USDA-ARS-NCAUR RPT, Peoria, USA.
University of California, Davis, Davis, USA.
J Ind Microbiol Biotechnol. 2019 Nov;46(11):1547-1556. doi: 10.1007/s10295-019-02209-y. Epub 2019 Jul 9.
Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.
布赫纳氏乳杆菌和酒香酵母是两种具有独特乙醇耐受性的革兰氏阳性细菌。基因组比较分析表明，布赫纳氏乳杆菌和酒香酵母都拥有一个 pntAB 基因座，而该基因座几乎不存在于所有其他乳酸菌（LAB）基因组中。我们的假设是，pntAB 基因座有助于这两种具有独特乙醇耐受性的生物体的乙醇耐受性特性。pntAB 基因座由 pntA 和 pntB 基因组成，编码 NADP(H)转氢酶亚基。这种膜结合的转氢酶催化 NADP+的还原，是维持细胞氧化还原平衡的重要酶。在这项研究中，克隆并分析了布赫纳氏乳杆菌 NRRL B-30929 和酒香酵母 PSU-1 的转氢酶操纵子。LbpntB 与酒香酵母（OopntB）的相似度为 71.0%。当与对照菌株相比时，乳糖乳球菌 ssp. lactis IL1403 中整个 pntAB 基因座的表达导致对乙醇（6%）、丁醇（1.8%）和异丙醇（1.8%）的耐受性增加。然而，携带整个 pntAB 基因座的重组大肠杆菌细胞并没有显示出任何提高的乙醇耐受性。在重组大肠杆菌 BL21(DE3)pLysS 宿主中独立表达 OopntB 和 LbpntB 时，与携带 pET28b 载体的对照大肠杆菌 BL21(DE3)pLysS 菌株相比，对乙醇的耐受性更高。携带 LbpntB 和 OopntB 的大肠杆菌菌株的乙醇耐受性比较表明，LbpntB 赋予更高的乙醇耐受性（4.5%）并导致更大的生物量，而 OopntB 赋予较低的乙醇耐受性（4.0%）并导致较低的生物量。因此，布赫纳氏乳杆菌的 pntB 基因是产生更高乙醇耐受性的更好选择。这是首次研究 pntAB 基因座对乙醇耐受性的作用。
