Identification Of Functional Genes In Butanol-tolerant Strain BW1857 And Construction Of Butanol-resistance Strain

With the aggravation of energy and environmental crisis,biobutanol,as an alternative clean energy resource and chemical,has been attracting much attention.Escherichia coli has been an important platform strain along with the development of synthetic biology.But it’s toxicity to cell is a bottleneck for improvement of butanol titer.Therefore,it is pivotal to identify butanol tolerance genes and develop butanoltolerant strains.Two butanol-resistant mutants named BW1847 and BW1857 were screened using error-prone whole genome shuffling in the previous work.Here,the growth evaluation under high butanol stress were performed,and the results showed that they can tolerate 2%(v/v)butanol.The identifications of SNPs in BW1857 indicated that the strain has 7 mutations and 17 deleted genes.The functional complementary experiments of these genes showed that the common deletion of dgsA and mdtJ inhibited the strain growth and decrease the butanol tolerance.The deletion of tqsA inhibited the strain growth in presence of 0 - 0.75%(v/v)butanol,but increased the butanol tolerance under 1 - 1.5%(v/v)butanol stress.Furthermore,the mutation of 491 T to 491 A in rplB gene also increased the butanol tolerance of BW1857 strain.The identification of the functional genes above provides a theoretical basis for analysis of butanol tolerance mechanism and construction of butanol tolerance strain.To study the impact of the combined expression of functional genes to butanol tolerance,the butanol tolerance genes in this study and reported butanol-tolerant genes were integrated into E.coli genome using site-specific mutagenesis or CRISPR-Cas9 genome editing technology.The growth of HBT1 strain,which deleted acrB and sitespecific mutated rob,is 22% - 26% higher than that of the acrB deletion mutant and the rob mutation strain under 0.75%(v/v)butanol stress.Furthermore,using the HBT4 as the strating strain,the butanol tolerance of HBT6 obtained by deletion of tqsA was improved by 10%.The resulting strain HBT6 has the same tolerance as BW1847 and BW1857 under 0.75%(v/v)butanol stress,but when the strain cultured with 1.25%(v/v)butanol,its growth is 80% and 26% higher than BW25113 and BW1847.The results indicate that the beneficial characters can be enriched using combined strategy.In conclusion,the identifications of butanol tolerance functional genes in BW1857 supply theoretical basis for reveal butanol tolerance mechanism.Furthermore,the effect of integration of functional genes on butanol tolerance were studied,and the study provides a theoretical basis for construction stress-resistance strain by inverse metabolic engineering.It also offers an excellent chassis bacteria for construction of butanol-producing strain.