| In the process of industrial manufacturing system, the microbiological metabolism andmechanical stirring produce a lot of heat, resulting in the temperature keeping rising up in thewhole fermentation system. The system temperature can’t decrease to cells’ optimummetabolic temperature voluntarily, requiring large amounts of cooling water to controltemperature, which leads to high power costs. Therefore, improving the heat resistance andbroadening the optimum temperature range of the fermentation strains will significantlyreduce the fermentation costs. Escherichia coli is one of the most common industrial strainthat wildly used in biochemicals fermentation system, hence, improving its heat resistance isvery important to obtain low-cost biological products. Currently, the traditional hightemperature acclimation and mutation has not only has been previously used to improve thethermotolerance of Escherichia coli. However, the method exists some disadvantages, such aslong period, heavy workload and lower thermotolerance effect, which can’t meet the needs ofgradually rising temperature of industrial fermentation. To solve these problems, this researchdesigned and constructed a serious of heat-resistant E. coli at the molecular level based on thethermal mechanism of Thermoanaerobacter tengcongensis MB4through synthetic biologymethods and techniques, and successfully applied heat-resistant E. coli to biotransformationof xylitol. This research provided a new idea for efficient and low-power bio-fermentationindustry. The main topics and results of this paper were listed as follows:In this research, we successfully cloned18heat resistant parts from Thermoanaerobactertengcongensis MB4, and constructed18corresponding heat resistant devices and2multifunctional heat resistant devices. The cell survival rate of engineered strainspreliminarily characterized the effect of heat resistant devices on E. coli. The results showedthat heat resistant devices of heat shock proteins significantly improved the heat resistance ofE. coli, and heat resistant devices of transcription factors take the second place.10good heatresistant parts were screened out to build heat resistant E. coli.With promoter PgapAas regulatory part, we successfully constructed10constitutivelyexpressed heat-resistant E. coli that could be used for high-temperature fermentation. Acomprehensive evaluation of heat resistant E. coli was analysed through constant hightemperature and gradient increasing temperature fermentation. The heat-resistance of10engineered strains were improved at varying degrees, widening the temperature tolerancerange during the fermentation process.7heat resistant engineered E.coli was screened out forfurther fermentation.The analysis of organic solvent resistance showed that some of the heat-resistantengineered strains had a variety of stress resistance. The butanol resistance of engineeried strains BL21-gapA-flia and BL21-gapA-groes increased by60%and43%. The1,3-propanediol tolerance of BL21-gapA-dnak, BL21-gapA-dnaj and BL21-gapA-rpoe7increased by53%,39%and50%, respectively. The Acetic acid of engineered strainsBL21-gapA-groel increased by16%. The result of improving ethanol tolerance of E.coli wasvery effective.Heat resistant devices and xylitol biotransfomation pathway were successfully integratedin E. coli. The results of fermentation at40℃showed that the xylitol production ofengineered strains BL21-xr-dnak, BL21-xr-dnaj, BL21-xr-thif and BL21-xr-flia were higherthan the control. Meanwhile, The specific production rate of the4engineered strains wereincreased by18.3%,27.7%,19.6%and26.3%, respectively. Simultaneously, hightemperature fermentation at40℃improved the space-time yield of cells, reducing the xylitolfermentation time that was in advance to48h. These results indicated that high temperaturefermentation could not only reduce the cooling water consumption, but also shorten thefermentation cycle, which provided a new idea for the production of bio-based products. |
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