Metabolic Engineering of Moesziomyces antarcticus - Disruption of Mat1 by application of the CRISPR-Cas9 system and conventional gene knock-out methods
Author
Nielsen, Cathrine Westergaard
Term
4. term
Education
Publication year
2019
Abstract
Mannosylerythritol lipids (MELs) are biosurfactants valued for high yields, diverse applications, and self-assembling properties, and the basidiomycetous yeast Moesziomyces antarcticus can produce up to 40 g/L, especially on vegetable oil. M. antarcticus secretes four MEL types (A–D), and this thesis aimed to obtain a homogeneous product of the deacetylated MEL-D by disrupting the acetyltransferase gene Mat1. The CRISPR-Cas9 system was explored for gene disruption and yielded several transformants, but Mat1 was not successfully targeted by Cas9 and the gene remained intact. Three alternative homologous recombination strategies were then tested: a plasmid-borne disruption cassette carrying a nourseothricin resistance marker flanked by Mat1 homology arms, delivery of the excised cassette, and a split-marker approach, all introduced by electroporation. To clarify fatty acyl handling in MEL biosynthesis, an in silico analysis of a potential partial β-oxidation chain-shortening pathway in M. antarcticus was conducted by comparison to peroxisomal β-oxidation in Homo sapiens and Saccharomyces cerevisiae; several putative genes were identified based on protein homology. Overall, targeted Mat1 disruption was not achieved within the scope of this work, but the study establishes transformation approaches and candidate metabolic genes that inform future efforts to tailor MEL composition in M. antarcticus.
Mannosylerythritol-lipider (MEL’er) er biosurfaktanter med høj udbytte, mange anvendelser og selvorganiserende egenskaber, og den basidiomycete gær Moesziomyces antarcticus kan producere op til 40 g/L, især ved dyrkning på vegetabilsk olie. M. antarcticus udskiller fire MEL-typer (A–D), og i denne afhandling var målet at skabe et homogent produkt af den deacetylerede MEL-D ved at slå acetyltransferase-genet Mat1 ud. CRISPR-Cas9-systemet blev afprøvet til genforstyrrelse og gav flere transformanter, men Mat1 blev ikke succesfuldt målrettet af Cas9, og genet blev derfor ikke forstyrret. Tre alternative tilgange baseret på homolog rekombination blev dernæst testet: et plasmid med en disruptionskassette med nourseothricin-resistens flankeret af Mat1-homologe regioner, levering af den udskårne kassette samt en split-marker-tilgang, alle introduceret ved elektroporation. For at belyse acylering og håndtering af fedtsyrekæder i MEL-biosyntesen blev der udført en in silico-analyse af en potentiel delvis β-oxidationsbaseret kædeforkortningsvej i M. antarcticus ved sammenligning med peroxisomal β-oxidation i Homo sapiens og Saccharomyces cerevisiae; flere potentielle gener blev identificeret på basis af proteinhomologi. Samlet set blev den målrettede disruption af Mat1 ikke opnået inden for rammerne af dette arbejde, men studiet leverer transformationstilgange og kandidatgener, der kan støtte fremtidig metabolisk ingeniørkunst til at skræddersy MEL-sammensætningen i M. antarcticus.
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