聚羟基脂肪酸酯（PHA）是微生物合成的一类结构和性能多样的高分子聚酯。作为环境友好型材料，将在解决由石油化石经济带来的能源和环境危机中作出贡献，在人类生活中有着广阔的应用前景，但是高昂的生产成本限制了PHA的大规模应用。从新疆盐湖中筛选出的盐单胞菌Halomonas sp.TD01是PHA低成本生产的一大突破，以此开发的无灭菌连续发酵生产PHA的新技术已用于工厂中试，能将PHA生产成本降低30%。但盐单胞菌TD01在PHA工业生产链中遇到了很多问题亟需解决。本论文旨在从遗传改造的角度寻求解决方案，在了解其遗传背景的基础上，进行了遗传平台搭建以及相应的代谢工程改造。 本论文完成了盐单胞菌TD01的全基因组测序，其基因组约4.09 Mb，GC含量52.57%，对胞内与PHA合成相关的重要代谢途径和重要酶进行了解析和鉴定。并以此为基础，系统地开发了适用于盐单胞菌TD01的遗传改造工具，包括接合转化方法，稳定高效的表达系统以及敲除系统，搭建起了盐单胞菌TD01的基础遗传改造平台。通过敲除盐单胞菌TD01的2-甲基柠檬酸合成酶，在最优丙酸浓度0.5 g/L下生产3-羟基丁酸和3-羟基戊酸共聚物PHBV，丙酸转化率和聚合物中3HV单体比例从10%和0.82 mol%分别提高至96.25%和12.03 mol%，不仅显著降低底物成本，更能提高PHBV的材料性能。进一步失活3个PHA降解酶并没有带来预期的胞内PHA含量的提高，但对于长期复杂的工业发酵中PHA产量的稳定有益。发酵实验也证明，敲除了2-甲基柠檬酸合成酶和3个PHA降解酶的盐单胞菌TD08是一株能高效且低成本生产PHB和PHBV 的优良工业菌株。在此基础上，构建了以葡萄糖为单一碳源、通过苏氨酸合成途径为生产PHBV提供内源性丙酰辅酶A的工程菌，在不添加丙酸时合成了3HV比例为4.08 mol%的PHBV聚合物，进一步降低了底物成本。同时，在生长稳定期诱导细胞分裂抑制因子MinCD的过表达，平均长度增加的盐单胞菌能够使胞内PHA含量从69 wt%提高至82 wt%，同时纤长化的菌体自沉降加快，能简化菌体下游处理。上述改造对于增强盐单胞菌TD01作为PHA工业生产菌的优势，进一步降低PHA的生产成本有重要理论和现实意义。
Polyhydroxyalkanoates (PHA) are environmental-friendly polyesters with diverse structures that can be synthesized by a wide range of bacteria as carbon and energy sources. PHA applications are limited due to the high cost of production. Halomonas sp. TD01, a halophile isolated from a salt lake in Xinjiang, is a hyper PHA producer able to grow in unsterile and continuous fermentation process. However during the pilot-scale production of PHA, low conversion rate of substrate to PHA, unstable PHA productivity and high cost of downstream processing are challenges. In this study, de novo sequencing of Halomonas sp. TD01 was completed and the 4.09 Mb genomic information with GC 52.57% was decoded, paving the road for developing genetic manipulations of Halomonas sp. TD01. Technologies have been developed including effective conjugation for plasmid transformation, efficient expression system for relatively high level expression of proteins and knockout method for markerless gene replacement or deletion. 2-Methylcitrate synthase of Halomonas sp. TD01 was successfully knocked out, which significantly increased the conversion rate of propionic acid to poly (3-hydroxybutyrate-co-3-hydroxyvalerate), short as PHBV. When fed with 0.5 g/L propionic acid, the knockout mutant accumulated PHBV with higher 3HV fraction from 0.82 mol% to 12.03 mol%, and even higher conversion rate of propionic acid from 10% to 96.25%, leading to remarkably reduction on the cost of substrate and improved properties of PHBV copolymer. Further inactivation of three PHA depolymerases of the strain did not increase PHA content, yet it will be helpful in the long lasting industrial fermentation for eliminating the possible influence of PHA depolymerases on PHA degradation. The recombinant strain with 2-methylcitrate synthase and three PHA depolymerases deleted exhibited excellent PHB and PHBV productivity in a 500 L fermentor study. Metabolic engineering was also conducted to directly produce PHBV from glucose in the resultant strain, and PHBV copolymer with 4.08 mol% 3HV was achieved in the absence of propionic acid when threonine synthesis pathway was overexpressed. In addition, over-expression of cell division inhibitor MinCD in stationary phage of cell growth led to elongated cell shapes, resulting in enhanced PHA accumulation from 69 wt% to 82 wt%, and longer cell shapes promoted faster self-flocculation of cells, simplifying the downstream processing of biomass. These works have contributed to the development of Halomonas sp. TD01 as a low-cost PHA production platform able to operate in an open, continuous and seawater based process.
