Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)
Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)
复制标题
用于生产 3-羟基丁酸酯和 3-羟基戊酸酯 (PHBV) 共聚物的蓝色盐单胞菌 TCA 循环的染色体工程
DOI：
10.1016/j.ymben.2019.03.006
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发表时间：
2019-07-01
影响因子：
8.4
通讯作者：
Chen, Guo-Qiang
中科院分区：
文献类型：
--
作者：
Chen, Yong;Chen, Xin-Yu;Chen, Guo-Qiang
聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）是一种具有良好力学性能和生物降解性的生物聚酯。PHBV的大规模生产仍然受到生产成本高的阻碍。利用CRISPR/Cas9技术对嗜蓝单胞菌染色体上的TCA循环进行改造，以葡萄糖为唯一碳源生产PHBV。在染色体上含有PHBV合成基因的H. bluephagenesis TD08AB中，分别缺失了编码琥珀酸脱氢酶组装因子2和异柠檬酸裂解酶的两个TCA循环相关基因sdhE和icl，以引导更多的通量，提高PHBV的3-羟戊酸（3HV）比率。由于突变菌株的生长行为较差，我们选择了具有中等强度P-porin-194启动子的H. bluephagenesis TY194进行进一步的研究。构建蓝发芽孢杆菌TY194的sdhE和/或icl突变株，细胞生长、PHBV合成和3HV摩尔比均有所增强。葡萄糖酸盐激活ED通路，从而激活TCA循环，增加3HV含量。发现H. bluephagenesis TY194 （Delta sdhE Delta icl）在PHBV中合成17mol% 3HV。利用插入H. bluephagenesis TY194 （Delta sdhE）染色体的ppc和vgb基因编码的磷酸烯醇丙酮酸羧化酶和玻璃体振荡菌血红蛋白的协同作用，增强TCA循环活性，以葡萄糖为唯一碳源，产生了一系列含有3-18mol% 3HV的PHBV菌株。摇瓶研究表明，H. bluephagenesis TY194 （Delta sdhE, G7::P-porin-ppc）在葡萄糖和葡萄糖酸盐中生长时，细胞干重（CDW）为6.3 g/L， CDW中PHBV为65%，PHBV为25mo1%。通过染色体表达系统报道的3HV最高为25mo1%。对不同3HV摩尔比的PHBV共聚物进行了提取和表征。新一代工业生物技术（NGIB）基于重组蓝芽生杆菌（H. bluephagenesis）在非无菌和连续条件下生长，可以以方便的方式生产P(3HB-0类似于25mol% 3HV)，降低了生产复杂性和成本。
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength P-porin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (Delta sdhE Delta icl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (Delta sdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (Delta sdhE, G7::P-porin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mo1% 3HV in PHBV when grown in glucose and gluconate. 25mo1% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0 similar to 25mol% 3HV) in a convenient way with reduced production complexity and cost.