近年来，石油基塑料造成的环境污染越来越严重，生物可降解塑料可能是解决这些问题的关键。其中，聚羟基脂肪酸酯 (Polyhydroxyalkanoates, PHA) 是一类可在微生物中合成和积累的天然聚酯，这些聚合物具有可与石油基塑料相媲美的物理和化学特性，为我们解决由石油基塑料带来的能源和环境危机中作出贡献。然而由于其成本过于高昂目前还无法替代石油基塑料进行大规模应用。其中，聚 (3-羟基丁酸) (PHB) 是最常见的聚羟基脂肪酸酯类型，本研究尝试利用重组需钠弧菌 (Vibrio natriegens) 合成PHB。需钠弧菌是目前已知生长最快的非致病菌，具有强大的代谢能力，能够利用多种底物作为能源生长，已成为合成生物学领域最具潜力的底盘菌株，然而需钠弧菌并不能天然合成PHB，并且能够应用于需钠弧菌的分子生物学方法也尚不并不完善。本论文表征了可用于需钠弧菌的组成型及诱导型启动子，同时通过在需钠弧菌中建立CRISPR-Cas9、CRISPR-AID、位点特异性重组等高效的基因编辑方法，在需钠弧菌中建立了PHB合成通路，成功地使需钠弧菌合成出PHB并初步探究不同基因组插入位点对PHB合成的影响。此外本论文还通过代谢工程改造、细胞形态学改造、辅因子工程改造以及发酵工艺的优化等手段，在摇瓶中将需钠弧菌12 h培养后PHB含量提升至55%，在7L-发酵罐中将需钠弧菌12 h发酵后的细胞干重提高至19 g/L，PHB最高产率约为0.97 g/L/h。
In recent years, the environmental pollution caused by petroleum-based plastics has become increasingly severe. Biodegradable plastics, polyhydroxyalkanoates (PHAs) may hold the key to solving these problems. PHAs are a class of natural polyesters that can be synthesized and accumulated in microorganisms, and possess physical and thermochemical material properties comparable to those of petroleum-based plastics, offering a sustainable alternative to the energy and environmental crises caused by the fossil economy. However, the high cost of PHAs currently prevents it from replacing petroleum-based plastics on a large scale. Poly-3-hydroxybutyrate (PHB) is the most prevalent type of PHAs. This study aims to produce PHB by Vibrio natriegens. Vibrio natriegens is currently recognized as the fastest-growing non-pathogenic bacterium, possessing a robust metabolic capacity that enables it to utilize various substrates for energy during growth. Consequently, it has become the most promising chassis strain in the field of synthetic biology. However, Vibrio natriegens cannot naturally produce PHB, and its molecular biology techniques are not yet fully developed. To overcome this limitation, the study characterized constitutive and inducible promoters that can be utilized in Vibrio natriegens. Furthermore, by establishing effective gene-editing methods such as CRISPR-Cas9, CRISPR-AID, and site-specific recombination in Vibrio natriegens, the heterologous PHB synthesis pathway has been successfully expressed, enabling the synthesis of PHB. Additionally, the study investigated the impact of different genomic insertion sites on PHB synthesis and utilized metabolic engineering, cell morphology engineering, co-factor engineering, and optimization of the fermentation process to increase the PHB content of Vibrio natriegens to 55% after 12 hours of cultivation in shake flasks. In a 7 L fermentation bioreactor, the cell dry weight of Vibrio natriegens after 12 hours of fermentation was increased to 19 g/L, and the maximum PHB productivity was approximately 0.97 g/L/h.
