| In this dissertation, a pyruvate-overproducing strain Saccharomyces cerevisiae TAM was selected as a model system to produce L-malic acid. The production of L-malic acid could be improved at four aspects by using the strategies of metabolic engineering: constructing fast and efficient cytoplasmic reductive TCA pathway(rTCA), enhancing the L-malic acid efflux system, removing the ubiquitin modification of C4-dicarboxylate transporters, optimizing different expression strengths. The main results were described as follows.1. Construction and optimization of L-malic acid synthesis pathway. A trp, his double null mutant of S. cerevisiae TAM was constructed by Cre/Loxp gene knockout system. In order to construct L-malic acid overproducing pathway, the pyruvate carboxylase(pyc) and malate dehydrogenase(mdh), both responsible for L-malic acid biosynthesis in rTCA pathway, were choosed from Aspergillus flavus and Rhizopus oryzae. Four genes Afpyc, Afmdh, Ropyc and Romdh were combinatorially introduced into auxotrophic strain thTAM. Strain W1001(Afpyc, Afmdh), which owns two genes from A. flavus, produced 1.85 g·L-1 L-malic aicd. However, strain W2001 and W3001, which obtained by introducting genes Ropyc, Romdh and Ropyc, Afmdh, respectively, has showed no accumulation of L-malic acid. But the best strain W4001, which owning the highest specific activity of Afpyc and Romdh, improved the L-malic acid title from 1.85 g·L-1 to 2.5 g·L-1.2. Effect of activating C4-dicarboxylate transporter and ubiquitination modification on the production of L-malic acid. DcuC gene from Eschrichia coli, Afmae from A. flavus and Spmae gene from Schizosaccharomyces cerevisiae were introduced into the strain W4001, which developed the strain W4101, W4201 and W4301, respectively. W4301 strain resulted in 5.15 g·L-1 L-malic acid, but the L-malic acid titers of strains W4101 and W4201 decreased 37.33%, 41.78%, respectively. To further improve the L-malic acid production, three high confidence lysine residues was mutated to relieve transporter Spmae* from ubiquitin modification. When protein Spmae was replaced by Spmae*, L-malic acid detected in W4401 up to 10.5 g·L-1 and it nearly 50% increase compared to the control strain W4301.3. Effect of different expression level of gene Afpyc, Romdh and Spmae*on L-malic acid production. Only two combinations increased the L-malic acid titer compared to the strain W4401. When Afpyc kept in low level, Romdh and Spmae* both in high level, W4409 was got the highest L-malic acid 13.2 g·L-1, with productivity of 0.16 g·L-1·h-1, DCW 2.42 g·L-1, and caused 26.2%, 33.3%, 3.0% increase than W4401, respectively. Thus, the reason of L-malic acid high-producing strain W4409 could be summarized that low Afpyc level caused more pyruvate flux into TCA to promoting biomass, and high Spmae* level could transported L-malic acid from intracellular to extracellular timely and improved the productivity of unit cell.4. Effect of inoculation quantity and thiamine levels on L-malic acid production. When inoculation quantity DCW was 2.31 g·L-1, the DCW, L-malic acid titer, productivity and yield of L-malic acid on glucose were 5.27 g·L-1, 25.14 g·L-1, 0.26 g·L-1·h-1 and 0.32 mo L·mo L-1, respectively. When thiamine level was 1.6 mg L-1, it allowed the highest production of L-malic acid up to 30.25 g L-1 and productivity 0.32 g L-1 h-1. Moreover, the alterant carbon flux led to a significantly decrease of metabolic intermediate pyruvate(43.25 g L-1) than that of control strain W2409-10-1(57.73 g L-1). |
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