Development of advanced genome-editing tools for basidiomycetous yeasts and engineering the biosynthetic pathway of mannosylerythritol lipids in Moesziomyces antarcticus
Author
Bobkov, Daniel
Term
4. term
Education
Publication year
2019
Submitted on
2019-06-10
Pages
61
Abstract
Efterspørgslen efter grønnere måder at fremstille kemikalier på har øget interessen for at bruge mikroorganismer til at producere biosurfaktanter, altså overfladeaktive stoffer. Mannosylerythritol-lipider (MEL) er særligt lovende, fordi de kan fremstilles med højt udbytte under milde betingelser og har mange anvendelser. I dette projekt blev den ikke-konventionelle gær Moesziomyces antarcticus anvendt som værtsorganisme til genetiske manipulationer og produktion af MEL. Gæren udskiller hovedsageligt MEL-A, MEL-B og MEL-C, mens den ikke-acetylerede MEL-D kun dannes i meget små mængder. Målet var at opnå en mere ensartet produktion af MEL-D ved at slå MAT1-genet ud. MAT1 koder for en acetyltransferase, som normalt acetylere MEL-D til de andre varianter. For at forstyrre MAT1 blev flere gen-ingeniørmetoder afprøvet: homolog rekombination med et PCR-forstærket DNA-fragment flankeret af sekvenser op- og nedstrøms for MAT1 og med en NatMX4-selektionskassette; en split-marker-tilgang med to afkortede, overlappende fragmenter; samt RNP-medieret genmålretning kombineret med tilførsel af ekstra DNA (dDNA) for at øge homolog rekombination. To transformationsmetoder blev sammenlignet: elektroporation (elektriske pulser åbner midlertidige porer i cellerne) og en kemisk PEG/LiAc/ssDNA-protokol. Derudover blev præbehandling af celler før elektroporation undersøgt. Split-marker-tilgangen med 70 bp overlap gav cirka dobbelt så høj indsætningsfrekvens af NatMX4-kassetten i kromosomet som brug af et enkelt PCR-fragment. En længere overlap på 231 bp gav en mindre yderligere forbedring. RNP-medierede forsøg (100× fortynding af RNP) gav enkelte mutanter, men ingen med målrettet disruption af MAT1; i stedet skete der tilfældige integrationer andre steder i genomet. Præbehandling af celler før elektroporation tredoblede transformationseffektiviteten. Overordnet set var elektroporation 26% mere effektiv end PEG/LiAc/ssDNA-metoden og blev derfor brugt i de efterfølgende RNP-forsøg. Selvom det ikke lykkedes at slå MAT1 ud og dermed opnå en homogen MEL-D-produktion, kortlægger studiet transformationsbetingelser, der forbedrer genredigering i M. antarcticus. Det er et vigtigt skridt mod mere målrettet og kontrolleret biosurfaktantproduktion.
Growing demand for greener chemical manufacturing has increased interest in using microorganisms to make biosurfactants, which are surface-active compounds. Mannosylerythritol lipids (MELs) are especially promising because they can be produced at high yield under mild conditions and have many applications. In this project, the non-conventional yeast Moesziomyces antarcticus was used as a host for genetic manipulation and MEL production. This yeast mainly secretes MEL-A, MEL-B, and MEL-C, while the non-acetylated MEL-D is made only in very small amounts. The goal was to obtain more uniform production of MEL-D by knocking out the MAT1 gene. MAT1 encodes an acetyltransferase that normally acetylates MEL-D into the other variants. Several gene engineering strategies were tested to disrupt MAT1: homologous recombination using a PCR-amplified DNA fragment flanked by sequences upstream and downstream of MAT1 and carrying a NatMX4 selection cassette; a split-marker approach using two truncated, overlapping fragments; and RNP-mediated gene targeting supplemented with additional DNA (dDNA) to increase homologous recombination. Two transformation methods were compared: electroporation (electric pulses briefly open pores in cell membranes) and a chemical PEG/LiAc/ssDNA protocol. Cell pretreatment before electroporation was also evaluated. The split-marker approach with a 70 bp overlap yielded roughly twice the chromosomal insertion efficiency of the NatMX4 cassette compared with the single PCR fragment. Extending the overlap to 231 bp gave a modest further improvement. RNP-mediated attempts (with a 100× dilution of RNP) produced a few mutants, but none with a targeted disruption of MAT1; instead, insertions occurred randomly elsewhere in the genome. Pretreating cells before electroporation increased transformation efficiency about threefold. Overall, electroporation was 26% more efficient than the PEG/LiAc/ssDNA method and was therefore used in subsequent RNP experiments. Although the MAT1 knockout was not achieved and homogeneous MEL-D production was not obtained, the study maps transformation conditions that improve gene editing in M. antarcticus. This is an important step toward more targeted and controlled biosurfactant production.
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