Construction Of An Ester Producing Platform In Rhodosporidium Toruloides

Non-model Rhodosporidium toruloides is one of the strains with the strongest lipid production capacity reported at present,and it is expected to become an economical and efficient natural fatty acid cell factory due to its high fermentation density and wide utilization of substrates.In this thesis,non-model R.toruloides is used as the chassis cell,and its strong fatty acid synthesis ability is used to efficiently synthesize wax esters（WEs）and fatty acid ethyl esters（FAEEs）,two widely used fatty acid derivatives.In the engineered strain,the ester synthesis pathways were constructed and optimized via genetic modifications of the chassis cell,which served as a more efficient,more economical and more environmentally friendly platform for the industrial production of oil chemicals.The main research results of this paper are as follows:Using R.toruloidesΔku70 as the starting strain,the fatty alcohol synthesis module was constructed by expressing the fatty acyl-Co A reductase gene Maqu derived from Marinobacter aquaeolei VT8,and then the wax ester synthase gene Ab WS*derived from Acinetobacter baylyi ADP1 was introduced to successfully constructed a de novo synthesis pathway for WEs.The chain lengths of the synthesized WEs were mainly C32,C34 and C36,and the WE titer in shake flask fermentation was 532mg/L.Subsequently,the fatty acid elongase Cg KCS derived from Cardamine graeca was heterologously expressed to successfully synthesize very long-chain wax esters（C38,C40 and C42）with a titer of88.14 mg/L,accounting for 15.8%of the total WEs produced.Then,overexpressing the Acyl-Co A synthetase（ACS）and ATP-citrate lyase（ACL）,which are related to endogenous fatty acid accumulation,significantly increased the WEs production,up to 1.149 g/L,which was more than two times higher.Subsequently,ACL1 and ACS1 were overexpressed and the titer was further increased to 1.251 g/L.Finally,by optimization of the fermentation conditions,a maximum of 1.64 g/L WEs was achieved by batch fermentation in a 1 L fermenter.By combined overexpression of five heterologous alcohol dehydrogenase（ADH）and endogenous pyruvate decarboxylase（Rt PDC）in R.toruloidesΔku70-Ab WS*,it was found that the recombinant strains overexpressing Bs ADHII and Rt PDC could de novo produce FAEEs,and the total amount reached 29.0 mg/L.In order to further improve the production of FAEEs,two kinds of heterologous pyruvate decarboxylase and Bs ADHII were co-overexpressed in this paper.Subsequently,the mutant alcohol dehydrogenase gene(adh E^G544D)with stronger alcohol-producing ability was overexpressed by combining with the expression of Rt PDC.In order to improve the stability of the engineered strain and improve the convenience of manipulation,an attempt was made to use the2A sequence for multi-gene expression,but the titer of FAEEs was not significantly improved in all the above attempts.