Production Of Acetol And Hydrogen From Glycerol By Recombinant Escherichia Coli

In this study,we focused on acetol and hydrogen production by Escherichia coli using glycerol as carbon source.Firstly,the metabolic pathways in Lin43 strain were modified to improve the level of intracellular coenzyme for the enhancement of acetol production.Secondly,we improved hydrogen production by the HW2 strain,which can catabolize glycerol rapidly under anaerobic condition.Both works were focused on improving the utilization of glycerol and the production of target products.(1)Production of acetol from glycerol by E.coli Lin43 strainE.coli strain Lin43 has mutations in glycerol kinase(GlpK)and the repressor for the glycerol 3-phosphate regulon(GlpR).When exposed to glycerol,it quickly accumulates lethal levels of methylglyoxal,which is a precursor of acetol.The convertion from methylglyoxal to acetol is controled by YqhD,so the yqhD gene was overexpressed to enhance production of acetol.Also,the gloA gene,which controls competitive bypass,was knocked out.After fermentating in M9 medium containing10 g/L glycerol for 48 hours,the Lin43?gloA pCA24N-yqhD strain produced 1.6 g/L acetol.In order to enhance acetol production,we increased the intracellular NADPH level which was used as coenzyme for acetol biosynthesis.Silencing of the gapA gene increased NADPH level and acetol production by 69.3%and 31.8%,respectively.Moreover,overexpression of nadK,pntAB and in combination of nadK and pntAB genes increased intracellular NADPH level by 123.7%,94.0%and 136.8%and increased acetol production by 61.0%,53.3%and 63.7%.Combined gapA gene silencing and nadK,pntAB genes overexpression,NADPH content and acetol production increased by 160.5%and 73.1%,respectively.Compared with the Lin43 strain,the gene gapA in strain Lin43?gloA pCA24N-yqhD pHN1009-gapA pBbB5K-nadK-pntAB was down-regulated by 4.8-fold,while the transcription levels of zwf,nadK,pntA,pntB were up-regulated by 2.0,2.3,1.8,and 2.2-fold,respectively.Finally,the Lin43?gloA pCA24N-yqhD pHN1009-gapA pBbB5K-nadK-pntAB strain produced 7.9 g/L acetol in the M9 medium containing 20 g/L of glycerol and 2 g/L glucose,with the initial OD₆₀₀ of20.(2)Production of hydrogen from glycerol by HW2 strainHW2 strain was obtained by adaptive evolution and can quickly catabolize glycerol under anaerobic conditions and produce hydrogen.In order to enhance hydrogen production,we knocked out the main bypass pathway in hydrogen production controlled by the ldhA and mgsA genes.Knockout of ldhA and mgsA strain increased hydrogen production by 1.5-fold and 1.4-fold,respectively,while ldhA/mgsA double knockout strain increased hydrogen production by 1.6-fold.Then dhaKL gene was overexpressed,which led to hydrogen production increased by 1.5-fold.DHAK enzyme activity increased by 12 times.Ultimately the HW2?ldhA?mgsA pCA24N-dhaKL allowed hydrogen production at(571.5±30.3)?mol/mg protein,which is 2.1 times of the original strain HW2.