Production Of Fumaric Acid By Recombinant Bacteria

Fumaric acid is an important chemical raw materials and fine chemical products, has a very wide range of applications in pharmaceuticals, chemicals, resins and other fields, and its biological production methods have received wide attention, such as microbial fermentation. In this paper, we took Pichia pastoris as the starting strain and investigated influence of the expression of fumarase gene from Rhizopus oryzae CGMCC3.2686 and pyruvate carboxylase gene from Pichia pastoris on the cytoplasmic pathway of fumarate. We also knocked out lactate dehydrogenase gene, which is a key enzyme in the metabolism of lactic acid, to make way for more carbon flow of cytoplasmic fumarate.In this study, we overexpressed PC gene in Pichia pastoris GS115, and investigated its influence on the diversion of carbon to the reductive TCA pathway and the yields of the oxaloacetate and L-malic acid. The expression vector pPIC3.5K-PC was constructed, and was introduced in Pichia pastoris GS115. A high-copy recombinant (pas-01) was screened out from 30 positive transformants by phenotypic identification, PCR analysis and G418 concentration gradient selection. After methanol induction, recombinant protein was analysed by SDS-PAGE and PC activity was detected. Results showed that PC was overexpressed in the Pichia pastoris, and the activity was increased 3-folds. Compared to the control (pPIC3.5K transformant), oxaloacetate production(177.4 mg.L-1) of the recombinant (pas-01) was increased by 109.6%, L-malic acid production (127.45 mg.L-1) increased by 33.7% and cell biomass increased by 10% through the fermentation. These results indicated that overexpression of PC is propitious to the accumulation of oxaloacetate and L-malic acid, which also play a significant role in promoting the growth of strain.In order to clone fumarase gene from Rhizopus oryzae CGMCC3.2686, in this experiment we designed twenty pairs of primers and tried to clone fumarase gene by reverse transcription polymerase chain reaction (RT-PCR), but failed. Subsequently, according to the conserved sequence of fumarase gene from Rhizopus oryzae, we designed a pair of primers and amplified 250 bp length conserved sequence, compared with the template is only 76% homology; that this strain is currently used by the cells of Rhizopus oryzae CGMCC 3.2686 The gene sequence of fumarase 1959 template sequence Israeli scholars have reported large differences in the fumR bacteria is a new gene. The LDH gene which encodes lactate dehydrogenase was cloned, and the bleomycin resistance gene zeocin was also cloned. At the same time, the knockout vector of LDH was constructed, named pMD18-T-LDHup-Zeocin-LDHdown, which has 900 bp upstream homologous arm and about 500 bp downstream homology arm in order to knockout the LDH genes from the genome of Pichia pastoris by homologous recombination.