Metabolic Engineering Of Yarrowia Lipolytica For Fatty Acid Derivatives Overproduction

Fatty acid derivatives have wide range of applications.For example,Fatty acid ethyl esters（FAEEs）,a kind of biofuels,can be directly used as transportational fuels due to sinilarities with petrodiesels.In addition,fatty alcohol and wax esters（WEs）can be used as material of surfactents and lubricants,which have a broad range of commercial areas.Tranditional methods for producing fatty acid derivatives usually rely on multistep extraction from plants,animals or organic chemical synthesis,which generally result in low yield,high cost and sometimes severe pollutions to the environment.Engineering microbial cell factories for the biosynthesis of fatty acid derivatives may be a suitable alternative to address these issues.However,most researches have been restricted to conventional organisms（like Escherichia coli and Saccharomyces cerevisiae）.Yarrowia lipolytica,an oleaginous non-conventional yeast,has gained increasing attention in recent years due to great lipid-producing capability.With the ease of genetic enginerring tool,Y.lipolytica is easily engineered for producing chemicals derived from acetyl-CoA,fatty acids and lipids.In this study,Y.lipolytica was used as a platform to efficiently produce fatty acid derivatives,including FAEEs,fatty acohols,very long-chain fatty acids（VLCFAs）and very long-chain WEs,which paved the way for further production of other fatty acid derivaties.For producing FAEEs in Y.lipolytica,wax ester synthase genes were expressed from Marinobacter hydrocarbonoclasticus DSM 8798（MhWS）,Acinetobacter baylyi ADP1（AbWS）,Euglena gracilis（EgWS）and Simmondsia chinensis（ScWS）.The most productive engineered strain expressed gene MhWS was found to produce 0.4 g/L FAEEs.To stepwisely increase FAEEs production,we optimized the promoter of gene MhWS by replacing hp4d with UAS4B-TEF promoter,and eliminated β-oxidation by deleting the PEX10 gene,and redirected metabolic flux toward acetyl-CoA by overexpression of ACS2.Coupled with an optimized ethanol concentration,the engineered strain GQY20 led to a maximum FAEEs titer of 1.18 g/L in shake flask cultures,approximately 5.5-fold increase compared to the wild type strain.The biosynthesis of Jojoba-like very long-chain WEs needs precursor VLCFAs.The most common fatty acids are C16 and C18 in Y.lipolytica.To enable VLCFAs（C22 or greater）fatty acids synthesis in Y.lipolytica,several strategies are used.The first step is the further elongation of fatty acids in Y.lipolytica up to a chain length of C20 and C22 by overexpressing an AtKCS from Arabidopsis thaliana.The second step is overexpression of the C16/18-elongase gene MaELO3 from Mortierella alpina to convert the fatty acids of 16 carbon chain into the fatty acids of 18 carbon chain.In the third step,to further elongate C20 to C22 or greater fatty acids,we have therefore screened and optimized Crayssinica elongase（CraKCS）.Cultivation of engineered strain GQ07,which harboring AtKCS,CraKCS and MaELO3 three expression cassettes have a significant impact on VLCFAs production.Replacement of the hp4d promoters with UAS4B-TEF coupled to the deletion of PEX10 and the overexpression of EcAldh led to a significant increase in VLCFAs content,with a maximum titer of 281.3 mg/L after 3 days in shake flask.Besides,as wax esters precursor,fatty alcohols can be produced in Y.lipolytica by using a functional fatty acyl-CoA reductase（FAR）to direct the conversion of fatty acyl-CoA to fatty alcohol.The two fatty alcohols-producing Y.lipolytica engineered strain was metabolically engineered in this study,which harbored the codon-optimized FARs from Marinobacter aquaeolei VT8 Maqu₂220（MaFAR）and barn owl Tyto alba（TaFAR）.Results suggested that TaFAR-harboring Y.lipolytica strains could produce the highest fatty alcohos titers and MaFAR-harboring Y.lipolytica strains have potential produce C18 and greater carbon chain fatty alcohols.Replacement of the hp4d promoters with UAS4B-TEF or deletion of PEX10 also led to an increased in fatty alcohols production.These two modifications were effective for the production of WEs and other fatty acid derivatives.Finally,a maximum fatty alcohols total titer of 406.1 mg/L with 370.4 mg/L C16 fatty alcohols was achieved in strain Polf-ΔPEX10-TaFAR.For the strain Polf-ΔPEX10-MaFAR,165.1 mg/L fatty alcohols was obtained at 3 day of cultivation,together with the highest C18 fatty alcohols production（107.8 mg/L）ever reported in microorganism.In an effort to ther produce very long-chain WEs in Y.lipolytica,we then expressed a previously characterized wax synthases,either Mh WS or AbWS together with fatty acyl-CoA reductase,either MaFAR or TaFAR in the VLCFAs produced background strain GQ07.In conclusion,the highest titer of very long-chain WEs was 647.82 mg/L with a chain length range from C32 to C40.The strain produced jojoba-like WEs achieved a titer of 159.41 mg/L.In these studies,Y.lipolytica was engineered to be an attractive platform cell factory to produce FAEEs,fatty alcohols,VLCFAs and very long-chain WEs efficiently.The strategies presented here could be employed to form the basis for further development of fatty acid derivatives production in Y.lipolytica.