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BLINCAR:一种可重复使用的生物发光和基于Cas9的遗传工具集,用于重复修改野生类型的Scheffersomyces stipitis

Walter D Reichard¹, Serenah E Smith¹, J Brian Robertson¹

¹Department of Biology, Middle Tennessee State University , Murfreesboro, Tennessee, USA.

|June 22, 2023

概括

新的分子工具使Scheffersomyces stipitis的高效基因工程成为可能,这种酵母对于基纤维素乙醇生产至关重要. 这些工具克服了转化酵母的挑战,并扩大了其对工业应用的潜力.

科学领域:

生物技术和合成生物学
生物燃料的微生物工程

背景情况:

Scheffersomyces stipitis是一种强大的酶发酵酵母,对于基纤维素乙醇生产有价值.
非正规的编码子使用和基因操纵障碍限制了S. stipitis的应用.
高效的乙醇生产需要工程酵母来利用各种各样的林氏纤维素衍生糖.

研究的目的:

开发一种多功能分子工具集,用于对Scheffersomyces stipitis进行基因工程.
克服现有的转换和操纵S. stipitis基因组的局限性.
为了增强S. stipitis对于基纤维素乙醇发酵的实用性.

主要方法:

开发BLINCAR (生物发光指标通过Cas9-激活的重组取消) 系统.
使用BLINCAR重复插入外源DNA有效载荷到S. stipitis基因组中.
使用BLINCAR从S.stipitis基因组中顺序删除本地基因.
使用短暂的Cas9表达来去除抗生素耐药性和生物发光标记物,以便重复使用.

主要成果:

成功生成了抗生素耐药,生物发光的S. stipitis殖民地,表明成功的基因修饰.
在DNA插入和基因删除方面,BLINCAR的重复使用已被证明.
通过Cas9实现了标记物去除,使其能够对抗生素和随后的遗传修饰重新敏感.

结论:

开发的BLINCAR工具集显著降低了对S. stipitis.遗传操纵的障碍.
这种扩展的工具集增强了S. stipitis在生产细胞醇乙醇方面的潜力.
促进S. stipitis的先进代谢工程,以提高生物燃料产量.

关键词:

在CUG群的酵母酵母.这就是Cas9的情况.在S. stipitis的研究中.生物发光是一种生物发光.

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