Analysis Of Key Physiologic And Regulatory Mechanism For Accumulation Of α-ketoglutarate In Yarrowia Lipolytica

a-Ketoglutarate(a-KG) is an important intermediate in the tricarboxylic acid cycle. As a key node in intracellular canborn and nitrogen pathways, a-KG participates in various cellular metabolism. ATP is generated as supply of energy when a-KG is utilizsed. Therefore, the carboxylate has broad applications in the fields of food and pharmaceuticals as supply of energy. The main concern of biosynthesis routes via microorganisms is low production yield. Yarrowia lipolytica is the most promising species for a-KG production due to its powerful ability to synthesize carboxylates. However, its wide utilization is obstructed by unclear accumulation mechanism and the formation of high concentration of pyruvate. In this thesis, comparative genomics analysis and comparative proteomics analysis were carried out to uncover a-KG accumulation mechanism. According to the mechanism, metabolic strategies were designed to improve a-KG production. The main results were achieved and present as follows:(1) The genomic DNA of Y. lipolytica WSH-Z06 was sequenced using Illumina Mi Seq technology and Pac Bio RS technology. 10,834,592 reads and 1,625 Mbp sequences were obtained from Illumina Mi Seq technology, 865,172 reads and 2,409 Mbp sequences were obtained from Pac Bio RS technology. The mechanism of accumulation of α-KG was uncovered through comparative genomics analysis on genome level, the content of acetyl-Co A were improved by deletion of 5 genes participated in triglyceride synthesis. Moreover,the α-KG production were improved by duplication of isocitrate dehydrogenase;(2) Six endogenous putative transporter genes, YALI0B19470 g, YALI0C15488 g, YALI0C21406 g, YALI0D24607 g, YALI0D20108 g and YALI0E32901 g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470 g, a-ketoglutarate accumulated to 46.7 g·L-1, whereas the concentration of pyruvate decreased to 12.3 g·L-1. Dupilication of these tranporter could be a potent inclue of accumulation of various carboxylates by Y. lipolytica WSH-Z06.(3) A comparative proteomics approach based on two-dimensional gel electrophoresis was used to investigate the cellular responses to environmental p H stimuli in the α-ketoglutarate overproducer Y. lipolytica WSH-Z06. Under condition of low p H circumstance, extrusion of intracellular protons, intracellular ATP and NADH synthesis were all stimulated, as was consumption of intracellular protons. Key enzymes in the tricarboxylate cycle and gluconeogenesis pathway were also up-regulated. Under conditions of thiamine deficiency, the activity of ketoglutarate dehydrogenase was severely impaired, which was a major contributor to accumulation of α-KG. It was concluded that the accumulation of α-KG was due to numerous push and pull effects of from various cell activities in response to acidic p H stimuli and thiamine deficiency.(4) To improve intracellular content of acety-Co A, efferts of overexpression of the a and b subunits of pyruvate dehydrogenase E1, E2 and E3 components was evalued on accumulation of a-KG. Due to overexpression of PDA1, the supply of acety-Co A was enhanced which was accompanied by elevated expressions of CIT1, CIT2, ACO2, IDP1, IDH1 and IDH2. The yield of pyruvate remained at a low level, decreasing from 34.2 g·L-1 to 20.1 g·L-1. Enhancement of the carbon flux from pyruvate to a-KG resulted in 19.9% more a-KG being synthesized, reaching a yield of 43.3 g·L-1.(5) Based on reduction of activity of KGDH, which resulted from separate overexpression of DLST, it was concluded that separate overexpression of DLST was benefited for accumulation of a-KG under condition of low p H stimuli. To reduce catabolism of a-KG via TCA cycle, two conserved active site residues of DLST, His419 and Asp423, were individually mutated. As result, site-mutagensis of these residues impaired the activity of KDGH. Compared to the parental strain, the extracellular a-KG content of the M6 mutant strain, in which the Asp423 residue was replaced by glutamate, was increased by 39.7% to 50.4 g·L-1. Analysis of the kinetic parameters of the enzyme variants indicated that Asp423 invovled both in substrate binding and catalysis.