Data underlying research on Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant co-factor. For instance, in <i>Yarrowia lipolytica</i>, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the <i>Ogataea parapolymorpha</i> Moco biosynthesis pathway associated with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in <i>Y. lipolytica</i>. This was achieved by coupling Moco biosynthesis to the Moco-dependent nitrate assimilation pathway of the same donor, <i>O. parapolymorpha</i>. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in <i>S. cerevisiae</i> demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

在微生物宿主中工程化构建全新代谢功能时，常受限于相关辅因子的可获取性。例如，在解脂耶氏酵母（*Yarrowia lipolytica*）中，功能性硝酸还原酶的表达因钼辅因子（molybdenum cofactor, Moco）生物合成通路的缺失而无法实现。本研究证实，将多形Ogataea（*Ogataea parapolymorpha*）的钼辅因子生物合成通路与高亲和力钼酸盐转运蛋白的表达相结合，可使解脂耶氏酵母实现钼辅因子的合成。该策略通过将钼辅因子的生物合成与供体菌多形Ogataea自身的钼辅因子依赖性硝酸同化通路相偶联得以完成。除引入11个异源基因外，实现以硝酸盐为底物的快速生长还需借助适应性实验室进化，该过程可使硝酸还原酶活性提升最高达100倍，生长速率提升最高达4倍。对进化分离株的基因组测序结果显示，注释的编码序列中仅存在少量非同义突变或小型插入/缺失变异。本研究在先前于酿酒酵母（*Saccharomyces cerevisiae*, *S. cerevisiae*）中成功构建钼辅因子合成途径的工作基础上进一步拓展，证实钼辅因子生物合成通路可成功转移至亲缘关系极远的酵母物种中，且理论上可推广至其他任何属级微生物，这将有望实现新型酶家族的功能表达，并拓宽工业酵母可利用的营养底物范围。