Metabolic Engineering Of Yarroma Lipolytica For α-ketoglutarate Production

In this sutdy, Yarrowia lipolytica CCTCC M207143, a thiamine-auxotrophic yeast and aproducer of-ketoglutarate, was chosen to study the effects of acetyl-CoA metabolism andpyruvate carboxylation pathway on redistribution of carbon flux and production of-ketoglutarate. To redirect carbon flux from pyruvate to the tricarboxylic acid cycle (TCAcycle) and improve the concentration ratio of-ketoglutarate to pyruvate, the acetyl-CoAavailability and pyruvate carboxylation pathway were enhanced by metabolic engineering.Main results were described as following:(1) The URA3gene in the plasmid p0was replaced by the hph gene encodinghygromycin phosphotransferase with enzyme digest and ligation. The resultant plasmid wasnamed as p0(hph), and had the resistance to hygromycin B. The p0(hph) plasmid wastransformed into Y. lipolytica WSH-Z06to contruct the recombinant strain Y. lipolytica-CON,which was used as the control in following experience.(2) Enhanced-ketoglutarate production in Y. lipolytica WSH-Z06by alteration of theacetyl-CoA metabolism. To enhance the acetyl-CoA availability, the ACS1gene, encodingacetyl-CoA synthetase from Saccharomyces cerevisiae CEN.PK2and the ACL gene, encodingATP-citrate lyase from Mus musculus, were overexpressed. The recombinant strain Y.lipolytica-ACS1and Y. lipolytica-ACL were screened on the YPD+HygB plate, respectively.Compared with the control strain, the specific acetyl-CoA synthetase activity of the Y.lipolytica-ACS1strain was increased10.5fold (0.92U/mg protein), and the specificATP-citrate lyase activity of the Y. lipolytica-ACL strain was increased11.6fold (1.51U/mgprotein). The concentration of acetyl-CoA in the Y. lipolytica-ACS1and Y. lipolytica-ACLwas131.2%and144.2%higher than that in the Y. lipolytica-CON strain, respectively. Inshake flasks, the final yield of-ketoglutarate increased from36.3g/L to42.2g/L and theyield of pyruvate decreased from21.2g/L to14.5g/L in Y. lipolytica-ACS1. Furthermore, thefinal yield of-ketoglutarate increased from36.3g/L to46.7g/L and the yield of pyruvatedecreased from21.2g/L to10.6g/L in Y. lipolytica-ACL. These results demonstrated that theincrease of acetyl-CoA concentration could promote carbon flux from pyruvate into-ketoglutarate production.(3) Enhanced-ketoglutarate production in Y. lipolytica WSH-Z06by regulation of thepyruvate carboxylation pathway. To increase the pyruvate carboxylase activity, the ScPYC1and RoPYC2gene, encoding the pyruvate carboxylase from S. cerevisiae CEN.PK2andRhizopus oryzae were overexpressed, respectively. The resultant strains were named as Y.lipolytica-ScPYC1and Y. lipolytica-RoPYC2. Compared with the control strain, the specificpyruvate carboxylase activity of the Y. lipolytica-ScPYC1and Y. lipolytica-RoPYC2strainwere increased6.5fold (0.59U/mg protein) and10.2fold (0.87U/mg protein), respectively.In shake flasks, the final yield of-ketoglutarate in the Y. lipolytica-ScPYC1and Y.lipolytica-RoPYC2strain were increased by24.5%and35.3%, from36.3g/L to45.2g/L and49.1g/L, and the yield of pyruvate were decreased by51.9%and69.8%, from21.2g/L to10.2g/L and6.4g/L, respectively. In order to demonstrate the effects of enhancing pyruvate carboxylation pathway on the intracellular carbon flux, RT-PCR and enzyme assays were usedto study changes of some key pathways involved in pyruvate metabolism and the TCA cycle.These results revealed that regulation of pyruvate carboxylation pathway successfullyincreased the carbon flux of the TCA cycle and redistributed it from pyruvate to-ketoglutarate formation.(4) Fermentation optimization and control of the recombinant strains.(1) By adding6g/L sodium acetate, the concentration of-ketoglutarate in Y. lipolytica-ACS1was increasedto49.2g/L, which was16.6%higher than the control (42.2g/L);(2) By adding4g/Ltrisodium citrate, the concentration of-ketoglutarate in Y. lipolytica-ACL was increased to53.1g/L, which was13.7%higher than the control (46.7g/L);(3) By adding biotin, theconcentration of-ketoglutarate in Y. lipolytica-ScPYC1was increased to51.2g/L, whichwas13.3%higher than the control (45.2g/L). Similarly, the addition of biotin led to13.2%increase in the concentration of-ketoglutarate (55.6g/L) in Y. lipolytica-RoPYC2;(4) Bycontrolling the pH in a3-L fermenter, the highest concentration of-ketoglutarate in Y.lipolytica-RoPYC2reached62.5g/L with a significant decrease of pyruvate yield from35.1g/L to13.5g/L.