TY - JOUR
T1 - Variation among metschnikowia pulcherrima isolates for genetic modification and homologous recombination
AU - Moreno-Beltrán, Mauro
AU - Gore-Lloyd, Deborah
AU - Chuck, Christopher
AU - Henk, Daniel
N1 - Funding Information:
Funding: This research was funded by the Industrial Biotechnology Catalyst (http://dx.doi.org/10.13039/501100006041, BBSRC and EPSRC to support the translation, development and commercialisation of innovative Industrial Biotechnology processes (EP/N013522/1) and via a URF studentship grant to M.M-B from the University of Bath.
Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/1/31
Y1 - 2021/1/31
N2 - Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in bi-otechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homolo-gous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 lo-cus, suggesting that the genome sequence or structure alters the efficacy of this system.
AB - Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in bi-otechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homolo-gous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 lo-cus, suggesting that the genome sequence or structure alters the efficacy of this system.
KW - Biotechnology
KW - Homologous recombination
KW - Nonconventional yeasts
UR - http://www.scopus.com/inward/record.url?scp=85100018921&partnerID=8YFLogxK
U2 - 10.3390/microorganisms9020290
DO - 10.3390/microorganisms9020290
M3 - Article
AN - SCOPUS:85100018921
VL - 9
SP - 1
EP - 11
JO - Microorganisms
JF - Microorganisms
SN - 2076-2607
IS - 2
M1 - 290
ER -