聚3-羟基丁酸-4-羟基丁酸酯的全新合成代谢通路的构建与优化

Polyhydroxyalkanoates (PHA) are a family of biodegradable bio-based plastic, among which poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) has the greatest potential for application. Production of P3HB4HB by natural microorganisms needs the addition of 4HB-related carbon sources, which are toxic to the cell. Meanwhile, the current metabolic pathway engineered for P3HB4HB synthesis from glucose competes for carbon flux with the TCA cycle and affects the cell growth, thus not suitable for industrial production. This project aims to construct a novel pathway for the synthesis of P3HB4HB from glucose without affecting the cell growth and is possible for high cell density and high productivity of P3HB4HB. A novel pathway for P4HB synthesis will be constructed in Escherichia coli. Then a library with different expression level of the genes in the pathway will be constructed. From the library, a series of recombinant E. coli will be screened which synthesize different content of P4HB in the cells. When the phaCAB operon for P3HB synthesis pathway is introduced into these recombinant strains, P3HB4HB with different 4HB fraction can be produced. When the P4HB synthesis pathway is introduced into the natural P3HB producer Halomonas TD01, the recombinant Halomonas strains can produced P3HB4HB. These recombinant Halomonas strains can be used as the platform for low-cost production of P3HB4HB, which is important for industrial application.

聚羟基脂肪酸酯是一类具有生物可降解性的生物基塑料，其中聚3-羟基丁酸-4-羟基丁酸酯P3HB4HB具有较大应用价值。天然微生物合成P3HB4HB需要添加4HB结构相关碳源，对细胞毒性大；而已有的葡萄糖到P3HB4HB的合成途径与三羧酸循环途径竞争碳源，影响细胞生长，不利于工业生产。本项目旨在构建新的P3HB4HB合成途径，不影响细胞生长，从而实现细胞高密度生长和高产量P3HB4HB。在大肠杆菌中构建新的P4HB合成途径，通过合成生物学手段构建该代谢途径中基因的不同表达水平的库，筛选得到一系列P4HB积累量不同的重组大肠杆菌，将P3HB合成途径导入这些重组菌即可得到一系列合成不同4HB组分比例的P3HB4HB生产菌株。另外，将该代谢途径导入P3HB生产菌盐单胞菌TD01，可得到合成P3HB4HB的重组盐单胞菌，该重组菌可作为低成本生产的平台菌株，对工业生产P3HB4HB具有重要意义。

聚羟基脂肪酸酯是一类具有生物可降解性的生物基塑料，其中聚3-羟基丁酸-4-羟基丁酸酯P3HB4HB具有较大应用价值。盐单胞菌作为低成本生产的平台菌株，将P3HB4HB合成途径导入盐单胞菌，对工业生产P3HB4HB具有重要意义。本研究包括4方面内容。第一，探索性尝试构建全新的P3HB4HB合成途径。第二，在盐单胞菌中构建了相关碳源为底物合成P3HB4HB的代谢途径，证明盐单胞菌作为底盘菌能够实现P3HB4HB的生产,在1000L中试发酵罐中重组盐单胞菌实现了细胞干重82.6 g/L，P3HB4HB含量60.62wt%，4HB比例16.10mol%。第三，构建了盐单胞菌中的基础元件——启动子库，并利用启动子库优化了相关碳源为底物合成P3HB4HB的重组盐单胞菌，在7L发酵罐上优化的重组盐单胞菌细胞干重达到75.61 g/L，P3HB4HB含量76.87wt%，4HB比例13.91mol%，高含量的P3HB4HB将更有利于工业生产和下游提纯。第四，开发了基于CRISPR/Cas9的盐单胞菌的基因编辑工具，并利用该工具确认了P3HB合成途径中的关键基因，该基因编辑工具为快速改造盐单胞菌提供了技术基础。相关碳源为底物合成P3HB4HB的重组盐单胞菌已经具备工业化生产的条件；而以启动子库和基因编辑工具为基础，可以实现在盐单胞菌中构建并优化非相关碳源到P3HB4HB的合成途径，对最终实现工业化，低成本生产P3HB4HB具有重要意义。

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