Metabolic Engineering Of Escherichia Coli And Bacillus Coagulans For The Bioactive Compounds Production

Bioactive compounds,such as N-acetylglucosamine(GlcNAc),have anti-inflammatory and anti-oxidation effects,and play an important role in human immune system.At present,most of these substances are produced by chemical methods,which could suffer from several potential drawbacks,such as raw material limitation and environmental pollution.Due to the rapid development of synthetic biology and metabolic engineering techniques,engineering of robust hosts for various bioactive compounds production become possible.As common prokaryotic microorganisms,Escherichia coli(E.coli)and Bacillus coagulans(B.coagulans)have been regarded as ideal chassis for bioactive compounds overproduction due to their rapid growth rate and ability for high cell density fermentation.However,inefficient transformation and/or gene editing efficiencies of E.coli and B.coagulans limit the genetic modification and application of these bacteria.In this study,the gene editing and transformation methods of E.coli and B.coagulans were optimized respectively,and a high GlcNAc producer was constructured based on E.coli MG1655(DE3)strain.Results obtained in this study are listed as follows:The gene editing efficiency of E.coli MG1655(DE3)was enhanced by optimizing the concentration of homology arms and the expression of g RNA and Cas9 protein.The single gene knockout and replacement experiments were carried out,and the efficiencies could reach 100%and 94%respectively.Also,the effects of antibiotic concentration and electroporation parameters on the plasmid transformation of B.coagulans were studied.Plasmid pNW33N and pJZ23-1 were successfully transformed into the host.Results showed that when the concentration of chloramphenicol in the screening plate was 7μg/mL,the highest electroporation and conjugation efficiencies reached 2.3×10~5cfu/μg and 2.2×10~3 cfu/m L respectively.Gene knockout results of B.coagulans indicated that mixed strains contain wild-type and mutant were obtained for isopropylmalate synthase gene(leuA)knockout.All other attempts of gene knockout in B.coagulans were unsuccessful.Furthermore,the optimized E.coli gene editing tool was used to engineer the E.coli MG1655(DE3)metabolic pathway and then the carbon source utilization and growth characteristics of obtained mutants were experimentally studied.By knocking out pfk A,pfk B and zwf which are key genes of the glycolysis(EMP)and pentose phosphate pathway(PPP),the fructose-6-P accumulating chassis was obtained.Then the GlcNAc producter was generated by overexpressing the glutamine fructose-6 phosphate transaminase gene glms(E.coli),the fructose-1-phosphatase gene ygaB(E.coli),the glucosamine6-phosphate N-acetyltransferase encoding gene gna-1(Caenorhabditis elegans),and the glycerol kinase encoding gene glpK(Pichia pastoris)in this fructose-6-P accumulating chassis cell.The expression level of upstream and downstream genes of metabolic breakpoint in metabolic pathways were analyzed by RT-qPCR.The experimental results show that the expression of fba B and zwf in the EMP blocking host were up-regulated by about 6 times compared to the wild type strain,indicating that carbon precursors bypassed the metabolic breakpoint and entered the downstream of EMP,and the flux of the PPP was also increased under the growth pressure.The fruK expression was up-regulated by 23 times in the dual-pathway knocked out host,indicating that fructose 1,6-diphosphate might be synthesized from the fructose-1-P pathway.When MK medium was used in the fermentation,the productions of GlcNAc of the EMP-blocked and EMP-PPP-blocked strains reached 31 mg/L and 14 mg/L respectively in shaking flask fermentation.The fermentation medium optimization results showed that adding 10%LB to MK medium(named MK1 medium)could effectively increase the growth rate and biomass of mutant strains.After 12 h fermetation,the biomass of mutant strains reached as the same as that of the wild-type strain.Compared to MK medium,MK1 medium increased the GlcNAc production of EMP-blocked strain by 22-fold,which reached about 0.69 g/L.The acetic acid production of the EMP-blocked strain was reduced by 55%compared to the wild-type strain.The MK1 medium also increased the GlcNAc production of EMP-PPP-blocked strain by 200-fold,which reached 2.8 g/L,while the acetic acid production decreased by 64%.Moreover,87.5%of acetic acid was reassimilated by the cell at the end of fermentation.In summary,based on the optimized CRISPR-Cas9 system,the EMP and PPP pathways of E.coli MG1655(DE3)was systematically engineered and a fructose-6-P accumulating chassis cell was developed.Through the introduction of GlcNAc synthesis pathway and glycerol utilization gene,the engineered strain could co-ferment glycerol and glucose for cell growth and GlcNAc production which also enhanced the yield of GlcNAc production.Results from this research provides essential references for the large-scale production of GlcNAc and its application in feed and medicine.At the same time,the metabolic engineering strategies used in this study has a broader applicability for the production of other bioactive compounds.In addition,this study also explored the metabolic modification methods for B.coagulans.The establishment and optimization of the plasmid transformation methods lay the foundation for the establishment of the metabolic modification methods of B.coagulans.