The Role Of Pyruvate Decarboxylase In The Synthesis Of Citric Acid In The Marine-derived Yeast Yarrowia Lipolytica

Citric acid (CA) is widely used in pharmaceutical and food industries as one of the important organic acid. Nowadays, CA is mainly produced by biological fermentation, using Aspergillus niger for commercial production. However, there are some environmental issues associated with this process, for instance, heavy metal pollution caused by this process. In addition, control over experimental condition of fermentation is complicated. Moreover, genetical modification on A.niger is unfavorable. Recently, it has been found that yeast Yarrowia lipolytica with certain advantages over A. niger can be used as alternative for commercial CA production.Pyruvate carboxylase (PYC) has been reported to play an important role in synthesis of organic acids such as succinic, a-ketoglutaric and malate. PYC can catalyze pyruvate to form oxaloacetate, which is a precussor of succinic, α-ketoglutaric, malate, CA and lipid accumulation. CA is then formed by the condensation of acetyl-CoA and oxaloacetate. Therefore, we consider PYC may play a vital role in synthesis of CA. In this study, the function of pyruvate carboxylase in CA biosynthesis was studied. We found that after PYC gene from organic acid high-yielding strains were overexpressed in the marine-derived yeast Y. lipolytica, CA accumulation was enhanced in the recombinant yeast strain.Vector pINA1312 has been widely used for expression of homologous and heterologous genes in Y. lipolytica. However, the multiple cloning sites of the vector are still limited for genetic modification so that its application is limited. Here we modified the vector pINA1312 into a new vector pINA1312-GY with three extra restriction sites so that the new vector can be easily used for homologous and heterologous gene expression in Y. lipolytica.The whole genomic DNA in the yeast Pichia guilliermondii has been sequenced and the yeast can produce a considerable amount of malic acid. After over-expression of the pyruvate carboxylase gene (PYC) from the marine yeast P. guillermondii in the yeast Y. lipoly/ica SWJ-1b, the transformant PG86 obtained had much higher PYC activity than Y. lipolytica SWJ-1b and the PYC gene expression and citric acid production by the transformant PG86 were also greatly enhanced. During 10-liter fed batch fermentation, the final concentration of CA was 101.0±1.3 g/l, the yield was 0.89 g/g and only 5.93 g/l reducing sugar was left in the fermented medium within 240 h of the fed batch fermentation. HPLC analysis showed that most of the fermentation products were CA. The results indeed demonstrated that overexpression of the pyruvate carboxylase gene in the yeast Y. lipolytica SWJ-1b could enhance the citric acid accumulation.Marine fungus Penicillium rubens is also a organic acid high-yielding strain. In this study, a pyruvate carboxylase gene (RPYC) from the marine fungus P. rubens 1607 was cloned and characterized. ORF of the gene (accession number:KM397349.1) had 3534 bp encoding 1177 amino acids with molecular weight of 127.531 kDa and PI of 6.20. The promoter of the gene was located at-1200 bp and contained a TATA A box, several CAAT boxes and the sequence 5’-SYGGRG-3’. The PYC deduced from the gene had no signal peptide, was a homotetramer (a4) and each protein had the four functional domains.After expression of the RPYC gene from the marine fungus P. rubens 1607 in the Y. lipolytica SWJ-1b, the transformant PR32 obtained had much higher specific pyruvate carboxylase activity (0.53 U/mg) than Y. lipolytica SWJ-1b (0.07 U/mg) and the RPYC gene expression and citric acid production by the transformant PR32 were also greatly enhanced compared to those by Y. lipolytica SWJ-lb. The result showed that the expression of the RPYC gene could further enhanced the CA production. During 10-liter fed batch fermentation, the final concentration of CA was 111.1±1.3 g/l, the yield was 0.93 g/g and only 1.72 g/l reducing sugar was left in the fermented medium within 240 h.