Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species

Dai, Ziyu; Deng, Shuang; Culley, David E.; Bruno, Kenneth S.; Magnuson, Jon K.

doi:10.1007/s00253-017-8357-7

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species

Journal Article · Mon Jun 19 04:00:00 EDT 2017 · Applied Microbiology and Biotechnology

DOI:https://doi.org/10.1007/s00253-017-8357-7· OSTI ID:1398168

Dai, Ziyu; Deng, Shuang; Culley, David E.; Bruno, Kenneth S.; Magnuson, Jon K.

Background: Because of interest in the production of renewable bio-hydrocarbon fuels, various living organisms have been explored for their potential use in producing fuels and chemicals. The oil-producing (oleaginous) yeast Lipomyces starkeyi is the subject of active research regarding the production of lipids using a wide variety of carbon and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements using the tools of synthetic biology and metabolic engineering. However, using these tools for strain improvement requires the establishment of effective and reliable transformation methods with suitable selectable markers (antibiotic resistance or auxotrophic marker genes) and the necessary genetic elements (promoters and terminators) for expression of introduced genes. Chemical-based methods have been published, but suffer from low efficiency or the requirement for targeting to rRNA loci. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. Results: In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species and that the introduced DNA can be reliably integrated into the chromosomes of these species. The gene deletion of Ku70 and Pex10 was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial -glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1 promoter was also stably expressed in seven different Lipomyces species. Conclusion: The results from this study clearly demonstrate that Agrobacterium-mediated transformation is a reliable genetic tool for gene deletion and integration and expression of heterologous genes in L. starkeyi and other Lipomyces species.

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC05-76RL01830

OSTI ID:: 1398168

Report Number(s):: PNNL-SA-123451; BM0101010

Journal Information:: Applied Microbiology and Biotechnology, Journal Name: Applied Microbiology and Biotechnology Journal Issue: 15 Vol. 101; ISSN 0175-7598

Publisher:: Springer

Country of Publication:: United States

Language:: English

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