Producing Strain Construction And Fermentation Condition Optimization Of Alpha-Ketobutyrate

Alpha-ketobutyrate has been widely used in medicine and food additive industry,because it serves as an active intermediate to synthesize lots of organic compounds,such as L-isoleucine,L-homoalanine and some additives.In this study,to construct a-ketobutyrate recombinant strains,the threonine producing strain Escherichia coli THRD was used as original strain under the guidance of metabolic engineering theory.The threoine dehydratase whose feedback inhibition were removed were overexpressed by plasmid pWSK29 into THRD,then obtained recombinant strains THRW.In the shake flask culture,the ?-ketobutyrate titer of THRW amounted to 13.43 g/L,while THRD have no accumulation of a-ketobutyrate.The presence of considerable L-threonine and pyruvate in the fermentation broth of the strain THRW,indicated the strong accumulation and export of the intermediate L-threonine.Therefore,we knocked out the gene rhtA and rhtC of THRW,which encode two strong threonine exporter,respectively,to weak the secretion.The L-threonine titer of knockout strains were all reduced,but the a-ketobutyrate yield became more only when the gene rhtA was detected individually,and amounted to 14.65 g/L,compared with THRW,the knockout of rhtA resulted in a 9.25%increase in a-ketobutyrate titer.In order to reduce the catabolism of a-ketobutyrate,we further knocked out the genes ilvIH,ilvBN,tdcE and avtA of THRW-1 individually and together.The a-ketobutyrate titer of THRW-4 was the highest and amounted to 16.44 g/L in shake flask cultures,increased by 12.22%.So we performed fermentation in a 7.5 L fermenter using the best strain THRW-4,the highest concentration of ?-ketobutyrate was achieved up to 27.91 g/L when IPTG was added at 10 h of fermentation,whereas 24.15g/L L-threonine still remained in the fermentation broth.THRW-4-S1 and THRW-4-S2 which all owning two biosynthesis pathways of a-ketobutyrate were constructed by using the plasmid pSTV28 to overexpress cimA and leuBCD.Compared with THRW-4,the a-ketobutyrate titers had both increase and the pyruvate concentrations were very low,demonstrated that the introduce of citramalate pathway was conductive to increasing a-ketobutyrate yield.Given the toxic effect of a-ketobutyrate,adaptive evolution was used to screen strains resistant to toxic product.The average biomasses of mutants increased by 60%,while the production of a-ketobutyrate decreased by 70%.So we tried another way that a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRD?rht?ilvIH to simplify operating steps and promote the expression of target gene,et al.The temperature-induced strain have the same genomes to THRW-4,named as THRB-4,it could accumulate a-ketobutyrate just via temperature shift,and allowed the production of 12.57 g/L a-ketobutyrate in flask.Finally,the initial temperature was maintained at 35? to guarantee normal cell growth,and elevated to 40? at 10 h of fermentation to induce the expression of threoine dehydratase.Under optimized conditions,the threonine was metabolized completely,and the a-ketobutyrate titer reached 40.88 g/L after 28 h of fermentation,with a productivity of 1.46 g/L/h and a yield of 0.22 g/g glucose,suggesting large-scale production potential.