Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae
Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae
复制标题
酿酒酵母固氮酶生物合成途径多基因的联合组装和靶向整合
DOI：
10.1021/acssynbio.9b00060
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发表时间：
2019-08-01
影响因子：
4.7
通讯作者：
Li, Jilun
中科院分区：
文献类型：
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作者：
Liu, Xiaomeng;Wang, Minyang;Li, Jilun
生物固氮是原核生物特有的固氮过程，由固氮酶复合体催化。长期以来，人们一直有兴趣在真核宿主中重建固氮酶的生物合成途径，最终目的是开发固氮谷类作物。在本研究中，我们报道了一条固氮酶生物合成途径(类似于38kb，包含15个基因)通过体内组装两种单独的一步方法组装，并整合在酿酒酵母染色体上的Delta和Ho位点。其中11个基因(nif B、nif H、nif D、nif K、nif E、nif N、nif X、HESA、nifV、groes、groEL)来自多粘类芽孢杆菌WLY78，4个基因(nif S、nif U、nif F、nif J)来自催产克雷伯菌。15个固氮酶的生物合成途径在重组酿酒酵母中被正确组装和转录。在酵母中形成了与多粘菌分子量相同的NifDK四聚体，表达的NifH具有铁蛋白的活性。本研究表明，在真核宿主中生产活性固氮酶是可能的。
Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N-2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (similar to 38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at delta and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nif B, nif H, nif D, nif K, nif E, nif N, nif X, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nif S, nif U, nif F, nif J) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.