Construction And Optimization Of Acetyl-CoA Biosynthetic Pathways In Pichia Pastoris

Acetyl-CoA is one of the most important central carbon metabolites in all organisms,and the insufficient supply of acetyl-CoA is the key factor that restricts the efficient synthesis of acetyl-CoA derivatives.To solve the problems above,this study aimed to construct and optimize the acetyl-CoA biosynthetic pathways in the cytoplasm of Pichia pastoris.The function of acetyl-CoA biosynthetic pathways was evaluated through growth compensation tests and target product synthesis.Firstly,the integration efficiency and integration stability of 6 specific sites(Int54,Int56,Int57,Int58,Int59,Int60)on P.pastoris chromosomes were identified.Using red fluorescent protein coding gene mCherry as a reporter gene,the fluorescent protein expression level of engineered strains was detected and the results showed that the integration efficiency of Int56,Int57,Int59 and Int60 reached 83.33%,91.67%,100%and 96.83%,respectively.These sites had high integration efficiency and good integration stability,and could be used as the insertion sites of genes related to exogenous acetyl-CoA synthesis pathway.Secondly,growth compensation tests for phosphoketolase and phosphotransacetylase pathway(PK),citrate lyase pathway(ACL)and formaldehyde synthesis acetyl-CoA pathway(SACA)were performed in P.pastoris.By knocking down the phosphofructokinase(PFK1)or pyruvate kinase(PYK1)in the glycolysis pathway,the synthesis of cytosolic acetyl-CoA in P.pastoris was blocked,and the PK pathway was introduced into the defective strain,and the cell growth was restored,which proved the effectiveness of the intracellular function of PK pathway.By knocking down acetyl-CoA synthase(ACS 1/ACS2),which catalyzes the synthesis of acetylCoA from acetic acid,the direct synthesis of cytosolic acetyl-CoA was blocked,and the engineered strain with the ACL pathway recovered growth in glucose,but still could not grow in ethanol,which proved the effectiveness of the intracellular function of ACL pathway.By simultaneously knocking out pyruvate decarboxylase(PDC),acetyl-CoA hydrolase(ACH1),and pyruvate dehydrogenase β subunit(PDB1),the direct synthesis of cytoplasmic acetyl-CoA through methanol assimilation was blocked,the engineered strain with the PK pathway recovered certain growth in methanol,whereas SACA functioned weaker or cannot function normally in intracellular.Finally,function verification was further constructed by taking triacetic acid lactone(TAL)as an example.The introduction of the PK pathway in TAL production strain,combined with the xylose utilization pathway to further increase the precursor supply of the PK pathway,led to efficient synthesis of acetyl-CoA using xylose.Compared with the control,the TAL fermentation yield of the engineered strain in SCX reached 825.6 mg/L,which was 1.6-fold higher than that of the strains without PK pathway.Combined PK and methanol metabolism could further improve the synthesis of acetyl-CoA.The engineered strain produced 57.1 mg/L in SCM,which was 2.8-fold higher than that of the control.The study showed that the strategy of combining PK pathway and xylose utilization pathway,combining PK pathway and methanol assimilation pathway,combining ACL pathway and glucose utilization pathway,could improve the overall synthesis level of intracellular acetyl-CoA in different carbon source utilization processes.In this study,the function of acetyl-CoA biosynthetic pathways was verified by growth compensation and synthesis of acetyl-CoA derivative TAL,we proved that the exogenous acetyl-CoA synthesis pathways,namely PK pathway and ACL pathway,are effective to improve the overall synthesis level of cytosolic acetyl-CoA by using different carbon sources.It is expected that the combination of the above pathways can construct P.pastoris as a platform strain for acetyl-CoA synthesis and provide technical basis for the efficient synthesis of acetylCoA derivatives.