| In this sutdy, Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast and a producer of pyruvate andα-ketoglutarate, was chosen as a model system to study the effect ofα-keto acid dehydrogenase (pyruvate dehydrogenase andα-ketoglutarate dehydrogenase) on redistribution of carbon flux and production ofα-ketoglutarate. To channel carbon flux from pyruvate to TCA cycle and improve the carbon mole ratio ofα-ketoglutarate to pyruvate, the activity of pyruvate dehydrogenase was improved while the activity ofα-ketoglutarate dehydrogenase was decreased by means of biochemical and gene engineering. The main results were described as follows:1. Hydrogen peroxide and methotrexate were chosen as the effectiveα-ketoglutarate dehydrogenase inhibitors of Torulopsis glabrata after investigation, in the production ofα-ketoglutarate: (1) By adding 6 mM hydrogen peroxide, the concentration ofα-ketoglutarate was increased to 21.8 g/L, which was 30.1% higher than the control (16.8 g/L); (2) Similarly, the addition of 0.08μM methotrexate led to 22.1% increase in the concentration ofα-ketoglutarate (20.5 g/L); (3) The activity ofα-ketoglutarate dehydrogenase decresed by 47% when these two inhibitors were presented in the broth, and as a result, the concentration ofα-ketoglutarate accumulated to 23.5 g/L, 40.1% higher than control; (4) The highest finalα- ketoglutarate concentration (28.4 g/L) was achieved when 0.04 mg/L thiamine was fed in the broth while the activity ofα-ketoglutarate dehydrogenase was blocked, which was 69.6% higher than the control.2. The gene kgd1, encoding the E1 subunit ofα-ketoglutarate dehydrogenase complex, was disrupted by a resistant mark of kan, and a recombination strain named T. glabrata kgd1::kan was screened. Compared with that of the parent strain, theα-ketoglutarate dehydrogenase activity was undetected while the activity of isocitrate lyase was improved by 70.7%, which demonstrated that the glyoxylate pathway was chosen to complete the metabolism of carbon source instead of TCA cycle. The inactivation ofα-ketoglutarate dehydrogenase also resulted in the descent of intracellular NADH/NAD+ (33.7%) and ATP/ADP (31.8%), to retrieve, the activities of enzymes related to NADH metabolism, such as pyruvate dehydrogenase, isocitrate dehydrogenase and malate dehydrogenase were increased by 58.1%, 33.3% and 32.5%, respectively. The concentration of intracellular pyruvate was decreased by 50.1%, while the concentrations of succinate, malate andα-ketoglutarate were increased by 172.7%, 66.1% and 41.1% than those of parent strain. Pyruvate family amino acids was lowered by 29.3%, while glutamate family amino acids and aspartate family amino acids were increased by 34.7% and 26.8%. From the above results, it can be concluded thatα-ketoglutarate dehydrogenase plays a noticeable role in the metabolism of engineering, carbon source and amino acids. 3. Study on the fermentation characteristrics of T. glabrata kgd1::kan showed that the increased thiamine concentration in culture broth could lead to a relatively high level ofα-ketoglutarate (22.0 g/L) and CKG/CPYR (1.14), which were 22.6% and 30.7% lower than those of parent strain under optimal inhibitors and the same thiamine concentration. Because of the inactivation ofα-ketoglutarate dehydrogenase, the carbon flux transfer to glyoxylate pathway andα-ketoglutarate node was missed, so, partial inhibition ofα-ketoglutarate dehydrogenase would be much more useful than gene deletion for manipulating carbon flux toα-ketoglutarate.4. To channel carbon flux from pyruvate to TCA cycle, the pda1 gene, encoding pyruvate dehydrogenase E1αsubunit, was overexpressed, a recombination strain T. glabrata-pda1 was screened. Compared with that of parent strain, the pyruvate dehydrogenase activity of the mutant strain was increased 3.8 fold (0.35 U/mg protein). In the culture of 3-L fermentor, the yield ofα-ketoglutarate reached the highest level of 31.7 g/L, which was 38.9% higher than that of parent strain (22.8 g/L); the value of CKG/CPYR reached 3.05 as the concentration of pyruvate decreased to a lowest level of 10.4 g/L, which made industrialization ofα-ketoglutarate production by T. glabrata much more possible.
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