Ruan Shupeng, Yang Yuxin, Zhang Xinying, Luo Guanjuan, Lin Ying, Liang Shuli
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Synth Syst Biotechnol. 2024 Jun 18;9(4):759-765. doi: 10.1016/j.synbio.2024.06.002. eCollection 2024 Dec.
, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the . cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10-100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of , such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of . This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of . This may lead to improved efficiency of genetic engineering in .
毕赤酵母是一种甲基营养型酵母，能够利用甲醇作为碳源和能源来合成高价值化学品，是生物制造的理想宿主。由于缺乏用于操纵多基因生物合成途径的遗传工具，构建毕赤酵母细胞工厂受到一定阻碍。为了拓宽其在代谢工程领域的应用，本研究利用CRISPR-Cpf1基因组编辑技术在毕赤酵母中鉴定并筛选了15个新的整合位点，以绿色荧光蛋白作为报告蛋白。这些整合位点的整合效率为10%-100%，且表达强度各异，能够根据需要基于基因表达水平进行选择。此外，这些整合位点应用于虾青素生物合成途径、卡尔文-本森-巴斯姆（CBB）循环的二氧化碳固定途径等在毕赤酵母中的异源生物合成。在三位点整合过程中，CBB循环的8个基因被整合到毕赤酵母基因组中。这表明这些整合位点在整合大片段方面的潜力，并暗示了它们在毕赤酵母代谢工程中的成功应用。这可能会提高毕赤酵母中基因工程的效率。
