Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan Universitygrid.258151.a, Wuxi, Jiangsu, China.
School of Biotechnology, Jiangnan Universitygrid.258151.a, Wuxi, Jiangsu, China.
Microbiol Spectr. 2022 Jun 29;10(3):e0005922. doi: 10.1128/spectrum.00059-22. Epub 2022 May 11.
Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNA sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene (green fluorescent protein) and the endogenous gene (phosphoribosylaminoimidazole carboxylase). Different regions of and were targeted with the gRNAs processed by the tRNA, and the transcription levels of and were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNA and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.
热带假丝酵母（Candida tropicalis）是一种非模式二倍体微生物，已被应用于工业底盘细胞。由于缺乏基因编辑和调控工具，热带假丝酵母的代谢工程具有挑战性。在这里，我们报告了一种 tRNA:guide RNA（gRNA）平台，用于提高热带假丝酵母的基因编辑和沉默效率。由于内源性 tRNA 加工系统能够自我切割，从而产生大量成熟的 gRNA，因此我们选择了来自热带假丝酵母 ATCC 20336 基因组的 tRNA 序列来构建 tRNA:gRNA 平台。在 CRISPR-Cas9 系统中，tRNA:gRNA 平台在单基因和多基因编辑中都被证明是有效的。此外，基于 tRNA:gRNA 平台，我们开发了一种 CRISPR 干扰（CRISPRi）系统，用于构建高效的 dCas9 介导的基因表达调控系统，用于调控热带假丝酵母的基因表达。CRISPRi 系统被用于调控外源基因（绿色荧光蛋白）和内源基因（磷酸核糖基氨基咪唑羧化酶）的表达。通过 tRNA 加工的 gRNA 靶向 和 的不同区域，成功地将 和 的转录水平下调至 23.9%±4.1%和 38.0%±7.4%。还研究了目标区域对基因调控的影响。此外，该调控系统还被用于沉默 （鲨烯合酶），以增强代谢修饰的热带假丝酵母菌株中的 β-胡萝卜素生物合成。结果表明，内源性 tRNA 和 CRISPRi 系统在热带假丝酵母的代谢工程中有很大的潜力。在非模式酵母假热带丝酵母中，缺乏可用的 RNA 聚合酶 III（Pol III）启动子，这阻碍了建立 CRISPR-Cas 介导的基因组编辑和沉默策略的 gRNA 表达平台的发展。在这里，构建了一个 tRNA:gRNA 平台。我们表明，该平台允许从单个多顺反子基因中高效和精确地表达和处理不同的 gRNA，该基因能够与 CRISPR-Cas9 一起介导多基因编辑。此外，与 dCas9 结合使用时，tRNA:gRNA 平台可有效地用于沉默外源和内源基因，代表了假热带丝酵母中的第一个 CRISPR 干扰工具（CRISPRi）。重要的是，建立的 CRISPRi-tRNA:gRNA 工具还通过调节假热带丝酵母中的 β-胡萝卜素生物合成用于代谢工程。结果表明，tRNA:gRNA 平台和 CRISPRi 系统将进一步推进基于 CRISPR-Cas 的编辑和 CRISPRi 系统在假热带丝酵母代谢工程中的应用。
