CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

作者：Mengwan Li, Xuye Lang, Marcos Moran Cabrera, Sawyer De Keyser, Xiyan Sun, Nancy Da Silva, Ian Wheeldon · 发表于：Biotechnology for Biofuels · 年份：2021 · DOI：10.1186/s13068-020-01852-3 · 被引用次数：75 · 研究领域：Microbial Metabolic Engineering and Bioproduction、Plant biochemistry and biosynthesis、Plant Gene Expression Analysis

Abstract Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus . First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4 , KmARO7 , and KmPHA2 , each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2 , with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ...