背景:突变技术已用于在实验室和工业微生物中实现全基因组变异。自然基因组进化的遗传多态性包括核苷酸变异和结构变异,这激发了我们的建议,这两种类型的基因型变异是潜在有用的提高性能的底盘细胞的工业应用。然而,同时产生结构和核苷酸变异的高效方法仍然缺乏。
结果:本研究的目的是开发一种通过结合核苷酸变异和结构变异(CNAS)来增加酵母中虾青素生物合成的方法,它们是通过大气和室温等离子体(ARTP)以及LoxP介导的进化(SCraMbLE)系统进行合成染色体重组和修饰而产生的。CNAS用于增加酵母中虾青素的生物合成,并使虾青素的产量提高了2.2倍和7.0倍。此外,该方法被证明能够生成结构(删除,重复,和倒置)以及核苷酸变异(SNP和InDels)。此外,对虾青素改良菌株的基因型变异的遗传分析表明,YJR116W的缺失和YOL084W的C2481G突变提高了虾青素的产量,提示基因型与表型的关系。
结论:这项研究表明,CNAS策略可以产生结构变异和核苷酸变异,允许通过酵母中的不同基因型提高虾青素产量。总的来说,这项研究为产生具有理想表型的基因组变异多样性以及了解进化过程中基因型和表型之间的关系提供了有价值的工具。
BACKGROUND: Mutational technology has been used to achieve genome-wide variations in laboratory and industrial microorganisms. Genetic polymorphisms of natural genome evolution include nucleotide variations and structural variations, which inspired us to suggest that both types of genotypic variations are potentially useful in improving the performance of chassis cells for industrial applications. However, highly efficient approaches that simultaneously generate structural and nucleotide variations are still lacking.
RESULTS: The aim of this study was to develop a method of increasing biosynthesis of astaxanthin in yeast by Combining Nucleotide variations And Structure variations (CNAS), which were generated by combinations of Atmospheric and room temperature plasma (ARTP) and Synthetic Chromosome Recombination and Modification by LoxP-Mediated Evolution (SCRaMbLE) system. CNAS was applied to increase the biosynthesis of astaxanthin in yeast and resulted in improvements of 2.2- and 7.0-fold in the yield of astaxanthin. Furthermore, this method was shown to be able to generate structures (deletion, duplication, and inversion) as well as nucleotide variations (SNPs and InDels) simultaneously. Additionally, genetic analysis of the genotypic variations of an astaxanthin improved strain revealed that the deletion of YJR116W and the C2481G mutation of YOL084W enhanced yield of astaxanthin, suggesting a genotype-to-phenotype relationship.
CONCLUSIONS: This study demonstrated that the CNAS strategy could generate both structure variations and nucleotide variations, allowing the enhancement of astaxanthin yield by different genotypes in yeast. Overall, this study provided a valuable tool for generating genomic variation diversity that has desirable phenotypes as well as for knowing the relationship between genotypes and phenotypes in evolutionary processes.