Construction Of Engineered Saccharomyces Cerevisiae Producing Long-chain Omega Hydroxy Fatty Acids And Optimization Of Fermentation Conditions

Hydroxy fatty acids(HFAs)are a class of chemical molecules having a carboxyl group and a hydroxyl group.They exhibit much higher reactivity,stability,and viscosity compared with normal fatty acids.Due to their special properties,HFAs are widely used in various fields,such as food,cosmetic and pharmaceutical industries,and as synthetic monomer in biodegradable polymer chemistry.In particular,the different positions of hydroxyl groups in the fatty acid chain play vital roles in various physiological mechanisms and chemical industries.And ω-hydroxy fatty acids(ω-OHFAs)are fatty acids with a hydroxyl group in the terminal end.These multifunctional compounds have a broad range of applications,including use as excellent green synthetic fibers,antimicrobial agents and pharmaceutical intermediates.Direct chemical synthesis of ω-OHFAs was limited owing to the desired specific hydroxylation and harsh reaction conditions.Alternatively,selective enzymatic hydroxylation is capable of producing specific HFAs through biological systems.Synthetic biology is a hotspot in recent year and it’s available to construct genetic engineered strains to achieve the synthesis of specific ω-OHFAs.In this study,synthetic biology has been used to construct engineered Saccharomyces cerevisiae strain with heterologous synthesis of long-chain HFAs at ω or ω-1 positions(ω-OHFAs).The metabolic pathway and fermentation conditions were optimized to achieve high production of long-chain ω-OHFAs.At first,the Saccharomyces cerevisiae BY4741 was used as a starting strain,and for the accumulation of precursor free fatty acids(FFAs),the strain B03 was constructed to disrupt the genes involved in the β-oxidation pathway in the cell,including the acylCo A oxidase gene POX1,the acyl-Co A activase genes FAA1 and FAA4.Subsequently,the cytochrome P450 monooxygenase Sb CYP52M1 was introduced to convert FFAs to HFAs,leading to the production of HFAs at ω or ω-1 positions.Next,CYP52M1 was reconstituted with the homologous reductase Sb CPR and the heterologous reductase cytochrome P450 reductase At CPR1.The results showed that the CYP52M1-At CPR1 system showed a significant increase in the hydroxylation of FFAs.Moreover,a self-sufficient P450 enzyme system was constructed by fusing CYP52M1 with At CPR1 to reach higher transformation efficiency,resulting in longchain ω/ω-1-OHFAs production up to 148.7 mg/L in shake flask.Fatty acid composition analysis showed that the HFAs consisted of ω-OH-C16:0,ω-1-OH-C16:0,ω-OH-C18:1,ω-1-OH-C18:1.Finally,the expression level was increased by knocking out the phosphatidic acid phosphatase gene PAH1,and the disruption of the fatty aldehyde dehydrogenase gene HFD1 and the fatty alcohol dehydrogenase gene ADH5 was finished to further achieved higher titer of long-chain ω-OHFAs.Briefly,we developed a biosynthetic pathway to directly produce de novo longchain ω-OHFAs in engineered Saccharomyces cerevisiae with glucose as a single carbon source.We then evaluated the yield in fed-batch fermentation in a 5 L bioreactor and further increased the titer to 347 mg/L.The results showed that this strategy was promising for the production of long-chain ω-OHFAs in Saccharomyces cerevisiae by using inexpensive glucose as the single carbon,without supplement of additional fatty acids.This is the highest yield reported so far.