The Construction Of E.Coli For Isobutanol Production By Coexpression With Multiple Promoters

Isobutanol has been approved as a next-generation renewable biofuel that can replace gasoline due to its distinguished advantages, such as higher energy density, above-average octane, lower hygroscopicity and lower reid vapor pressure. Biosynthesis of isobutanol becomes research focus with the fast development of synthetic biology and molecular biology. This study aimed at the construction of a recombinant E.coli with good isobutanol productivity.Here, isobutanol pathway was coexpressed in E.coli BL21(DE3). Two-promoter vectors, pCDFDuet, pRSFDuet and pACYCDuet were utilized. Gene kivd and alsS were cloned into pCDFDuet, contributing to vector pCDFDuet-kivd-alsS. Gene yqhD and ilvCD were cloned into pRSFDuet or pACYCDuet, contributing to vector pRSFDuet-yqhD-ilvCD and pACYCDuet-yqhD-ilvCD, respectively. Thus, two kinds of coexpression systems were constructed and the resultant strains were Eco(CDF+RSF) and Eco(CDF+ACYC). The strain Eco(CDF+RSF) produced up to2.7g/L isobutanol, while Eco(CDF+ACYC) produced more,3.5g/L for24h with the yield,0.11g/g glucose. So the strain Eco(CDF+ACYC) was choosed for further engineering.To identify the rate-limiting enzyme, we increased gene copy number of single enzyme in the strain Eco(CDF+ACYC) by introducing a third compatible vector carrying single enzyme gene of isobutanol pathway each time, producing five corresponding strains, Eco(alsS+), Eco(ilvC+), Eco(ilvD+), Eco(kivd+) and Eco(yqhD+). In all of five strains, the enzyme with gene copy number increased expressed better with higher activity, while others’expression were out of any improvement. Compared with Eco(CDF+ACYC), only the strain Eco(kivd+) with higher KDCA activity got better productivity,4.0g/L for24h with yield0.12g/g glucose. These results suggested that KDCA was the rate-limiting enzyme of isobutanol pathway in the strain Eco(CDF+ACYC).To improve the expression of the rate-limiting enzyme KDCA, gene kivd was coloned into pET30a(+) or pRSFDuet, contributing to vector pET30-kivd and pRSFDuet-kivd. Each of them were then cotransformed with vector pACYCDuet-yqhD-ilvCD and pCDFDuet-alsS, constructing strains Eco01and Eco02respectively. The expression of KDCA was improved with enzyme activity increased5.6times in Eco01and half times in Eco02, compared with the strain Eco(CDF+ACYC). Both of the two new strains produced more isobutanol, Eco01up to4.5g/L (its yield was0.13g/g glucose), while Eco02up to5.4g/L, which was54%higher than Eco(CDF+ACYC)(the yield was0.15g/g glucose). This result showed that the improved strain Eco02was the most promising strain used for isobutanol production.