Enzyme Technology Laboratory, Bioresource Technology Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani, 12120, Thailand.
Appl Microbiol Biotechnol. 2012 Nov;96(3):697-709. doi: 10.1007/s00253-011-3866-2. Epub 2012 Jan 17.
In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.
在这项研究中，我们从耐热甲基营养酵母汉逊酵母（Hansenula polymorpha）中鉴定并表征了线粒体醇脱氢酶 3（HpADH3）。HpADH3 的氨基酸序列与其他酵母的醇脱氢酶有超过 70%的同源性，与 H. polymorpha 的醇脱氢酶 1 的相似度最高达 91%。然而，与胞质的 HpADH1 不同，HpADH3 似乎是一种线粒体酶，因为其 N 端存在一个线粒体靶向延伸。在大肠杆菌中过表达的重组 HpADH3 对乙醇氧化和乙醛还原表现出相似的催化效率。HpADH3 对底物具有特异性，对中链长度的伯醇和乙醛分别表现出明显的偏好，分别用于氧化反应和还原反应。尽管 H. polymorpha 的 ADH3 基因在培养基中被乙醇诱导，但 ADH 同工酶模式分析和 ADH 活性测定均表明 HpADH3 不是 H. polymorpha DL-1 中的主要 ADH。此外，HpADH3 缺失并不影响细胞在不同碳源上的生长。然而，当 HpADH3 突变体被与强组成型启动子融合的 HpADH3 表达盒互补时，与野生型菌株相比，该菌株在葡萄糖培养基中产生的乙醇量显著增加。相比之下，在木糖培养基中，HpADH3 过表达菌株的乙醇产量与野生型菌株相比显著降低。综上所述，我们的研究结果表明，表达 HpADH3 将是开发汉逊酵母作为特定底物生物乙醇生产菌株的理想工程目标。
