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

• Main Authors: Da Silva, N. (Author), De Keyser, S. (Author), Lang, X. (Author), Li, M. (Author), Moran Cabrera, M. (Author), Sun, X. (Author), Wheeldon, I. (Author)
• Format: Article
• Language: English
• Published: BioMed Central Ltd 2021
• Subjects: Amino acids; Biochemistry; Biofuels; Biosynthesis; Economical production; Expression regulation; Fed-batch operation; Feedback-insensitive; Flavor and fragrances; Flavor compounds; Flavors and fragrances; Fuel additives; gene expression; Gene expression; genetic engineering; genome; Integration; Kluyveromyces marxianus; Metabolic engineering; organic compound; Pathway engineerings; Personal care products; Shake-flask cultures; Strain; Thermotolerance; yeast
• Online Access: View Fulltext in Publisher

 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 33-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 KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, 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. © 2021, The Author(s).