A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2024-07-05 DOI:10.1016/j.mec.2024.e00245
Priti Regmi , Melanie Knesebeck , Eckhard Boles , Dirk Weuster-Botz , Mislav Oreb
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Abstract

Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.

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酿酒酵母中 NADPH 供应策略的比较分析:以从二木糖生产二木糖醇为例进行研究
提高代谢工程细胞中氧化还原辅助因子 NADPH 的供应量是优化脂肪酸或萜类化合物等多种产品合成的关键目标。在 S. cerevisiae 中,人们在不同的实验环境中开发出了几种成功的方法。但是,还没有对这些方法进行系统比较的报道。在这里,我们将依赖 NADPH 的木糖还原酶将木糖还原成木糖醇的反应作为一个模型反应,以比较在批量发酵过程中不同 NADPH 供应策略的功效,在批量发酵过程中,葡萄糖和乙醇依次被用作碳源和氧化还原供体。我们的研究表明,过量表达葡萄糖-6-磷酸脱氢酶 Zwf1 的菌株表现最佳,在搅拌罐生物反应器中可从 20 克/升木糖中产生高达 16.9 克/升的木糖醇。在乙醇消耗阶段,Zwf1 活性增加的有利影响尤其明显。醛脱氢酶 ALD6 基因的缺失也是同样的道理,尽管其数量水平较低。磷酸葡萄糖异构酶 Pgi1 的表达量减少,以及来自乳酸克鲁维酵母菌的依赖 NADP+ 的甘油醛-3-磷酸脱氢酶 Gdp1 的异源表达,在葡萄糖存在的情况下与 ZWF1 的过量表达具有协同作用,但在二重转移后则产生了不利影响。同样,在细胞质中表达线粒体 NADH 激酶 Pos5 也只能在葡萄糖条件下提高木糖醇的产量,但不能与 Zwf1 活性的增强相结合。为了证明我们的观察结果具有普遍性,我们发现最有前景的策略--ZWF1 的过表达和 ALD6 的缺失--也能改善由 d-半乳糖醛酸产生 l-半乳糖醛酸的情况。因此,我们希望这些发现不仅能为木糖醇的生产工程提供有价值的指导,也能为需要 NADPH 的其他各种途径的生产工程提供有价值的指导。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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