A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast,ACS Synthetic Biology

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A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-10-29 00:00:00 , DOI: 10.1021/acssynbio.8b00339
Leanne Bourgeois _{1,

2} , Michael E. Pyne _{1,

2} , Vincent J. J. Martin _{1,

2}

Affiliation

A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of (S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.

中文翻译：

用于酵母中精确多拷贝基因整合的高特性合成着陆垫系统

代谢工程的一项基本工作涉及识别和解决因通量通量不平衡而引起的代谢瓶颈。为了加快与DNA拷贝数调节相结合的直系同源酶的系统筛选，在此我们开发了一个简单且特性良好的酿酒酵母中CRISPR-Cas9整合系统。我们的工程策略将一系列合成DNA着陆垫（LP）引入酿酒酵母中基因组充当高级基因整合的位点。LP促进一次转化中一个，两个，三个或四个DNA拷贝的多拷贝基因整合，从而提供对DNA拷贝数的精确控制。我们将我们的LP系统应用于降尿酸月桂酸合酶（NCS），这是一种动力学性能较差的酶，参与了高价值苄基异喹啉生物碱的生产的第一步。该平台通过将十个NCS直系同源物整合到四个基因拷贝中，从而能够快速构建40株NCS的文库。确定了六个活跃的NCS变体，通过增加NCS可以进一步提高（S）-去甲紫花氨酸的产量副本编号。我们预计LP系统将通过严格控制基因拷贝数并加快菌株和途径工程活动来协助代谢工程工作。

更新日期：2018-10-29

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