| Lignocellulose is the most abundant biomass in the nature, 75% of which is composed of cellulose and hemicellulose. Utilization of these substrates efficiently for production of fuels and chemicals through microbial fermentation could decrease the cost of the process and substitute the traditional petrochemical process as an sustainable alternative.Xylose is the main component of hemicellulose. However, B. subtilis could not utilization xylose as the sole carbon source. In this study evolutionary engineering was performed and a mutant E72 which could grow rapidly with xylose as the sole carbon source was obtained. In addition, through whole-genome sequencing and inverse metabolic engineering, three mutations, which were beneficial for xylose utilization, were identified. Mutation in gene araR enabled B. subtilis to use xylose directly. Mutation in gene sinR improved the specific growth rate on xylose to 0.437 h-1. And mutation in gene comP extended the exponential phase of cell growth on xylose. The final reconstructed strain 168 ARSRCP harboring these mutations could use xylose efficiently similar to strain E72. Moreover, in contrast to strain E72, strain 168 ARSRCP had shorter lag phase and higher transformation efficiency.Through deletion of gene acoA and bdhA, strain 168 ARSRCP could produce acetoin from xylose with high yield. Furthermore, by overexpressing constitutively araE from B. subtilis and xylAB from Escherichia coli, strain 168ARSRCPΔacoAΔbdhA(pHP3-PA-PAB) could utilization glucose, xylose and arabinose simultaneously for acetoin production. In bioreactor, this strain could produce 57.4 g/l acetoin in 72 h from 200 g/l glucose-xylose-arabinose mixture with a yield of 0.30 g/g, which is 61% of the theoretical yield.Production of fuels and chemicals from cellulose and hemicellulose through consolidated bioprocessing is an economical process. However, the obstacle is that most organisms could not produce enough cellulase and hemicellulase for utilization of cellulose and hemicellulose, including B. subtilis. Therefore, hemicellulase was overexpressed for improving xylan utilization by B. subtilis, and the engineered strain 168ARSRCPΔacoAΔbdhA(p9CR-P43-xynA) was able to produce acetoin from xylan. In addition, the expression of cellulase in B. subtilis was optimized from the aspects of transcription, translation and protein secretion. Firstly, signal peptide was screened through in silico prediction and SywnC was picked for cellulase EglS. As a result, the extracellular cellulase activity was increased by 22%. Secondly, promoters was screened and PcspD was picked for cellulase transcription, which was 58% stronger than the commonly used promoter P43. Thirdly, a mRNA stabilizer was added in front of the RBS region of gene eglS and the extracellular cellulase activity was successfully increased by 35%. Finally, through expression cellulase on a plasmid with higher copy number, B. subtilis could grow with cellulose as the sole carbon source on solid plate. |
