| Glucaric acid(also called Saccharic acid, GA), an organic acid with important biological functions, is widely used in the fields of pharmaceutical, chemical and food industries. Currently, the major method for preparing glucaric acid is chemical oxidation of glucose. However, this chemo-catalytic process is difficult to expand due to the high cost, low yield, and environmental harms. Microbial fermentation for producing glucaric acid is an alternative method and has attracted much attention. In this study, a glucaric acid synthetic pathway was firstly constructed in Pichia pastoris. The glucaric acid production was futher improved by a series of strategy, such as fusion expression of enzymes in the pathway, application of different peptides, optimization of cultivation conditions in the shake flasks, and fed-batch fermentaion in a 3-L fermenter. The main results were described as follows:(1) Functional validation of a potential MIOX encoding gene in P. pastoris. By multiple-sequence alignment of the potential myo-inositol oxygenase(ppMIOX) and other well-studied MIOXs, we found that the highly conserved iron or substrate binding residues were also conserved in ppMIOX. Then, the ppMIOX was overexpressed in P. pastoris, the corresponding recombinant strain P. pastoris GS115-pM was constructed. P. pastoris GS115-pM was cultivated in medium supplemented with myo-inositol, and glucuronic acid was detected in the culture supernatant. The above results demonstrated that hypothetical protein ppMIOX is a myo-inositol oxygenase.(2) Construction of a glucaric acid biosynthetic pathway in P. pastoris. By co-expression of MIOX( ppMIOX or mMIOX) and udh, a glucaric acid biosynthetic pathway was constructed. After 60 h of fermentation, P. pastoris GS115-U-mM accumulated obvious glucaric acid when cultivated in either YPD(107.19 ± 11.91 mg·L-1) or YPDM(785.4 ± 1.41 mg·L-1) medium. P. pastoris GS115-U-pM accumulated glucaric acid just when cultivated in YPDM only(90.46 ± 0.04 mg·L-1). No detectable glucaric acid was observed in P. pastoris GS115-U and the control P. pastoris GS115. The activities of MIOX and Udh were also analyzed. The activity of mouse MIOX(mMIOX) was higher than that of ppMIOX, and was influenced by addition of myo-inositol. But mMIOX was an unstable protein. Udh was stable, and expressed well.(3) Enhanced production of glucaric acid by fusion expression of Udh and mMIOX. In this study, Udh was fused in the N-terminal of mMIOX by adding different kinds of linkers. P. pastoris GS115-U-(G4S)1-mM, P. pastoris GS115-U-(EA3K)3-mM and P. pastoris GS115-(U-mM) accumulated higher concentrations of glucaric acid, 944.67 ± 5.31 mg·L-1, 930.63 ± 3.35 mg·L-1 and 921.30 ± 45.86 mg·L-1, respectively. The results indicate that the linkers(GGGGS)1 and(EAAAK)3 in the enzyme fusion were advantageous for synthesis of glucaric acid. As we expected, the linkers(GGGGS)1 and(EAAAK)3 generated higher specific activities of MIOX(5.46 ± 0.32 U·mg-1 and 5. 16 ± 0.17 U·mg-1) than free MIOX(3.21 ± 0.17 U·mg-1) in the control strain.(4) Optimization of shake flask cultivation of P. pastoris GS115-U-(G4S)1-mM and fed-batch fermentation in a 3-L fermenter. By fermentation optimization of P. pastoris GS115-U-(G4S)1-mM in shake flasks, the results showed the optimum carbon source was glucose(60 g·L-1 was the best initial concentration), the optimum initial pH was 5.5, the optimum seed time was 24 h, and the optimum inoculation amount was 25%. After optimization, the glucaric acid production was improved to 1697.60 ± 74.61 mg·L-1, which was increased by 70.85% than that without optimnization. Given the shaken flask cultivation results, P. pastoris GS115-U-(G4S)1-mM was cultivated in a 3-L fermenter. After 96 h of fermentation, the production of glucaric acid by P. pastoris GS115-U-(G4S)1-mM reached 6.61 ± 0.30 g·L-1 by addition of glucose and myo-inositol. |
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