Liu Minzhi, Zhou Sihan, Cao Yunsong, Yang Keqin, Xiao Yao, Wang Wei
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
Key Laboratory of Biosynthesis of Natural Products of National Health Commission of the Peoples Republic of China, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
Microb Cell Fact. 2024 Aug 8;23(1):224. doi: 10.1186/s12934-024-02496-w.
Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity.
At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (P and P) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (P) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated P enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully.
We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.
选择标记在巴斯德毕赤酵母的基因改造中很有用。然而，启动子的渗漏会导致选择标记的意外表达，尤其是那些有毒蛋白，如MazF，从而阻止细胞生长并阻碍原核系统中的基因操作。在本研究中，建立了一种基于新的反向选择标记的策略，用于高效、低毒的无缝改造。
首先，分别在两种宿主大肠杆菌和巴斯德毕赤酵母中研究了增强型绿色荧光蛋白（EGFP）作为报告基因在巴斯德毕赤酵母六个诱导型启动子控制下的渗漏表达。结果表明，DAS1和FDH1启动子（P和P）在原核生物和真核生物中具有最高的渗漏表达活性，而DAS2启动子（P）具有中等强度的诱导性但渗漏表达活性较低，所有这些都被选择用于进一步研究。接下来，挖掘了紫茉莉抗病毒蛋白（MAPs）c21873-1、c21873-1T（c21873-1的截短形式）和c23467作为新的反向选择标记，并分别将潮霉素B（Hyg B）抗性基因用作正向选择标记。然后，构建了具有MAP-靶基因-Hyg B盒的模块化质粒，并在线性化后用于转化巴斯德毕赤酵母细胞，靶基因通过单交换同源重组（HR）整合到其基因组的BmT1位点。在甲醇培养基诱导的反向选择后，标记c21873-1和c21873-1T被有效地回收。但c23467由于对巴斯德毕赤酵母细胞有毒性作用而未能被回收。最后，在严格调控的P下的反向选择标记c21873-1使报告基因EGFP和测试蛋白的编码基因能够整合到靶位点并成功表达。
我们开发了MAP c21873-1作为一种新型反向选择标记，它可以通过定点HR进行高效的基因敲入。在反向选择后，该标记可以回收重复使用，除了靶基因外不会引入不需要的序列。这种无标记的基因改造策略未来可能会扩展到其他基因改造，包括但不限于基因敲除和定点诱变。
