Construction Of A Zymomonas Mobilis Genetic Manipulation System And Its Applications In Biorefinery Processes

Zymomonas mobilis is a gram-negative facultative bacterium with great potentials for biorefinery processes for its extraordinary ethanol fermentation performance and unique metabolic features. The research on the metabolic engineering of Z. mobilis will benefit this promising microorganism into the biorefinery applications. However, the present genetic manipulation systems for Z. mobilis have many problems, including low transformation efficiency, high false positive rate, relative big plasmid size, and extremely limited cloning sites in the vector etc. These problems severely restricted the extensive implementation of this unique strain in the field of metabolic engineering. In this thesis, the genetic manipulation system of Z. mobilis was improved and some heterologous and homologous genes were expressed in Z. mobilis ZM4 with improved shuttle vector, pHW20a. The physiology and metabolism of Z. mobilis were further studied with the constructed recombinant strains and several key problems hindering the application of Z. mobilis in biorefinary process were also studied.Genetic engineering was an effective method for understanding the physiology of microorganism. The construction of a high efficient and convenient genetic manipulation system sufficient to genetic manipulation is also very important for reconstructing microorganism metabolic pathways. In this thesis, a shuttle vector, pHW20a, for Z. mobilis was constructed from the existed mobilizable pLOI193 plasmid by removing the redundant genes and integrating DNA fragments from various sources. Transconjugation experiments proved pHW20a was a efficient plasmid and its transformation efficiency was about two magnitudes higher than the previous pLOI193 shuttle vector. Using the newly constructed pHW20a, the Escherichia coli BL21 (DE3) mdh gene, the Saccharomyces cerevisiae FDH1 gene, and Z. mobilis native gfo gene and ppc gene, which encode MDH, FDH, GFOR and PEPCase respectively, were expressed by pHW20a in Z. mobilis ZM4. The plasmid transformation efficiency and stability studies with constructed pHW20a derivatives unveiled the key factors influencing transconjugation efficiency and segregational stability.Based on the previous expression results of mdh in Z. mobilis (pHW20a-mdh), the function of ppc gene in Z. mobilis ZM4 metabolism was further analyzed. Phosphoenolpyruvate carboxylase normally acts as an anaplerotic pathway enzyme for oxaloacetic acid production in some microorganisms. In this study, the ppc gene of Z. mobilis wild strain was disrupted by homologous recombination and the PEPCase pathway of Z. mobilis ZM4 was confirmed as the sole pathway for oxaloacetic acid synthesis from glucose, which was indicated by the distinct growth response of the mutant strain to ppc knock-out. The important role of PEPCase in the anabolism of Z. mobilis indicated the unusual metabolism of this ethanologenic bacterium. In the following study, the regulations of ppc gene to the carbon metabolism and energy metabolism in Z. mobilis at transcription level were preliminarily confirmed by ppc gene transcription study, the growth improvements of Z. mobilis with OAA supply and influence to recombinant cell growth from ppc expression at different levels. The foregoing experimental results proved the PEPCase pathway was important in Z. mobilis anabolism and partially participated in its uncoupled growth.Based on the over-expression of gfo in Z. mobilis recombinant strain, the cells for sorbitol biotransformation were prepared and the potential facters hindering the gene over-expression in Z. mobilis ZM4 was also discovered by analyzing the key fermentation parameters. The utilization of gfo over-expression cells and metal ion inhibitors significantly improved the sorbitol productivity and yield. The problems existed in the sorbitol biorefining was relieved by the strategies proposed in this study.Based on the expression of FDH1 the formate metabolic pathway and NADH regeneration system were established simultaneously in Z. mobilis ZM4. The expression of FDH1 in Z. mobilis had the formate metabolism coupled with NADH regeneration in the constructed Z. mobilis recombinant cells and the tolerance or (and) metabolism of the recombinant strain to formate and furan inhibitors were improved consequently. The corn stover hydrolysate fermentation demonstrated the expression of FDH1 in Z. mobilis recombinant cells was effective in improving the cell growth, ethanol productivity, and ethanol yields. At the same operation conditions, both the cell density and ethanol yield of the FDH1 expressing recombinant strain were twice that much of the control strain at the end of Z. mobilis (pHW2Oa-fdh) fermentation. According to the previous reports, the improved supply of NADH can enhance the tolerances and metabolisms of some microorganism to furan inhibitors. The encouraging results confirmed this rule also to be effective in Z. mobilis. The improved tolerance to 4-hydroxybenzaldehyde, a lignin derivative compound, indicated the NADH regeneration system might also be involved in improving the tolerances (or even metabolisms) of Z. mobilis to aromatic inhibitors.Some general genetic and metabolic rules of Z. mobilis were sumerized during improving its genetic manipulation system and analyzing its physiology and metabolism in this thesis. The inspiring results enhanced the understanding of this ethanologenic strain and will definitely pave the way of applying this bacterium in the biorefinary process.