Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose
Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose
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DOI：
10.1002/biot.201700584
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发表时间：
2018-09-01
影响因子：
4.7
通讯作者：
Wheeldon, Ian
中科院分区：
文献类型：
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作者：
Schwartz, Cory;Curtis, Nicholas;Wheeldon, Ian
酵母解脂耶氏菌因其高水平合成和积累细胞内脂质的能力而被广泛研究。最近的研究已经确定了能够促进生物质糖生长的天然基因，但这些基因的表达不足以促进强劲的代谢。在这项工作中，研究人员开发了一种聚脂y菌中的CRISPR-dCas9激活（CRISPRa）系统，并利用它激活天然-葡萄糖苷酶的表达，以支持纤维二糖的生长。一系列不同的转录激活剂比较了它们在脂肪瘤中的有效性，合成的三方激活剂VPR产生最高的激活。然后将VPR-dCas9融合到驱动hrGFP表达的合成启动子中的不同位置，并实现激活。随后，CRISPRa系统被用来激活两种不同的天然-葡萄糖苷酶基因的转录，促进以纤维素二糖为唯一碳源的生长。这项工作扩展了脂肪瘤菌代谢工程的合成生物学工具箱，并展示了CRISPR-Cas9系统的可编程性如何能够方便地研究基因组的转录沉默区域。
The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica is developed and is used it to activate native -glucosidase expression to support growth on cellobiose. A series of different transcriptional activators are compared for their effectiveness in Y. lipolytica, with the synthetic tripartite activator VPR yielding the highest activation. A VPR-dCas9 fusion is then targeted to various locations in a synthetic promoter driving hrGFP expression, and activation is achieved. Subsequently, the CRISPRa system is used to activate transcription of two different native -glucosidase genes, facilitating enhanced growth on cellobiose as the sole carbon source. This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.