海洋嗜鹽古菌功能物質(zhì)利用的遺傳工程技術(shù)研究報告

向華

摘 要：極端嗜鹽古菌是古菌域的一個重要生理類群，可以產(chǎn)生具有重要價值的產(chǎn)品，如生物可降解塑料PHBV和生物納米材料紫膜等，但相關(guān)基礎(chǔ)研究及生物技術(shù)亟待加強。該研究以重要海洋嗜鹽古菌為材料，在基因組水平開展了其重要功能物質(zhì)生物可降解塑料PHBV合成關(guān)鍵基因和途徑的解析，以及其遺傳、代謝及生物工程利用技術(shù)的研究，已取得如下研究成果：（1）完成了兩株產(chǎn)PHA的極端嗜鹽古菌的基因組注釋和分析工作，通過基因敲除等手段，鑒定100多個與PHA代謝、碳源利用及調(diào)控相關(guān)的新基因，包括合成PHBV的關(guān)鍵酶基因phaA、bktB、phaB、phaE和phaC；PHA顆粒結(jié)構(gòu)蛋白編碼基因phaP；以及一系列PHBV途徑特異性基因等，形成了對嗜鹽古菌PHA合成生物學(xué)的系統(tǒng)認(rèn)識。（2）結(jié)合功能基因組及分子生物學(xué)技術(shù)，首次揭示極端嗜鹽古菌合成PHBV獨特的代謝途徑，尤其是發(fā)現(xiàn)了4條可利用非相關(guān)碳源合成丙酰-CoA，進而為合成高質(zhì)量PHBV提供3HV前體的獨特的代謝途徑。還利用功能基因組學(xué)等方法，在基因組水平研究了嗜鹽古菌PHA可能的代謝調(diào)控機制。（3）構(gòu)建了兩株極端嗜鹽古菌高效遺傳操作系統(tǒng)，尤其是高效的基因敲除體系和基因表達系統(tǒng)。通過敲除富鹽菌胞外多糖的編碼基因，獲得了PHBV產(chǎn)能優(yōu)化的工程菌株。（4）建立了富鹽菌及其工程菌生產(chǎn)生物塑料PHBV的5L發(fā)酵缸發(fā)酵工藝，發(fā)酵水平由前期的2 g/L提高到 25.15 g/L （占細胞干重的50%）；同時，發(fā)現(xiàn)該菌可利用更加廉價的幾丁質(zhì)和水解的纖維素作為碳源積累較高水平的PHBV。發(fā)酵條件的優(yōu)化和廉價碳源的使用明顯降低了PHBV的生產(chǎn)成本。該研究開辟了PHBV在古菌域中的遺傳工程研究與開發(fā)的新領(lǐng)域，揭示了極端嗜鹽古菌PHBV合成關(guān)鍵基因及其獨特的代謝途徑，研發(fā)了相關(guān)遺傳操作與發(fā)酵技術(shù)，獲得了性能提高的工程菌。這不僅為生產(chǎn)優(yōu)良性能的PHBV提供了理論指導(dǎo)，還為降低PHBV生產(chǎn)成本，向?qū)崿F(xiàn)其工業(yè)化生產(chǎn)邁進了重要的一步。

關(guān)鍵詞：嗜鹽古菌 基因組 關(guān)鍵基因 代謝途徑 生物可降解塑料 PHBV 工程菌 發(fā)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 unrelated 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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