Rauter Marion, Kasprzak Jakub, Becker Karin, Riechen Jan, Worch Sebastian, Hartmann Anja, Mascher Martin, Scholz Uwe, Baronian Kim, Bode Rüdiger, Schauer Frieder, Matthias Vorbrodt H, Kunze Gotthard
Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany.
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
Microb Cell Fact. 2016 Oct 12;15(1):175. doi: 10.1186/s12934-016-0573-9.
The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation.
In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption.
Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.
非常规酵母嗜腺嘌呤阿氏酵母以1-丁醇作为碳源，最近作为一种有前景的1-丁醇生产生物受到关注。醇脱氢酶（adhp）是1-丁醇代谢中的重要催化剂，但嗜腺嘌呤阿氏酵母中只有Aadh1p得到了表征。该酶参与乙醇合成，但对1-丁醇降解的影响较小。
在本研究中，我们鉴定并表征了嗜腺嘌呤阿氏酵母的第二种adhp（Aadh2p）。与酿酒酵母ADHs（ScAdh）的蛋白质序列相比，它与ScAdh6p、7p和4p起源于同一个祖先节点。它也定位于细胞质中，并以NAD(H)作为辅因子。该酶对中链长度醇类具有最高活性，对1-丁醇具有最大活性，纯化酶的催化效率分别比乙醇和乙醛高42倍和43000倍。构建了嗜腺嘌呤阿氏酵母菌株G1212/YRC102-AADH2，其在强组成型TEF1启动子的控制下表达AADH2基因。它的ADH活性高达8000 U/L和500 U/g干细胞重量（dcw），而对照菌株G1212/YRC102的ADH活性高达1400 U/L和200 U/g dcw。基因表达分析表明，使用乙醇、丙酮酸、甘油或1-丁醇等不可发酵碳源时，AADH2确实会发生去阻遏或诱导。与G1212/YRC102相比，AADH2敲除菌株在以1-丁醇作为唯一碳源时生长速率较慢且1-丁醇消耗较低，而AADH2转化体在相同条件下根本无法生长。然而，添加支链氨基酸亮氨酸、异亮氨酸和缬氨酸可使转化体利用1-丁醇作为碳源。向对照菌株和Δaadh2突变体培养物中添加这些氨基酸具有加速1-丁醇消耗的作用。
我们的结果证实Aadh2p在嗜腺嘌呤阿氏酵母的1-丁醇代谢中起主要作用。当细胞在1-丁醇上生长时，它的上调高达60倍，而Aadh1p的相对表达水平仅发现有微小变化。因此，AADH2基因的组成型过表达可能有助于嗜腺嘌呤阿氏酵母生产1-丁醇，尽管可能需要敲除其他ADHs以防止1-丁醇氧化。
