嗜盐单胞菌利用乙酸盐合成PHB的研究

Studies on PHB production in Halomonas using acetate as substrate

首先测试了嗜盐单胞菌Halomonas sp.TD1.0对乙酸钠的耐受性,结果显示乙酸钠浓度由25 g·L^(-1) 提高到100 g·L^(-1) 时对TD1.0生长的抑制率只有45.8%。在摇瓶培养中,TD1.0在36 h内消耗完27.3 g·L^(-1) 乙酸钠,细胞干质量达到9.6 g·L^(-1) ,其中PHB质量分数为61%,表明该菌株具有很好的高浓度乙酸钠耐受性和利用乙酸合成PHB的特性。随后为进一步提高乙酸钠的利用速度,在TD1.0中分别用高拷贝和低拷贝表达载体表达了来自枯草芽孢杆菌的乙酰辅酶A合成酶基因acs,结果显示,含有高拷贝表达质粒的菌株TD-PN59的乙酸盐平均利用速率为0.91 g·L^(-1) ·h^(-1),比TD1.0提高了19.7%。TD-PN59的细胞干质量和PHB质量分数分别达到9.98 g·L^(-1) 和65%,PHB产量达到6.49 g·L^(-1) ,比TD1.0提高了约10.8%。在以葡萄糖和乙酸钠为混合碳源(10 g·L^(-1) Glu和10 g·L^(-1) NaAC)的培养基中,利用低拷贝载体表达acs的TD-PN85菌株的乙酸钠利用速率显著高于TD1.0,并且在一定程度上缓解了碳分解代谢物阻遏现象(CCR),促进了葡萄糖和乙酸钠的共利用。

In this study,we firstly tested the tolerance of wild-type Halomonas sp.TD1.0 to acetate sodium(NaAC),the cell growth of TD1.0 was only inhibited by 45.8%when the concentration of NaAC was increased from 25 to 100 g·L^(-1).TD1.0 consumed 27.3 g·L^(-1) NaAC in 36 h and reached a dry cell weight(DCW)of 9.6 g·L^(-1) with 61%PHB content in shake flask cultivation.The results indicated that the strain has a good tolerance to NaAC with high concentration,and can efficiently produce PHB from acetate.In order to further improve its acetate utilization rate,the acetyl-Coenzyme A synthetase gene acs from Bacillus subtilis was over-expressed in TD1.0 using two plasmids with high and low copy number.The average of acetate utilization rate in strain TD-PN59 harboring a high-copy expression plasmid was 0.91 g·L^(-1)·h^(-1),19.7%higher than that of control strain TD1.0.The dry cell weight and PHB content of TD-PN59 were 9. 98 and 65%, respectively, and the PHB production was 6. 49 g·L^(-1) , which was increasedby 10. 8% compared to TD1. 0. In the medium supplemented with 10 g·L^(-1) glucose and 10 g·L^(-1)acetate sodium as mixed carbon sources, the acetate sodium utilization rate of strain TD-PN85 expressingacs gene with a low-copy number vector was significantly higher than that of TD1. 0. What’s more, thecarbon catabolite repression (CCR) was alleviated, which promoted the co-utilization of glucose and acetatesodium.