Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering.
Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering.
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解脂耶氏酵母中两种 NADPH 依赖性赤藓糖还原酶的鉴定、表征以及利用代谢工程提高赤藓糖醇生产力
DOI：
10.1186/s12934-018-0982-z
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发表时间：
2018-08-29
影响因子：
6.4
通讯作者：
Cheng H
中科院分区：
文献类型：
--
作者：
Cheng H;Wang S;Bilal M;Ge X;Zhang C;Fickers P;Cheng H
赤藓糖醇是一种具有甜味特性的四碳糖醇，被农业食品工业用作食品添加剂。在解脂雅罗维氏酵母中，赤藓糖醇合成的最后一步涉及到特定的红细胞还原酶还原红细胞(S)。在早期的报告中，从产赤藓糖醇的解脂耶尔森菌MK1中鉴定出一个红细胞还原酶基因(YALI0F18590G)(Janek等人)。在Microb Cell Fact 16：118中，)。然而，溶脂耶尔森菌MK1中该基因的缺失仅导致红素醇产量降低，但其合成过程仍然保持不变，这表明解脂耶尔森菌基因组中可能存在另一种红素还原酶基因(S)。在本研究中，我们分离到编码两个新的红细胞还原酶(ER)的基因g141.t1(YALI0D07634g)和g3023.t1(YALI0C13508g)。纯化的酶的生化特性表明，它们对红血球有很强的亲和力。两个ER基因加上g801.t1(YALI0F18590G)的缺失并没有阻止赤藓糖醇的合成，这表明在该酵母中仍未发现其他ER或类似ER的酶。与野生型菌株相比，新分离的两个基因(ER10或ER25)的过表达导致赤藓糖醇产量平均提高14.7%，生产率提高31.2%。最后，通过过量表达编码葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶的基因ZWF1和GND1来改造NADPH辅因子代谢，与野生型菌株相比，赤藓糖醇产量提高了23.5%，生产率提高了50%。我们构建的最好的菌株在以葡萄糖为主要碳源的折流摇瓶中产生的赤藓糖醇效价为190g/L。我们的结果突出表明，在解脂耶尔森菌基因组中，有几个基因编码能够将红血球还原为赤藓糖醇的酶。这些酶的催化性质和对辅因子的依赖性与已知的解脂耶尔森菌的红细胞还原酶不同。新分离基因的结构性表达和NADPH辅因子代谢工程导致了赤藓糖醇效价的增加。发酵策略的发展将使这一生产率在未来得到进一步提高。本文的在线版本(10.1186/s12934-0180982-z)包含补充材料，可供授权用户使用。
Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast Yarrowia lipolytica, the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene (YALI0F18590g) from erythritol-producing yeast Y. lipolytica MK1 was identified (Janek et al. in Microb Cell Fact 16:118,). However, deletion of the gene in Y. lipolytica MK1 only resulted in some lower erythritol production but the erythritol synthesis process was still maintained, indicating that other erythrose reductase gene(s) might exist in the genome of Y. lipolytica. In this study, we have isolated genes g141.t1 (YALI0D07634g) and g3023.t1 (YALI0C13508g) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 (YALI0F18590g) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source. Our results highlight that in the Y. lipolytica genome several genes encode enzymes able to reduce erythrose into erythritol. The catalytic properties of these enzymes and their cofactor dependency are different from that of already known erythrose reductase of Y. lipolytica. Constitutive expression of the newly isolated genes and engineering of NADPH cofactor metabolism led to an increase in erythritol titer. Development of fermentation strategies will allow further improvement of this productivity in the future. The online version of this article (10.1186/s12934-018-0982-z) contains supplementary material, which is available to authorized users.