Rewriting The Acetyl-Coa Synthetic Pathway In Yarrowia Lipolytica And Its Application In Enhancing ?-Ionone Biosynthesis

Acetyl-CoA is an important precursor for numerous of chemicals and biofuels production in microorganism,such as the fatty acid,organic acid,the terpenoid.Yarrowia lipolytica is widely used in synthesis of these chemicals and biofuels due to its potential in high flux through of acetyl-CoA.Further improvement of acetyl-CoA supply can increase the production of high value-added fine chemicals.In this study,we constructed the phosphoketolase pathway in Yarrowia lipolytica to improve the acetyl-CoA supply and apply on the biosynthesis of?-ionone.This study combined the phosphoketolase?from the Leuconostoc mesenteroides and Bifidobacterium bifidum?with phosphotransacetylase?from Clostridium kluyveri and Bacillus subtilis?into four phosphoketolase pathways?LmPK-BsPTA,LmPK-CkPTA,BbPK-BsPTA,BbPK-CkPTA?.The phosphoketolase pathway expression cassettes were constructed by Gibson assembly and were integrated into the rDNA loci of Y.lipolytica by CRISPR-Cas9,respectively.The four recombinant strain showed phosphoketolase and phosphotransacetylase activity,through the in vitro enzyme activity assessment.Specifically,BbPK is of higher activity than LmPK and CkPTA is of similar activity to BsPTA.The RT-qPCR results detecting the transcriptional expression are in accordance with enzyme activity assessment.Through shake flask fermentation,the acetate production of four recombinant strains are1.6-2.2 times of the original strain and the strain with BbPK are higher than the strain with LmPK.The highest?-ionone yield is 246.4 mg/L produced by the Bb PK-BsPTA integrated strainYLBI3109,which is 1.9 times of Bb PK-CkPTA integrated strain YLBI3108 and 3.8times of original strain YLBI3001.Selecting rDNA,pox4,D17 and Leu2 loci to integrate PHK?BbPK-BsPTA?pathway to detect the effect of different integration loci on the production of?-ionone,the results showed no obvious difference by t-test and the strain YLBI3111with PHK?BbPK-BsPTA?pathway integrated to Leu2 locus showed highest production 268.4 mg/L.Further constructed xpr2-pox4,rDNA-pox4,rDNA-xpr2 double-copy PHK?BbPK-BsPTA?pathway strain and,rDNA-xpr2-pox4 triple-copy PHK?BbPK-BsPTA?pathway strain to study the impact of double and triple copy number of PHK?BbPK-Bs PTA?pathway on the procution of?-ionone.More copy number of PHK pathway resulted lower?-ionone titer,while the acetate titer increased.Thus,one copy number of PHK can support enough acetyl-CoA for the production of?-ionone in this case.And then,carbon source and nitrogen source were optimized in shake-flask by using YLBI3111.D-glucose is the best carbon source and the production of?-ionone can reach 335.8 mg/L when using a glucose initial concentration of 60 g/L.Tryptone is best nitrogen source rather than urea?3.8 times?and?NH₄?₂SO₄?4.1 times?.In 3 L bioreactor fermentation,the production of?-ionone is 369.5mg/L under the fermertation condition of 20%DO,20 ^oC and pH 6.0.