海洋嗜盐古菌功能物质利用的遗传工程技术研究报告

摘 要：极端嗜盐古菌是古菌域的一个重要生理类群，可以产生具有重要价值的产品，如生物可降解塑料PHBV和生物纳米材料紫膜等，但相关基础研究及生物技术亟待加强。该研究以重要海洋嗜盐古菌为材料，在基因组水平开展了其重要功能物质生物可降解塑料PHBV合成关键基因和途径的解析，以及其遗传、代谢及生物工程利用技术的研究，已取得如下研究成果：（1）完成了两株产PHA的极端嗜盐古菌的基因组注释和分析工作，通过基因敲除等手段，鉴定100多个与PHA代谢、碳源利用及调控相关的新基因，包括合成PHBV的关键酶基因phaA、bktB、phaB、phaE和phaC；PHA颗粒结构蛋白编码基因phaP；以及一系列PHBV途径特异性基因等，形成了对嗜盐古菌PHA合成生物学的系统认识。（2）结合功能基因组及分子生物学技术，首次揭示极端嗜盐古菌合成PHBV独特的代谢途径，尤其是发现了4条可利用非相关碳源合成丙酰-CoA，进而为合成高质量PHBV提供3HV前体的独特的代谢途径。还利用功能基因组学等方法，在基因组水平研究了嗜盐古菌PHA可能的代谢调控机制。（3）构建了两株极端嗜盐古菌高效遗传操作系统，尤其是高效的基因敲除体系和基因表达系统。通过敲除富盐菌胞外多糖的编码基因，获得了PHBV产能优化的工程菌株。（4）建立了富盐菌及其工程菌生产生物塑料PHBV的5L发酵缸发酵工艺，发酵水平由前期的2 g/L提高到 25.15 g/L （占细胞干重的50%）；同时，发现该菌可利用更加廉价的几丁质和水解的纤维素作为碳源积累较高水平的PHBV。发酵条件的优化和廉价碳源的使用明显降低了PHBV的生产成本。该研究开辟了PHBV在古菌域中的遗传工程研究与开发的新领域，揭示了极端嗜盐古菌PHBV合成关键基因及其独特的代谢途径，研发了相关遗传操作与发酵技术，获得了性能提高的工程菌。这不仅为生产优良性能的PHBV提供了理论指导，还为降低PHBV生产成本，向实现其工业化生产迈进了重要的一步。

关键词：嗜盐古菌 基因组 关键基因 代谢途径 生物可降解塑料 PHBV 工程菌 发酵

Abstract：Haloarchaea， a distinctive physiological group in the domain Archaea， can produce high-valued products such as biodegradable plastic of poly（3-hydroxybutyrate-co-3-hydroxyvalerate （PHBV）. However， the key genes and pathways for PHBV biosynthesis in haloarchaea are not clear and the related bioengineering technology remains to be established. In this study， we have identified and characterized the key genes and metabolic pathways for PHBV biosynthesis in two halophilic archaea from sea salterns，and studied their related genetics， metabolism and biotechnology. The major research findings are listed as follows.（1）Genome sequencing of these two haloarchaea and identification of the genes involved in polyhydroxyalkanoates （PHA） biosynthesis in haloarchaea.Based on the genome sequence， we have identified more than 100 novel genes in PHA metabolism，carbon utilization，and metabolic regulation via gene knockout， including the key genes involved in PHBV biosynthesis，phaA，bktB，phaB，phaE and phaC；the PHA granule-associated protein encoding gene phaP； and other genes involved in PHBV precursor supplying.（2）By using a combinatorial approach of functional genomics and molecular biology， we have demonstrated the four propionyl-CoA supplying pathways from uelated carbon sources， which supply the 3HV precursor for biosynthesis of the desirable bioplastic PHBV in Haloferax mediterranei. Additionally， we have investigated the possible mechanism of haloarchaeal PHA metabolic regulation on the genomic scale by functional genomics methods.（3）We have established the highly efficient genetic manipulation system for two haloarchaea， especially the highly efficient gene knockout and gene expression systems. By knocking out of the genes for extracellular polysaccharide synthesis in H. mediterranei， an engineered strain ES1 with stronger PHBV-producing capability has been obtained.（4）The fermentation process for PHBV production by H. mediterranei strain ES1 has been established in a 5-liter bioreactor. The PHBV production has remarkably increased from 2 g/L to 25.15 g/L （accounting for 50% of the cell dry weight）. Besides， H. mediterranei could use cheap carbon sources including chitin and cellulose hydrolysate to accumulate PHBV. Optimization of fermentation conditions and utilization of cheap carbon sources can significantly reduce the PHBV production cost. In conclusion， we have demonstrated the key genes and unique metabolic pathways for haloarchaeal PHBV synthesis， established the genetic manipulation system and fermentation technology， and obtained an engineered strain with improved PHBV production capacity. This study would not only provide theoretical guidance for production of desirable bioplastic PHBV， but also reduce its production cost， thus striding an important step toward its industrial production.

Key Words：Haloarchaea；Genome；Key Gene；Metabolic Pathway；Biodegradable Plastic；Poly（3-hydroxybutyrate-co-3-hydroxyvalerate）；Engineered Strain；Fermentation

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