Zhang Baixi, Cao Jiacan
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, China.
J Fungi (Basel). 2024 Jan 12;10(1):63. doi: 10.3390/jof10010063.
The oleaginous yeast is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for to meet the high demand for metabolic engineering studies. However, these systems often necessitate an additional outgrowth step to achieve high gene editing efficiency. In this study, we introduced the eSpCas9 protein, derived from the Cas9(SpCas9) protein, into the genome to enhance gene editing efficiency and fidelity, and subsequently explored the optimal expression level of gene by utilizing different promoters and selecting various growth periods for yeast transformation. The results demonstrated that the integrated gene editing system significantly enhanced gene editing efficiency, increasing from 16.61% to 86.09% on and from 33.61% to 95.19% on , all without the need for a time-consuming outgrowth step. Furthermore, growth curves and dilution assays indicated that the consistent expression of eSpCas9 protein slightly suppressed the growth of , revealing that strong inducible promoters may be a potential avenue for future research. This work simplifies the gene editing process in , thus advancing its potential as a natural product synthesis chassis and providing valuable insights for other comparable microorganisms.
由于产油、外源蛋白分泌以及利用各种廉价碳源方面的卓越表现，产油酵母成为生物炼制研究的一个突出对象。为满足代谢工程研究的高需求，已开发出许多CRISPR/Cas9基因组编辑系统。然而，这些系统通常需要额外的生长步骤才能实现高基因编辑效率。在本研究中，我们将源自Cas9(SpCas9)蛋白的eSpCas9蛋白导入产油酵母基因组，以提高基因编辑效率和保真度，随后通过利用不同启动子并为酵母转化选择不同生长时期来探索产油酵母基因的最佳表达水平。结果表明，整合的产油酵母基因编辑系统显著提高了基因编辑效率，在[具体条件1]上从16.61%提高到86.09%，在[具体条件2]上从33.61%提高到95.19%，且所有这些都无需耗时的生长步骤。此外，生长曲线和稀释试验表明，eSpCas9蛋白的持续表达略微抑制了产油酵母的生长，这表明强诱导型启动子可能是未来研究的一个潜在方向。这项工作简化了产油酵母中的基因编辑过程，从而提升了其作为天然产物合成底盘的潜力，并为其他类似微生物提供了有价值的见解。
