Methanol Bioconversion By Engineered Methylotrophic Corynebacterium Glutamicum

Methanol is an organic Cl compound with high energy density,which can be produced from coal,biomass,carbon dioxide and so on.It is an ideal raw material for biomanufacturing.There are a variety of natural methylotrophs that can use methanol in nature and researchers have realized the bioconversion of methanol to various chemicals by mutation screening and genetic modification.Meanwhile,based on the understanding of natural methylotrophs,synthetic methylotrophs,which integrates methanol assimilation pathway into platform strains for methanol bioconversion,become a research hotspot.However,the low methanol assimilation efficiency,the poor methanol tolerance and unclear limiting factors in synthetic methylotrophs have been widely reported.In this study,protein engineering,omics analysis,tolerance engineering and other technologies were combined to explore the key factors affecting methanol metabolism in synthetic methylotrophs and improve the bioconversion efficiency of methanol.This research is mainly divided into the following parts:In the first chapter,firstly,the production and application of methanol and its advantages in biotransformation were briefly described.Secondly,the methanol assimilation pathways in native methylotrophs and the preliminary attempts of synthetic methylotrophs in platform strains were presented.Then,the current strategies to improve the methanol utilization efficiency and the research progress on bioconversion of methanol to various fuels and chemicals of synthetic methylotrophs were summarized.Finally,existing problems and main content of this paper were discussing.In the second chapter,in order to solve the problems of poor methanol oxidation rate and highly toxic intermediate formaldehyde,the fusion proteins of Mdh,Hps and Phi were designed and constructed to improve methanol bioconversion efficiency by shortening the spatial distance.Firstly,NAD+-Mdh from Bacillus stearothermophilus DSM 2334 and Hps and Phi from B.methanolicus MGA3with high catalytic activity were screened.Then,bifunctional and trifunctional fusion proteins were constructed by using different lengths of flexible linkers.The results showed that fusing Mdh with Hps or Hps-Phi displayed a 4.8-fold increase of the Vmax and enhanced methanol conversion to fructose-6-phosphate by 30%.Finally,the analyses of SEC、DLS and TEM revealed changes in the polymerization state of proteins caused by fusion engineering and produced larger multimers,which may be responsible for the changed catalytic characteristics.This study provides an effective component for accelerating methanol oxidation.In the third chapter,in order to explore the key targets affecting methanol utilization.Transcriptome analysis was performed based on the methanol dependent ancestral strain and evolved mutant strain.The results showed that many genes involved in glycolysis,amino acid biosynthesis and respiratory chain were regulated.Further experimental verification showed that C.glutamicum could use nitrate as an additional electron accepter to balance the excess reducing power generated by methanol oxidation during aerobic methanol metabolism,and the biosynthesis of several amino acids was limited when strain co-utilize xylose and methanol.the sedoheptulose bisphosphatase pathway for generating formaldehyde acceptor ribulose-5-phosphate was found effective in C.glutamicum and beneficial for methanol utilization.In this study,the key factors affecting methanol utilization were found and identified,which provided effective targets for developing superior synthetic methylotrophs.In the fourth chapter,in order to solve the problem of poor methanol tolerance of methanol-dependent C.glutamicum.MX-11 was subjected to adaptive laboratory evolution with elevated methanol content.The mutants with improved methanol tolerance(20 g/L),growth rate and methanol bioconversion efficiency were obtained.The best mutant MX-14 showed that the co-utilization ratio of methanol and xylose reached 7.04:1,demonstrating that methanol was the major carbon source.Transcriptome analysis suggested that glycolysis was downregulated under high methanol concentrations,while amino acid synthesis,oxidative phosphorylation,and part TCA cycle were upregulated.The expression regulation of these genes may be responsible for the accelerated growth of MX-14 under high methanol concentrations.Then,the mutations in O-acetyl-L-homoserine sulfhydrylase cgl0653G1256A and methanol-induced membrane-bound transporter cgl0833C1439T were proven crucial for methanol tolerance.Knock-down or knock-out cgl0653 and cgl0833 were effective to improve cellular tolerance to methanol.On the one hand,the methanol tolerance mechanism discovered in this study deepens the understanding of the methanol tolerance and provides a theoretical basis for engineering cellular tolerance to methanol.On the other hand,the evolved mutant obtained in this study can provide a chassis for engineering superior synthetic methylotrophs.In all,the application of fusion protein in methanol bioconversion was explored in this study.Furthermore,the limiting factors that affect methanol utilization,methanol toxicity and the mechanism of cellular resistance to high methanol concentrations were discussed.Moreover,a methanol-dependent C.glutamicum with improved methanol tolerance and methanol utilization efficiency was obtained.This study established a foundation for further modification of synthetic methylotrophs and improved methanol bioconversion efficiency.