Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

Structural Analysis and Docking of Stilbene Synthase Protein from Chinese Grape Vine Vitis pseudoreticulata.

Aims: The present work aims to perform the molecular modeling of stilbene synthase protein from Chinese grape vine Vitis pseudoreticulata. Place and Duration of Study: The study has been performed in the Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam, India for a period of 8 months. Methodology: The sequence of Vitis STS protein was obtained by BLAST search from DFCI web server using Arabidopsis Stilbene synthase sequence. To read the amino acid pattern among these sequences, Multiple Sequence alignment have been performed using clustal W. The secondary and 3D structures were predicted for the protein and the stability of the structures was determined through Ramachandran plot and PROSA analysis. 3D structure obtained using Swiss model workspace was utilized for docking studies. Results: In the multiple sequence alignment except Gossypium and Ipomea remaining sequences were aligning well. The secondary structure of the protein is possessing helices, coils and sheets respectively and most of the protein structure is coiled. The predicted model was subjected to evaluation by PROSA with a Z score of -10.1. Ramachandran plot revealed that the predicted that 96.6% residues were in favoured region, 2.6% were in allowed region and 0.8% were in outlier region proving that the predicted model is acceptable. Docking STS protein with secondary metabolite ligands elucidated that anethole, ascorbic acid and arbutin have good binding affinity. Conclusion: The structural model of Vitis pseudoreticulata stilbene synthase has been determined, and in silico docking studies have elucidated that this protein has docked with some of the essential secondary metabolites like anethole, ascorbic acid and arbutin which might enhance the performance when they enter into a biological system.