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03650nam a2200529Ia 4500 |
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10.1186-s13068-020-01852-3 |
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220427s2021 CNT 000 0 und d |
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|a 17546834 (ISSN)
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|a CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus
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|b BioMed Central Ltd
|c 2021
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|z View Fulltext in Publisher
|u https://doi.org/10.1186/s13068-020-01852-3
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|a 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).
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|a Amino acids
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|a Biochemistry
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|a Biofuels
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|a Biosynthesis
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|a Economical production
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|a Expression regulation
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|a Fed-batch operation
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|a Feedback-insensitive
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|a Flavor and fragrances
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|a Flavor compounds
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|a Flavors and fragrances
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|a Fuel additives
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|a gene expression
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|a Gene expression
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|a genetic engineering
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|a genome
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|a Integration
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|a Kluyveromyces marxianus
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|a Kluyveromyces marxianus
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|a Metabolic engineering
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|a organic compound
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|a Pathway engineerings
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|a Personal care products
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|a Shake-flask cultures
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|a Strain
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|a Thermotolerance
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|a yeast
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|a Da Silva, N.
|e author
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|a De Keyser, S.
|e author
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|a Lang, X.
|e author
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|a Li, M.
|e author
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|a Moran Cabrera, M.
|e author
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|a Sun, X.
|e author
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|a Wheeldon, I.
|e author
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|t Biotechnology for Biofuels
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