Metabolic Engineering Of Clostridium Beijerinckii For Enhanced Butanol Production

Biobutanol,as a substitute for gasoline,has promising physical features such as high energy density,low volatility,and less corrosivity.Clostridium beijerinckii,a promising industrial microorganism for butanol production,suffers from some drawbacks such as low butanol titer,high ratio of by-product acetone,low efficiency of xylose utilization and lack of high-efficiency genetical engineering toolkit.Therefore,this study was mainly focus on improving butanol titer,butanol ratio and xylose utilization.Histidine kinases（HKs）responsible for Spo0A phosphorylation have been demonstrated as functionally important components in regulating butanol biosynthesis in solventogenic clostridia such as C.acetobutylicum,but no study about HKs has been conducted in C.beijerinckii.Six annotated but uncharacterized candidate histidine kinase（HK）genes（including cbei2073,cbei4484,cbei2087,cbei2435,cbei4925 and cbei1553）sharing partial homologies（no less than 30%）with those in C.acetobutylicum were selected based on sequence alignment.The encoding region of these HK genes were deleted with CRISPR-Cas9n-based genome editing technology.The deletion of cbei2073 and cbei4484resulted in significant change in butanol biosynthesis,with butanol production increased by40.8%and 17.3%（13.8 g/L and 11.5 g/L vs 9.8 g/L）,respectively,compared to the wild-type.Faster butanol production rates were observed,with butanol productivity greatly increased by40.0%and 20.0%,respectively,indicating these two HKs were important in regulating cellular metabolism in C.beijerinckii.In addition,the sporulation frequencies of two HKs inactivated strains decreased by 96.9%and 77.4%,respectively.The other four HK-deletion（including cbei2087,cbei2435,cbei4925 and cbei1553）mutant strains showed few phenotypic changes compared with the wild-type.In order to improve butanol ratio,the gene encoding acetoacetate decarboxylase was deleted.The mutant strain produced no acetone with decreased ethanol and butanol.The butanol ratio increased from 69.8%to 95.0%,which was the highest level ever achieved in C.beijerinckii.In addition,acetic and butyric acids increased from 0.5 g/L and 0.6 g/L to 4.0 g/L and 1.9 g/L,resulting in severe inhibition of cell growth with OD₆₀₀ decreased from 3.0 to 2.2.To restore the butanol titer,we attempted to delete histidine kinase gene cbei2073,add methyl viologen dichloride,overexpress acid assimilation related genes cbei0305 and ald/ctfAB,and inhibit the expression of gene adc by dCas9.However,these methods failed to improve butanol titer.The xylose transport in C.beijerinckii was through a xylose proton-symporter.To improve utilization of xylose,we overexpressed gene xylT in plasmid pIMP1_thl under the thiolase promoter.The growth kinetics of xylT overexpressed strain was similar with parental strain while the xylose consumption increased from 19.3 g/L to 23.6 g/L with 22.3%improvement when xylose was used as the carbon source.Meanwhile,the acetone,butanol and ethanol titers were increased by 71.2%,14.3%and 50.0%with butyric acid increased by about 3-fold.Therefore,the overexpression of xylT gene was beneficial for improving the xylose transport capacity and metabolic ability of C.beijerinckii.This study demonstrated the role of HKs on solventogenesis and sporulation in C.beijerinckii,and provided a novel engineering strategy of HKs for improving metabolite production.The strains generated in this study with high butanol titer,high butanol ratio and improved xylose utilization have great potentials in industrial biobutanol production.