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

Background2-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.ResultsWe 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 519% 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 +/- 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 +/- 6 mg/L. The best strain achieves 1943 +/- 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures.Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Automated summary

The study focuses on engineering the Shikimate and Ehrlich pathways in yeast Kluyveromyces marxianus for the biosynthesis of 2-phenylethanol (2-PE). A CRISPR-mediated multigene integration system was developed to integrate multiple gene expression cassettes efficiently, leading to an increase in 2-PE production. The best strain achieved 1943 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures, demonstrating the potential of this approach for sustainable and economical production.