地中海富盐菌中phaB基因的鉴定及PHBV前体供应途径的分析

Identification of the phaB genes and analysis of the PHBV precursor supplying pathway in Haloferax mediterranei

摘要:

摘要：【目的】进一步揭示地中海富盐菌（Haloferax mediterranei）中聚羟基丁酸羟基戊酸酯[poly(3-hydroxybutyrate-co-3-hydroxyvalerate)，PHBV]前体供应的途径并鉴定其中的关键基因。【方法】将细菌及其他古菌中已鉴定的与聚羟基脂肪酸酯前体（3-羟基脂酰-CoA）供应相关的酶与地中海富盐菌预测的全蛋白质组进行同源性比对，得到相似性比较高的5个基因，分别命名为：phaB1，phaB2，phaJ1，phaJ2和phaJ3。首先利用RT-PCR检测了5个基因在产PHBV的条件下的转录情况。然后利用同源重组双交换的方法，将5个基因分别或组合敲除，得到突变株：ΔphaB1，ΔphaB2，ΔphaJ1，ΔphaJ2，ΔphaJ3，ΔphaB1phaB2，ΔphaJ1phaJ2和ΔphaJ1phaJ2phaJ3。并在突变株ΔphaB1phaB2中分别互补phaB1和phaB2基因。【结果】无论是将3个phaJ基因单独敲除，还是组合敲除，对地中海富盐菌PHBV的积累都没有明显影响。单独敲除phaB1基因对PHBV的积累没有明显影响，单独敲除phaB2基因导致突变株PHBV产量明显下降，而且3-HV单体组分所占的摩尔比例也有所下降。 将phaB1和phaB2基因同时敲除后，得到的突变株不再产生PHBV。【结论】在地中海富盐菌中可能主要存在由乙酰-CoA和丙酰-CoA提供PHBV前体的途径，其中编码乙酰乙酰-CoA还原酶的两个基因即为phaB1和phaB2。

Abstract:

Abstract: [Objective] Identification and characterization of the genes involved in precursor supplying for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in the haloarchaeon Haloferax mediterranei. [Methods] By using BLAST (Basic Local Alignment Search Tool) search methodology, we obtained five genes (phaB1, phaB2, phaJ1, phaJ2 and phaJ3) that were possibly involved in the 3-hydroxyacyl-CoA precursor supplying for PHBV biosynthesis in H. mediterranei. Firstly, we proved that these five genes were all transcribed under the PHBV-accumulating condition in H. mediterranei. Then, we knocked out these genes individually or in combination, by double-crossover homologous recombination, resulting in the following mutants: ΔphaB1, ΔphaB2, ΔphaJ1, ΔphaJ2, ΔphaJ3, ΔphaB1phaB2, ΔphaJ1phaJ2 and ΔphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the ΔphaB1phaB2 strain, with the phaB1 and phaB2 genes, respectively. [Results] Whenever the three phaJ genes were knocked out individually or in combination, there was no obvious influence on PHBV accumulation in H. mediterranei. Knockout of phaB1 also did not affect the PHBV accumulation obviously. However, when phaB2 was knocked out, the yield of PHBV and the fraction of the 3-HV monomer decreased significantly. Notably, when the phaB1 and phaB2 were knocked out in combination, the mutant ΔphaB1phaB2 no longer produced PHBV. [Conclusions] The PHBV-specific acetoacetyl-CoA reductases (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.