Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for β-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of β-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

中文翻译：

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使用双重 CRISPR/Cas9 策略构建野生型解脂耶氏酵母 IMUFRJ 50682 营养缺陷型突变体，用于新型生物技术方法


解脂耶氏酵母 IMUFRJ 50682 是一种巴西野生型菌株，具有在生物转化过程中的潜在应用，可以通过合成生物学进行改进。在这项研究中，我们重点研究了 CRISPR/Cas9 介导的组合双切割，用于构建不可逆营养缺陷型突变体 IMUFRJ 50682，该突变体的基因组信息不可用，但认为是针对 URA3 基因上游和下游位点的配对 sgRNA。对于更接近 URA3 的起始密码子和终止密码子的 sgRNA 组合，破坏效率范围为 5% 至 28%，并且营养缺陷型突变体丢失了包含所有编码序列的约 970 bp，验证了该方法用于野生型菌株的基因组编辑。此外，我们引入了荧光表型，并实现了 80% 至 100% 的克隆率。 ura3Δ 菌株 IMUFRJ 50682 也被设计用于 β-胡萝卜素合成，作为概念证明。与野生型菌株相比，类胡萝卜素生产菌株表现出相似的生长曲线，并且能够在 YPD 和 YNB 烧瓶培养物中合成 30.54-50.06 mg/L（高达 4.8 mg/g DCW）的 β-胡萝卜素，表明有希望营养缺陷型突变体 IMUFRJ 50682 的未来作为生产新型增值化学品的基础。