained best mutant pmirLAADM4 are respectively increased by 5.2 times and 5.7 times. The yield of α-ketoisovaleric acid can reach 96.5 g/L, and the transformation rate is greater than 97%. By adopting the method of the disclosure, the cost can be greatly reduced, and the industrialization process of production of α-ketoisovaleric acid by an enzymatic transformation method is accelerated.
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