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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 ± 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.

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