Abstract
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 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.ConclusionsThe 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.
Highlights
Like many esters and alcohols produced during yeast fermentation, the aromatic alcohol 2-phenylethanol (2-PE) is used in a wide variety of applications
Clustered regularly interspaced short palindromic repeats (CRISPR)‐mediated one‐step, multigene integration Given the importance of the Shikimate pathway to 2-PE production, we sought to increase flux to phenylpyruvate (PP), the precursor to the Ehrlich pathway, via Shikimate pathway refactoring
Based on the design of a CRISPR-CRISPR-associated protein 9 (Cas9)-mediated gene integration tool that we previously created for the oleaginous yeast Y. lipolytica, we designed a two-plasmid system for targeted gene integration in K. marxianus [41]
Summary
Like many esters and alcohols produced during yeast fermentation, the aromatic alcohol 2-phenylethanol (2-PE) is used in a wide variety of applications. Food-grade 2-PE is still produced mainly by isolation from rose petals, which presents a technically challenging separation problem and the supply is subject to annual fluctuations in crop yields [4]. The end result is a high market price of ~ $US1000 per kg. Synthesis by chemical catalysis is possible, but typically produces 2-PE that sells for less than $US4 per kg because it is not suitable for human consumption or use [5]. The chemical catalysis route creates environmental and health challenges due to the reliance on petrochemical reagents [6]. As a metabolic intermediate formed via the Shikimate and Ehrlich pathways, 2-PE produced by microbial systems is a potentially sustainable alternative to fossil fuel-based production and isolation from native plants
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