Construction And Transcriptomic Analysis Of E.coli Alanine Producing Strains

L-alanine has a wide range of applications in industries such as medicine and he alth products,daily chemicals,food and food additives.Therefore,high-yield strains of L-alanine are urgently needed in the market.The alanine-producing bacterium MG1655-31(?ldhA?pflB?dadX::alaD)was successfully constructed in the early stage of our laboratory,and its L-alanine production was measured to be higher than that of the wild type,which provided the research basis for this study.In this study,strain MG1655-31 was used as the starting strain,and CRISPR/Cas9 technology was used to knock out and knock in genes at the chromosomal level.L-alanine producing bacteria were preliminarily constructed by blocking other competing metabolic pathways,overexpressing exogenous key enzymes with high activity and alanine transporter.The results showed that:(1)CRISPR/Cas9,as a widely used geneed iting tool,could perform simple,efficient,multi-locus gene knockout and knock-in of the E.coli genome;(2)The L-alanine production of E.coli strain MG655-714(?ldhA?pf lB?dadX::alaD?frdA::alaD?adhE::alaE?ackA-pta)obtained by multiple rounds of gene editing was increased from 5.54mg/mL to 15.01mg/mL by 22 h shake flask anaerobic fermentation,and from 7.37mg/mL to 20.01mg/mL by 48 h anaerobic fermentation in5 L fermentor.(3)The mRNA fragments of E.coli K-12 wild-type strain MG1655 and genetically edited E.coli strain MG1655-714 at different timepoints during fermentation were extracted and transcriptomic analysis was performed.By means of Volcano Plot analysis of differentially expressed genes and functional enrichment analysis of differe ntially expressed genes,the changes of genes related to L-alanine biosynthesis in the two strains at the transcriptional level were compared and analyzed,metA and sad were knocked out,and the copy numbers of alaD and alaE were continuously increased by gene knocking in order to ensure more carbon flow to L-alanine biosynthesis and secretion.