| Gluconobacteroxydans could transform varies of sugars or alcohols via the membrane bound dehydrogenases, which presents its significant role in green production of ketones, aaldehydes, and organic acids. When glycerol was used as the substrate, corresponding products could be dominantly dihydroxyacetone and rarely glyceric acid. Dihydroxyactone was widely used in food, cosmetic and chemical intermediates fields. Rest-cell catalyzing was introduced in dihydroxyacetone production for the substrate and product inhibition. But it is limited in green synthesis for the reasons of time andcell culture cost. It is necessary to get a fermentation strain for industrial dihydroxyacetone production.This thesis using adaptive evolution strategies with the sole carbon source of glycerol and which strains were grown in different concentration gradients to create selective pressure.An evolved strain G. oxydans-adaptivewas screened in a continuous batch and follow-up studies have shown it evolved stable genetic traits.Glycerol conversion and dihydroxyacetone yield greatly improved and the growth in glycerol medium was determined wellthan the original strain.In order to find the reasons for improved glycerol utilization and DHA formation of G. oxydans-adaptive, different levels of the molecular basis such as morphology, genomics, and proteomics were studied. Combined with differences in gene verification and protein functional effects were summarized as follows:First, ransparent ring-like capsular surrounding cells were observed under an optical microscope with the capsular staining experiments in both G. oxydansDS>M2003and G. oxydans-adaptivecells. Real-time quantitative PCR and showed that capsule-related genes (gox1483, gox1485, gox1486, gox1477, gox1846) transcript levels were increased more than10times in G. oxydans-adaptive. Flow cytometry cell catalysis resting cell viability after24h, the results show the adaptive evolution of bacteria cell survival was1.46times starting bacteria. More dispersed and rounded cells of G. oxydans.adaptive were observed by scanning electron microscopy, Transmission electron microscopy revealed the thickness of cell membrane in G. oxydans-adaptivewas more than2times of G. oxydansDSM2003. Adaptive evolution on cell morphology reflects thickening of the capsule and membrane were cell response mechanism to resist the external environment and tolerate high concentrations of glycerol.Secondly, genome re-sequencing was performed forG. oxydans-adaptive-The single nucleotide sequencingachieved an average depth of370. Genome-wide coverage was98.58%. Genome length was2702173bp and GC content of61.73%.2631open reading frames and genome encodes density was of90.82%. Comparative genomics research tool was used to find11SNP loci,15short segments indel sites,105differentially expressed genes, and gene rearrangement phenomenon was not found. For adaptive evolution may affect the analysis of differentially expressed genes, ABC transporter system has five related genes changed in G. oxydans.adaptive, two pairs of component regulator gene mutation, which showed the ability to use glycerol be promoted.Aldehyde dehydrogenase cytochrome c subunit (gox0585) gene located on the cell membrane produced17mismatchesand10gaps, the gene was considered to be responsible for the conversion of glycerol to generate glycerol acid.Glyceric acid in G. oxydans. adaptivecatalysis system was only little, which may be associated with mutation of gene gox0585.Third, glycerol dehydrogenase is a key enzyme responsible for the conversion of glycerol to dihydroxyacetone.Real-time quantitative PCR showed that two subunits of glycerol dehydrogenaseinG. oxydans.adaptive increased more than ten-fold. iTRAQ showed that glycerol metabolism related enzymes such as membrane-bound dihydroxyacetone transporter protein expression and intracellular protein dihydroxyacetone kinase increased. Adaptive evolution is an important factor in efficient conversion of glycerol and glycerol metabolism. Glycerol3-phosphate dehydrogenase, two-component regulatory upregulated.Peroxidase and thioredoxin reductase protein are cellular antioxidant response to the oxidant pressure. ATP synthesis-related protein, inorganic pyrophosphatase associated with the energy production, and NADPH generating enzyme intracellular transhydrogenase improved inG. oxydans-adaptive, suggesting an efficient mechanism for reducing power production within the cell. Two-dimensional electrophoresis of membrane proteins showed that iron-sulfur protein subunit in G. oxydans.adaptiveincreased, which involved in electron transfer process and reflected the rapid oxidation capacity. Take the overall protein level differences together; the level of intracellular reduction is the most obvious difference between G. oxydans DSM2003and G. oxydans-adaptive in protein levels, latter showing a higher level of energy synthesis.Fourth, according to the resolved evolutionary mechanism, cell redox balance required to maintain homeostasis and DHA high-yielding strains were constructed by promoting intracellular reducing power generatedon the cell membrane to promote the oxidation process. Thiamine pyrophosphate (TPP) was added in medium and resting cells catalysis system respectively, both growth and glycerol utilization were increasedin G. oxydans DSM2003. Recombinant bacteria with TPP single phosphokinase gox2416was determined with improved growth and glycerol catalysis. While using G. oxy dans-adaptiveas the host, recombinant gox2416showed that cell concentration increased by25%, DHA fermentation yield increased by5%,and glycerol complete conversion time shortened by about one hour.The recombinant strain with thiamine monophosphate synthase gox0229was determined22%decreasedcell concentration, whileglycerol completely conversion time shortening2hours and DHA improved yield of15%.Series coexpression gox2416and gox0229in G. oxydans-adaptive, the resultingrecombinant was determined with DHA yield increased by about20%, although the biomass reduced by 38%. The result showed the recombinant strain unit cell production capacity has been significantly improved.Fifth, in the genome re-sequencing and protein expression profile studies, we found the different expression levels of variability or hypothetical protein withinG. oxydans. adaptivecytoplasm. The reducing power level change will affectphysiologicalfunctions of oxidoreductases which depend on NAD (H) or NADP (H). This paper chose one of three enzymes anddetermined catalytic function. Gox2181,a strictly dependent NADH dehydrogenase, showed a weak oxidant activity toward glycerol and dihydroxyacetone reduction activity. InG. oxy dans-adaptive, the expression levelof the protein Gox2181showed a12%increase compared with original strain, indicating that the enzyme with dihydroxyacetone intracellular metabolites associated with the evolution of bacteria while reducing power intracellular NADH high demand related. Gox2036and Gox0644was demonstrated none activity toward glycerol or dihydroxyacetone, and not directly related to the process of adaptive evolution, but its broad substrate spectrum reflects the potential applications. In addition to the physiological function of these enzymes, the catalytic substrate spectrum study also showed the potential application of these three enzymes in organic synthesis or drug synthesis, so we resolved their crystal structure, for subsequent functional development and rational reform. |
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