Data underlying research on expression of heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation genes enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast <i>Saccharomyces</i> <i>cerevisiae</i>. In this study, we identified 7 Moco biosynthesis genes in the non-conventional yeast <i>Ogataea parapolymorpha</i> by <i>Spy</i>cas9-mediated mutational analysis and expressed them in <i>S. cerevisiae</i>. Functionality of the heterologously expressed<i> </i>Moco biosynthesis pathway in <i>S. cerevisiae</i> was assessed by co-expressing <i>O. parapolymorpha </i>nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered <i>S. cerevisiae</i> was observed on nitrate as sole nitrogen source. Relative to the engineered, evolved nitrate-assimilating <i>S. cerevisiae</i> strains isolated from these cultures showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a <i>Chlamydomonas reinhardtii</i> high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered <i>S. cerevisiae</i> was rapidly outcompeted by the spoilage yeast <i>Brettanomyces bruxellensis. </i>In contrast, an engineered and evolved nitrate-assimilating <i>S. cerevisiae</i> strains persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness industrial processes upon contamination with spoilage yeasts. Since over 50 Moco-dependent enzymes have been described, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of <i>S. cerevisiae</i>.

细胞的代谢能力不仅取决于其酶与代谢物的组成谱，还受酶辅因子的可用性影响。钼辅因子（Moco）在真核生物中分布广泛，但工业模式酵母酿酒酵母（Saccharomyces cerevisiae）中并不存在该辅因子。本研究通过SpyCas9介导的突变分析，在非常规酵母副形Ogataea（Ogataea parapolymorpha）中鉴定出7个Moco生物合成基因，并将其在酿酒酵母中异源表达。我们通过共表达副形Ogataea的硝酸盐同化酶（包括依赖Moco的硝酸还原酶（nitrate reductase）），评估了异源表达的Moco生物合成通路在酿酒酵母中的功能活性。经过两周培养后，工程化酿酒酵母可在以硝酸盐为唯一氮源的培养基中生长。相较于工程化亲本菌株，从该培养体系中分离得到的经定向进化的硝酸盐同化型酿酒酵母菌株，其异源基因拷贝数更高、编码蛋白的表达水平更高，且细胞提取物中的硝酸还原酶活性提升5倍。通过共表达莱茵衣藻（Chlamydomonas reinhardtii）的高亲和力钼酸盐转运蛋白，工程菌可在纳摩尔浓度的钼酸盐培养基中生长。在含硝酸盐培养基的连续分批培养体系中，非工程化酿酒酵母会被致腐酵母布雷特酵母（Brettanomyces bruxellensis）快速竞争性淘汰。与之相反，经工程化改造并定向进化的硝酸盐同化型酿酒酵母菌株在35代共培养过程中始终存活。该结果表明，工程菌株利用硝酸盐的能力，有望在污染性酵母污染工业生产过程时，提升工程菌的竞争优势。鉴于目前已报道的Moco依赖性酶超过50种，引入功能完整的Moco生物合成通路，可为进一步拓展酿酒酵母的生物催化功能谱提供极具潜力的研究方向。