Mechanistic analysis of high-yield protein production in E. coli for the production of natural products

Research Project

Project/Area Number	17K07726
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Applied microbiology
Research Institution	University of Shizuoka
Principal Investigator	FUna Nobutaka 静岡県立大学, 食品栄養科学部, 准教授 (70361574)
Co-Investigator(Kenkyū-buntansha)	長谷部文人静岡県立大学, 食品栄養科学部, 助教 (30781801)
Project Period (FY)	2017-04-01 – 2020-03-31
Project Status	Completed (Fiscal Year 2019)
Budget Amount *help	¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000) Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000) Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Keywords	大腸菌 / malonyl-CoA / 二次代謝産物 / 蛋白質 / 微生物
Outline of Final Research Achievements	The fatty acid biosynthetic pathway was blocked by gene disruptions of fabF, fabB, and fabH, KS enzymes that resume the intracellular malonyl-CoA to elongate the fatty acyl intermediates. As a result, the intracellular malonyl-CoA level was greatly increased in the fab knockout strains. Overexpression of RppA in fab knockout strains resulted in the enhanced production of polyketides, indicating that these strains would be useful for the production of important natural products where the intracellular malonyl-CoA level is the rate-limiting factor. Interestingly, the transcriptional level of fadR, a transcriptional regulation of the fatty acid biosynthesis and metabolism, was increased compared to the parent strain, suggesting that the inherent negative feedback system accompanied with fatty acid biosynthesis was alleviated. Surprisingly, the intracellular level of acetyl-CoA was also increased, suggesting the existence of an unprecedented feedback regulation system.
Academic Significance and Societal Importance of the Research Achievements	微生物には一つの代謝経路が遮断されても、別の経路が活性化することにより物質を作り出すロバスト性がある。本研究では、大腸菌E. coliにおいて、生命維持に必須な脂肪酸合成経路の遺伝子を破壊することにより、中枢代謝経路の亢進および抑制を誘発した。脂肪酸合成経路の破壊株ではmalonyl-CoAおよびacetyl-CoA（二次代謝産物の前駆体）の過剰蓄積が起こる。我々は、脂肪酸合成経路の破壊株で如何なる代謝経路が亢進または抑制させるのかを解明し、何故malonyl-CoAが蓄積するのかを解明した。本研究課題は、植物二次代謝産物を効率良く大腸菌で生産させるための宿主改良の基盤となる。