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.

Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula.

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Display of heterologous proteins on the surface of E. coli by Red recombineering / 中华微生物学和免疫学杂志

Objective To display of heterologous proteins on the surface of E. coli . Methods The 1653 bp luciferase report gene was knocked in Ipp and ompA genes of chromosome by Red recombine system and selection-counter selection kan/sacB. Results The quantitative analysis results of exogenous lu-ciferase expression displayed that it could be expressed as fusion with the outer membrane proteins on the cell surface. The fusion protein of foreign protein and outer membrane protein Lpp-OmpA-Luc could be high-effi-ciently displayed on cell surface. Conclusion The stable expression of exogenous gene on the surface of E. coli had no effect on the bacterial growth and propagation.