其他摘要 | Although de novo biosynthesis of FAEE in the genetically engineered microbes has been considered to be an alternative approach to the traditional FAEE production, the FAEE yield of the engineered microbes is at present not economical enough for industrial application. In this study, the author has performed works on metabolic engineering of E. coli, based on the previously constructed FAEE-producing strain of E. coli KC3, for the purpose of improving the FAEE production in E. coli. The experiments and results are summarized as follows:First, three plant thioesterases, including Cc FatB1 from Cinnamomum camphorum, Ch FatB2 from Cuphea hookeriana and Uc FatB1 from Umbellularia californica, were obtained and introduced into E. coli. And Cc FatB1 was proved to possess better performance than the commonly used tesA’ from E. coli for FAEE production. The optimized FAEE-producing strain KC4 of E. coli, with 21.4 mg/L/OD600 FAEE production under flask condition, was constructed by co-expression of Cc FatB1 and tesA’. Compared with the reported FAEE-producing strain KC3, KC4 possesses the higher FAEE producing ability.Second, the effects of fatty acyl-CoA synthetase on FAEE production were evaluated by introducing another copy of fadD from E. coli or a FAA2 gene from Saccharomyces cerevisiae into the previously constructed FAEE-producing strain KC3. However, increasing the copy numbers of fadD gene has not significant effect on FAEE production, while introducing a FAA2 gene resulted in a 26% decrease in FAEE production compared to the starting strain KC3. Meanwhile, the yield of free fatty acids was increased to be 11.3-fold and 9.1-fold higher than the KC3 respectively. These results indicate that the excessive fatty acyl-CoA synthetase has the negative effect on FAEE production and the transformation of free fatty acid to fatty acyl-CoA catalyzing by fatty acyl-CoA synthetase might not be the limited step for FAEE production.Third, maqu-ws2 gene from Marinobacter aquaeolei VT8, ces1 gene from rat, FaeeS3 gene from human myocardial cells, eth1 and eeb1 genes from Saccharomyces cerevisiae, which can in vivo catalyze fatty acid and ethanol into FAEE, were obtained and expressed in E. coli for evaluating their catalytic efficiency on FAEE production. It was found that only maqu-ws2 gene had a relative good ability to synthesize FAEE in E. coli. But FAEE yield in maqu-ws2 expressing strain is still lower than that expressing atfA encoding WS/DGAT enzyme. And the strain YL15 with double copies atfA gene was constructed for the next experiment using glycerol as the sole carbon resource.Four, fermentations of optimized mutant Escherichia coli strains such as KC3, KC4 and YL15 were performed using minimal medium with glycerol, the low-cost byproduct of biodiesel production, as the sole carbon source. Specially, the production of fatty acid ethyl esters in the mutant strain YL15 was found to be 1.7-fold higher than that of the control strain KC3. The yield of FAEE per OD600 (YFAEE/OD) of strain KC4 approched to 31.16 mg/L/OD600 under 5L fermentation condition. In addition, the conversion rates in the fermentations of the KC3 strain using glucose or glycerol as the sole carbon source showed that the glycerol ultilization of E. coli need a further improvement. For further improving the utilization of glycerol in FAEE-producing strain, gldA and dhaKLM genes were over-expressed. And the glycerol utilization rate in the resultant strain was 3-fold higher than the control strain.CO2 emission is one of the major environmental impacts caused by production of fuels and chemicals based on petroleum resource. An attractive and effective way to reduce our dependence on petroleum resource and thus CO2 emission is to develop biomass resource-based technologies for making biofuels and biochemicals. Microbial fermentation is one of the most important conversion technologies for the production of bio-products from biomass or biomass-derived sugars besides chemical catalysis. However, microbial cells inevitably release CO2 when producing biofuels and chemicals through fermentation, particularly for ethanol producing pathway with equal molar ratio of ethanol to CO2. Here we constructed a modified Escherichia coli E2, co-expressing phosphoribulokinase (prk) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Synechocystis sp. PCC6803, in which a 35.7% reduction of carbon emissions can be achieved. It indicates that autotrophic CO2 fixation pathways can be genetically introduced into heterotrophic microbes to effectively reduce carbon emissions.This study investigated the potential on microbial fixation of carbon dioxide during the fermentation process of Escherichia coli and improved the de novo synthesis of biodiesel by metabolically engineering Escherichia coli. |
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