A novel process for obtaining pinosylvin using combinatorial bioengineering in Escherichia coli.
Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli. Firstly, we evaluated different construct environments to give a highly efficient constitutive system for enzymes of pinosylvin pathway expression: 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). Secondly, malonyl coenzyme A (malonyl-CoA) is a key precursor of pinosylvin bioproduction and at low level in E. coli cell. Thus clustered regularly interspaced short palindromic repeats interference (CRISPRi) was explored to inactivate malonyl-CoA consumption pathway to increase its availability. The resulting pinosylvin content in engineered E. coli was obtained a 1.9-fold increase depending on the repression of fabD (encoding malonyl-CoA-ACP transacylase) gene. Eventually, a phenylalanine over-producing E. coli consisting phenylalanine ammonia lyase was introduced to produce the precursor of pinosylvin, trans-cinnamic acid, the crude extraction of cultural medium was used as supplementation for pinosylvin bioproduction. Using these combinatorial processes, 47.49 mg/L pinosylvin was produced from glycerol.
Department of Bioengineering, College of Food Science and Institute of Food Biotechnology, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou, 510640, China.|Joint Research and Development Center for Natural Products of Alchemy Biotechnology Co. Ltd. and South China Agricultural University, Guangzhou, 510640, China.
Jing-Long
JL
Liang
Department of Bioengineering, College of Food Science and Institute of Food Biotechnology, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou, 510640, China.|Joint Research and Development Center for Natural Products of Alchemy Biotechnology Co. Ltd. and South China Agricultural University, Guangzhou, 510640, China.
Li-Qiong
LQ
Guo
Department of Bioengineering, College of Food Science and Institute of Food Biotechnology, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou, 510640, China. linjf@scau.edu.cn.|Joint Research and Development Center for Natural Products of Alchemy Biotechnology Co. Ltd. and South China Agricultural University, Guangzhou, 510640, China. linjf@scau.edu.cn.
Jun-Fang
JF
Lin
Department of Bioengineering, College of Food Science and Institute of Food Biotechnology, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou, 510640, China.
Ze-Qi
ZQ
He
Department of Bioengineering, College of Food Science and Institute of Food Biotechnology, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou, 510640, China.
Fa-Ji
FJ
Cai
The School of Chemistry and Pharmaceutical Engineering, Qi-Lu University of Technology, Jinan, 250353, China.
Jun-Fei
JF
Chen
D051080:Acyl-Carrier Protein S-Malonyltransferase / Q000096:biosynthesis / Q000235:genetics; D000217:Acyltransferases / Q000378:metabolism; D057005:Bioengineering / Q000379:methods*; D002934:Cinnamates / Q000737:chemistry; D003066:Coenzyme A Ligases / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000096:biosynthesis / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis; D005990:Glycerol / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D013267:Stilbenes / Q000737:chemistry / Q000191:economics / Q000378:metabolism*; D000094162:Phytoalexins
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases; D002934:Cinnamates; D003066:Coenzyme A Ligases; D003373:Coumaric Acids; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D005227:Fatty Acids; D005990:Glycerol; D008316:Malonyl Coenzyme A; D010649:Phenylalanine; D013267:Stilbenes; C029010:cinnamic acid; C049032:pinosylvin; D000094162:Phytoalexins; C022674:stilbene synthase; C494968:fabD protein, E coli
Biosynthesis; CRISPRi; Combinatorial bioengineering; Pinosylvin