Joint BioEnergy Institute, Emeryville, California, USA
Biomass Science and Conversion Technologies, Sandia National Laboratories, Livermore, California, USA.
mSphere. 2019 Mar 20;4(2):e00099-19. doi: 10.1128/mSphere.00099-19.
Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as and Significant advancements in the past few years have bolstered ' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of exhibiting successful targeted disruption of the native gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, , with much greater success; editing efficiencies of deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both and in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in : CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in .
微生物生产生物燃料和生物制品为石油基燃料和化学品提供了一种可持续且经济的替代方案。担子菌酵母 是一种很有前途的生物制品生产平台生物，因为它能够消耗广泛的碳源（包括源自木质纤维素生物质的碳源），并自然积累高水平的脂质和类胡萝卜素，这两种生物合成途径可以用来生产广泛的生物制品。虽然 具有很大的潜力，但与更先进的酵母平台生物（如 和 相比，它的遗传工程工具集更为有限。在过去的几年中，取得了重大进展，增强了 的工程能力。在这里，我们通过展示第一个使用基于 CRISPR-Cas9 的基因敲除在 中，扩展了这一能力。转化包含潮霉素抗性的 Cas9 表达盒，并在该抗生素上选择转化体，导致 菌株成功靶向敲除了天然的 基因。虽然编辑效率最初很低（0.002%），但对盒的优化将效率提高了 364 倍（达到 0.6%）。应用这些优化的设计条件，使另一个参与类胡萝卜素生物合成的天然基因 的敲除更加成功； 的缺失编辑效率达到约 50%。最后，我们通过在单个转化中同时敲除 和 ，证明了高效的多重基因组编辑。总之，我们的结果为在 中应用 CRISPR-Cas9 提供了一个框架，将促进在这个具有工业相关性的生物体中快速、高通量的基因组工程。微生物生物燃料和生物制品平台提供了对清洁和可再生碳源的获取，这些碳源比石油基碳源更可持续、更环保。此外，它们还可以作为合成难以通过纯化学方法生产的先进分子的有用途径。 已成为将可再生木质纤维素材料转化为有价值的燃料和化学品的有前途的潜在宿主。然而，由于缺乏先进的基因组工程工具，工程努力来提高酵母的生产能力受到了阻碍。虽然这种情况正在迅速改变，但一种关键工具在 中仍未得到探索：CRISPR-Cas9。这里概述的结果首次展示了有效的多重 CRISPR-Cas9 基因敲除如何为其他研究人员提供一个框架，使其能够有效地在 中利用这一革命性的基因组编辑工具。
