| Ethanol produced from biomass is an important renewable energy that can meet the increasing energy demand and alleviate the energy crisis.The key technological issues for industrial production of ethanol from biomass include efficient hydrolysis of biomass and maximum utilization of released sugars,novel ethanol-producing strains and optimized fermentation processes.To improve the ethanol production efficiency and economic feasibility from biomass,in this thesis,a set of metabolically engineered strains derived from Escherichia coli were developed and their ethanol productivities as well as novel ethanol fermentation processes were investigated.The main results are as follows:1)Ethanol yields were significantly improved from glucose in recombinant E.coli cells by blocking synthetic pathways for main organic acids.Ethanologenic E.coli B0013-1030PA was developed in which the pathways for formation of acetate,lactate and formate were deleted.Ethanol fermentation in shaking flasks with E.coli B0013-1030PA showed that it was able to metabolize 25 g·L-1 of glucose to 11.25 g·L-1 of ethanol with86.0%of the theoretical yield,which is 15.7%higher than that of the control.As predicted,no detectable lactic acid and formic acid were formed and the dramatic reduction of acetic acid formation(0.18 g·L-1 of)was detected.However,significant accumulation of succinic acid(2.37 g·L-1)was observed.To block succinic acid formation,the frdA encoding for fumaric acid reductase was further deleted and strain E.coli B0013-1031PA was constructed.In shaking flask fermentation,the strain B0013-1031PA was able to ferment 25 g·L-1 of glucose to produce 11.90 g·L-1 of ethanol with 93.0%of the theoretical yield.Meanwhile,only 0.12g·L-1 of succinate was formed.2)The genetic basis of slow growth phenotype of E.coli B0013 on xylose was elucidated,and the xylose metabolism was restored by homologous recombination.The specific growth rate of E.coli B0013 on xylose was 0.17 h-1,which was 48.6%of that of E.coli K12(0.35 h-1).Expression of xylFGH from E.coli K12 in E.coli B0013 increased its specific growth rate on xylose up to 0.32 h-1.Cloning and sequencing of xylFGH from E.coli B0013 showed that the second base in 126th codon of xylH was mutated and resulted in a nonsense mutation TAG,which caused premature termination of the xylH reading frame.The reversed mutant of E.coli B0013,named E.coli B0013H,was obtained by cloning and transforming part of the xylH?117 bp-462 bp?from E.coli K12 into E.coli B0013 and selecting on xylose as sole carbon source.Nucleotide sequencing of xylH in E.coli B0013H showed that its 126th codon TAG was reversely mutated to TGG.The specific growth rate of E.coli B0013H on xylose reached 0.32 h-1,which was similar to that of E.coli K12.The results indicated that a nonsense mutation in xylH caused disruption of the major xylose transporter in E.coli B0013,and restoration of its function by reverse mutation resulted in normal growth on xylose.3)The metabolic imbalance between glucose and xylose in E.coli was investigated,glucose-and xylose-selective ethanologenic E.coli strains were then developed and their metabolic characteristics for glucose and xylose were examined by co-fermentation.The metabolism of xylose was significantly delayed compared with that of glucose in E.coli B0013-1031H growing on the mixture of glucose and xylose.Through gene deletion,the xylose-nonutilizing strain E.coli B0013-2010 and the glucose-nonutilizing strain E.coli B0013-2011H were developed.By introducing the ethanol synthesis pathway,strains E.coli B0013-2010PA and E.coli B0013-2011HPA were obtained.By combining E.coli B0013-2010PA and E.coli B0013-2011HPA in a single process in shaking flasks,25 g·L-1 of xylose and 25 g·L-1 of glucose were converted to 24.2 g·L-1 of ethanol,with ethanol formation rates of 1.01 g·L-1·h-1 and consumption rates of 2.02 g glucose/?L·h?and 1.05 g xylose/?L·h?.The ratio of xylose/glucose consumption rates was 0.52:1,which was 1.4 times of that of the control?0.37:1?.The simultaneous utilization of xylose and glucose were significantly improved using co-fermentation.4)A novel E.coli strain with regulated glucose utilization was developed,and technical feasibility for ethanol fermentation from sugar mixture was investigated.A strain,E.coli B0013-2021H,was developed by inserting the ptsG expression cassette under the control of the temperature-inducible?PR and PL promoters into the frdA locus in E.coli B0013-2020H,a glucose nonutilizing strain.E.coli B0013-2021H was able to metabolize xylose,galactose and arabinose and failed to metabolize glucose at 34oC.Its glucose utilization was restored at induction temperatures of 37oC and 42oC.The ethanol-producing strain,E.coli B0013-2021HPA,was constructed and ethanol fermentation on the sugar mixture was conducted in a 7-L fermentor using a“two-phase-two-temperature"strategy?Cell growth under aerobic condition at 34oC and ethanol fermentation under anaerobic condition at elevated 42oC?.Mixed sugars were converted to 40.6 g·L-1 of ethanol with the ethanol formation rate of 2.03 g·L-1·h-1,which was 22.3%higher than that of E.coli B0013-1031HPA[1.66 g·L-1·h-1].The sugars consumption rate was 5.90 g·L-1·h-1,which was24.5%higher than that of the control[4.74 g·L-1·h-1].The results indicated that the strain with regulated glucose utilization was able to metabolize xylose,galactose and arabinose and not glucose during cell propagation and converted all sugars to ethanol under fermentation,and its ethanol productivity on the mixed sugars was significantly improved.5)Xylose-nonutilizing E.coli strain was developed by disrupting xylose transport and catabolism,and a new process for ethanol fermentation with in situ recovery of xylose on mixed sugars was investigated.Xylose-nonutilizing E.coli B0013-2012 was constructed from E.coli B0013-2010 by further deletion of xylE.It is capable of metabolizing glucose,arabinose and galactose but not xylose.In a 7-L bioreactor,the ethanologenic E.coli B0013-2012PA derived from strain E.coli B0013-2012 fermented glucose,galactose and arabinose and produced 53.4 g·L-1 ethanol and the xylose remained in the broth,which represented 98.6%of the total xylose in the sugar syrup added. |
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