Efficient Production Of Novel Aviation Fuel EIZ And Several Chemicals By Engineered Saccharomyces Cerevisiae

With the gradual depletion of traditional fossil energy sources and increasing attention to environmental pollution,the development of renewable bioenergy and chemicals has become a research hotspot in the current energy and chemical industries.In this paper,the research on the metabolic engineering of Saccharomyces cerevisiae to synthesize a new type of bio-aviation fuel epi-isozizaene and important chemicals(7-dehydrocholesterol and S-adenosyl-L-methionine)is carried out.The sustainable development of new aviation biofuels and related chemicals production has important theoretical and application values.Epi-isozizaene(EIZ)is a potential new type of aviation fuel.Firstly,by introducing a high-copy 2?plasmid containing the epi-isozizaene synthase gene into industrial Saccharomyces cerevisiae,epi-isozizaene biosynthesis was successfully achieved.Further,by harnessing CRISPR/Cas9 genome editing technology,the expression of ERG9 was dynamically adjusted by replacing the original promoter,and the production level of epi-isozizaene was increased by 151%(80.76 mg/L)by using Gelidium amansii hydrolysate as a carbon source.Next,the genes LPP1 and DPP1 in the bypass metabolic pathway of the key intermediate FPP of Saccharomyces cerevisiae were sequentially knocked out,and multiple genes involved in the synthetic pathway of FPP were integrated at the same time,which greatly improved the production of epi-isozizaene and the yield reached 228.82 mg/L.Finally,in a 10 L fermentor,the cell dry weight can reach 134 g/L and the yield of epi-isozizaene reached 2.99 g/L with in situ extraction of n-dodecane.The possibility of biosynthesis of other important chemicals by Saccharomyces cerevisiae was further investigated.By analyzing the chemical synthesis pathways of vitamin D₃ and hydroxylated VD₃ and combining the endogenous biosynthetic pathways of Saccharomyces cerevisiae,metabolic engineering of Saccharomyces cerevisiae was carried out to synthesize 7-dehydrocholesterol(7-DHC)and 25-hydroxy-7-dehydrocholesterol(25(OH)7-DHC),which are then transformed to VD3 or hydroxylated VD₃ with chemical or biological methods,respectively.First,CRISPR/Cas9 genome editing technology was used to knock out the ERG5and ERG6 genes in the ergosterol synthesis pathway of industrial Saccharomyces cerevisiae,and then simultaneously overexpressed the 18 synthetic genes upstream of cholesterol-5,7,24-trienol.Finally,the heterologous 7-dehydrocholesterol reductase gene was introduced to obtain engineering yeast HD-DHC002,and the 7-dehydrocholesterol biosynthesis was achieved.By further optimizing the promoter,using ENO2p as a promoter,the engineered strain containing a single copy of the expression plasmid can synthesize a higher level of 7-DHC,and the highest yield can reach 0.11 mg/L.Finally,by significantly increasing the copy number of the plasmid containing 7-dehydrocholesterol reductase,the yield of 7-DHC in the shake flask was greatly improved,reaching 69.9 mg/L.Finally,in a 10 L fermenter,the inorganic salt medium was used for feed culture.After 64.5 h of cultivation,the dry weight of the engineered Saccharomyces cerevisiae reached a maximum of 134.8 g/L and 7-DHC reached 2.46 g/L.This is the highest report in the literature to date.In order to realize the biosynthesis of 25-hydroxy-7-dehydrocholesterol from Saccharomyces cerevisiae,a screening method of microorganisms with hydroxylation ability was carried out.First,an organic contaminated soil samples were subject to the screening model,and a strain zju4-2 capable of hydroxylating vitamin D₃ at carbon 25was successfully screened out.After morphological and 16S r DNA identification analysis,it was identified as Bacillus cereus.The single-factor and multi-factor tests on the co-solvent system and the conversion conditions obtained the optimal conversion reaction conditions.On this basis,the conversion system was scaled up on a 5 L fermenter.After 60 h of conversion,the concentration of 25(OH)VD₃reached 830 mg/L,and the yield was 41.5%.This research not only obtained a microbial strain with a high probability of 7-dehydrocholesterol hydroxylase gene,but also this new transformation strain and new technology has many advantages over corresponding chemical methods and has important application prospects.In addition,during the screening process,it was also found that a Streptomyces 748 strain deposited in the laboratory had the ability to hydroxylate 7-DHC to synthesize 25(OH)7-DHC.The above work laid a good foundation to engineer Saccharomyces cerevisiae to bioproduce 25(OH)7-DHC using our metabolic engineering strategy.A certain amount of S-adenosyl-L-methionine(SAM)can be synthesized in the process of Saccharomyces cerevisiae's biofuels EIZ and 7-DHC,which provides a theoretical basis for the research of Saccharomyces cerevisiae's metabolic engineering to synthesize multiple biofuels and biochemicals.Based on the high-yield SAM produced by Saccharomyces cerevisiae in our laboratory,further research on how to control the key impurity 5'-adenosyl-methylthiopropylamine in the culture process was carried out.A plasmid containing SAM decarboxylase was introduced into E.coli,a recombinant strain was constructed,and a high-purity standard was prepared by adding L-methionine,which was successfully used for the quality control of industrial production of SAM by Saccharomyces cerevisiae.In summary,this paper focuses on the research direction of Saccharomyces cerevisiae metabolic engineering to transform synthetic biological aviation fuel and produce multiple chemicals in parallel,and realizes the efficient biosynthesis of new terpenoid aviation fuel and multi-vitamin D₃ precursor compounds.Several related exploratory studies on metabolic pathways and quality control have achieved some preliminary results.