Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica.

In-Seung Jang,Ji Yeon Jang,Jonggeon Jegal,Byung Jo Yu,Ju Young Lee,Alvaro Galli

doi:10.1371/journal.pone.0194954

In-Seung Jang, Ji Yeon Jang + Show 4 more

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https://doi.org/10.1371/journal.pone.0194954

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Abstract

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the β-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.

Highlights

The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive host with multiple biotechnological and industrial applications for production of various chemicals and heterologous proteins [1,2,3]
The Yeast Extract Peptone Dextrose (YPD) medium was prepared with 20 g/L bacto peptone (Difco Laboratories), 10 g/L yeast extract (Difco), 20 g/L glucose (Sigma-Aldrich), the Yeast Nitrogen Base (YNB) medium was made with 6.7 g/L yeast nitrogen base (Difco), 0.69 g/L complete amino acid supplement mixture (CSM)-uracil supplement (MP Biomedicals), and 20 g/L glucose
We designed a URA3-blaster cassette for an efficient polymerase chain reaction (PCR)-based gene targeting for the Y. lipolytica strain, using short primers (70 bp) to flank both sides of the URA3 marker gene that were 50 bp homology arm to the target gene (Fig 1) that is based on the S. cerevisiae URA3-blaster system [40, 44]

Summary

Introduction

The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive host with multiple biotechnological and industrial applications for production of various chemicals and heterologous proteins (i.e. proteases, lipases and RNase) [1,2,3]. The availability of the genome sequence of Y. lipolytica [12, 13] and development of genetic tools such as transformation methods [14,15,16] for gene deletion [17] and integration [18,19,20,21,22,23] enable the redesign of a synthetic metabolic pathway [24].

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