The Study On Regulating T10,c12-Conjugated Linoleic Acid Accumulation In Yarrowia Lipolitca Based On Fatty Acid Oxidation Pathway

Conjugated linoleic acid(t10,c12-CLA)is a kind of octadecadienoic acid with a variety of physiological functions,which can produce by the linoleic acid isomerase(PAI)derived from Propionibacterium acnes catalyzing linoleic acid(LA).The recombinant strain with expression of pai was constructed in our laboratory,which explored a new way for the biosynthesis of t10,c12-CLA.However,the substrate and the product are rapidly consumed due to the substrate LA and the product t10,c12-CLA as a carbon source involved in the fatty acid ? oxidation pathway in the peroxisomes.Therefore,in order to stabilize the accumulation of t10,c12-CLA monomer,this study mainly regulated the metabolic pathway of fatty acid oxidation using genetic engineering.The results are as follows:(1)Four recombinant strains with ?-oxidation-related gene knockout were successfully constructed by Cre-lox system,and successfully integrated and expression pai gene,named Polf-?pox2/1312 opai,Polf-?pox3/1312 opai,Polf-?pox2?pox3/1312 opai,Polf-?pex10/1312 opai,respectively..The titer of t10,c12-CLA was about 3 mg/L,indicating that there was no significant difference in t10,c12-CLA production between mono-copy strains.(2)Gene-knockout affected the growth of the strain.Compared with the control strain,the growth phase of four knockout strains were delayed by 5 h in YPD medium,and the biomass of PEX10 knockdown strain was disturbed.From the side of lipid carbon source utilization,Polf-?pox2?pox3/1312 opai,Polf-?pex10/1312 opai strains can use volatile fatty acids to grow and stabilize lipid accumulation.By exogenous adding 20 g/L LA at stable early period,the biomass of Polf-?pox2?pox3/1312 opai and Polf-?pex10/1312 opai strains were increased by only 2 g/L,indicating that capacity of untilization LA was limited.Among four knockout strains,the lowest degradation rate of t10,c12-CLA of was Polf-?pex10/1312 opai,which was 20 mg/L/h,significantly lower than that of untreated strain(97.2 mg/L/h),which means improved the stability of t10,c12-CLA accumulation.Therefore,PEX10 knockout strain has the best effect for alleviating the degradation of substrate LA and stabilizing CLA production.(3)PAI multi-copy integrated strain was constructed based on Polf-?pex10 strains.PAI copy number,western and GC analysis showed that a high titer t10,c12-CLA strain Polf-?pex10/1292 opai-6 was obtained with 22.3 mg/L.By addition SaO at stable early period in Polf-?pex10/1292 opai-6,The total titer of t10,c12-CLA was 7.4 g/L,which was 3 times higher than the mono-copy strain.The CLA degradation rate was 23.0 mg/L/h,comparing to 126.3 mg/L/h of the unmodified multi-copy strain,the knockout strain significantly stabilized the CLA monomer production.(4)At the level of fermentor,two-stage batch feeding strategy was adopted.VFAs were added after glucose was used out.The biomass(18 g/L),the t10,c12-CLA titer(9.8 g/L)or the conversion rate(49%)were all better than the shake flask level.It also proved that this strain can use VFAs to grow and accumulate lipid in order to save the cost.