The Study On Construction Of Engineered Strain E.Coli PET28A (+)-paw And Its Using Wastes As Raw Materials For Biodiesel Production

Biodiesel was considered as a renewable, non-toxic and biodegradable diesel fuel, which was consisted of the alkyl monoesters of fatty acids with short chain alcohols, such as fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). Nowadays, more than95%of the world's biodiesel is produced from edible vegetable oils, which resulted in increased cost of production of biodiesel by approximately1.5times higher than that of traditional diesel. Search for new sources of cheaply available fatty acids and sugar substitute to reduce the cost of biodiesel production is urgently necessary. The methods of biodiesel production by microbias showed cleaner and no environment pollution than the methods of biodiesel production by chemical catalysts. The microbiology could satisfy the needs for the compositions of biodiesel for human. Therefore, the fermentation of biodiesel by engineered strain has important practical significance.The main results are as follows:(1) The construction of engineered strain for biodiesel productionThe genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) were amplifed from total genomic DNA of Z. mobilis ZM4. Gene atfA which encodes wax ester synthesis/acyl coenzyme A:diacylglycerol acyltransferase (WS/DGAT) was amplifed from total genomic DNA of Acinetobacter sp. A DNA fragment PA that comprising of pdc and adh was amplified by overlap extension PCR, yielding a fusion gene with the function of ethanol production. Both genes of pdc and adh share a T7promoter through the deletion of TAA of pdc. In order to help the right fold and guanteed the catalyst activities of the fusion protein PA, the linker was inserted between the pdc and adh fragments. Furthermore, a DNA fragment PAW was amplified by combining fusion gene with an additional T7promoter and atfA using overlap extension PCR. The engineered E. coli pET28a (+)-PAW was obtained by the technology of molecular clone. Both the PDC/ADH activity of the fusion protein and the WS/DGAT activity have been detected by SDS-PAGE and the detection of enzyme activity indicating that the heterologous protein could be expressed efficiently by the engineered strain.(2) The study of the biodiesel production by the engineered strain The recombinant strain was cultivated in LB medium with sugar source and fatty acids resulted in significant FAEEs and ethanol formation. The results showed that xylose in the fermentation medium was favorable for the FAEEs synthesis in the E. coli pET28a (+)-PAW, and glucose was favorable for the ethanol production by the engineered strain. The maximum yield of ethanol reached8.12g l-1and1.81g l-1of FAEEs was getting in the medium with glucose. The cellular FAEEs content could reach62%during the cultivation. The maximum yield of FAEEs reached2.79g l-1and3.73g l-1of ethanol was getting in the medium with xylose. The cellular FAEEs content in the xylose medium could reach82.2%during the cultivation. An evident intracellular accumulation of lipid droplets could be clearly observed by transmission electron microscopy (TEM) in the recombinant E. coli pET28a (+)-PAW cells.(3) The study of the biodiesel production from waste restaurant oil by the engineered strainThe production of biodiesel with waste restaurant oil as the substitutes of fatty acids was investigated. The minimal yield of FAEEs0.047g l-1and1.05g l-1of ethanol was obtained in the waste restaurant oil without the addition of sugar sources. The maximum yield of FAEEs0.249g l-1was obtained with2%xylose in the waste restaurant oil medium. The result showed that xylose was favorable for biodiesel synthesis which was in accordance with the results of the fermentation in the LB medium. The ethanol yield was found to be positively and significantly correlated with the sugar content in the fermentation medium by Pearson correlation analysis (R=0.844, p=0.017). Three quadric equations were obtained and glucose was found not only have positive influence on the sugar utilization, but also favorable for the ethanol production. Xylose was favorable for the FAEEs production by the analysis of equation. The analysis of GC/MS showed that most of fatty acids in the waste restaurant oil were transesterified to fatty acids ethyl ester by the fermentation of the engineered strain. The CFPPs of the biodiesel was from-5℃to10℃that means the biodiesel synthesized by the engineered strain could be utilized in the cold environment.(4) The study of the biodiesel production from straw hydrolysate by the engineered strain The content of cellulose in the corn straw (49.20%) was higher than the cellulose of rice straw (46.64%) and wheat straw (44.13%). The content of hemicellulose in the corn straw (20.92%) was higher than the hemicellulose of the wheat straw (20.74%) and rice straw (17.08%). The glucose concentration in the corn straw hydrolysate (10.40g l-1) was higher than in the rice straw hydroly sate (7.93g l-1) and the wheat straw hydrolysate (4.55g l-1). The xylose concentration in the wheat straw hydrolysate (40.56g l-1) was higher than in the corn straw hydrolysate (37.15g l-1) and the rice straw hydrolysate (31.23g l-1). When straw hydrolysates were utilized as sugar substitutes for biodiesel production by E. coli pET28a (+)-PAW, the maximum FAEEs yield of0.30g l-1(wheat) was obtained which is higher than the yield of FAEEs0.25g l-1(corn) and0.19g l-1(rice) respectively. The results showed that the straw hydrolysate, especially wheat straw hydrolysate, could be utilized by E. coli as the substrates for biodiesel production.