Abstract
BackgroundThe thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology.ResultsIn this study, we developed a CRISPR–Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette PScTEF1-TAL-PScTPI1-4CL-PScTEF2-STS were integrated into the genome. Resveratrol production was increased ~ 20 fold compared to the one copy integrant and reached 97.23 ± 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae.ConclusionsAn efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species.
Highlights
The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications
Scheme for CRISPR–Cas9‐assisted genome editing in O. polymorpha Due to unavailability of a stable episomal vector in O. polymorpha, the Cas9 and gRNA expression cassettes were integrated into the chromosome using recombinant plasmids pWYE3208 and pWYEN
The plasmid pWYE3208 harboring the PScTEF1-controlled cas9 gene and the up- and downstream homologous arms (UHA and DHA) of the OpMET2 gene was linearized with SpeI and transformed into O. polymorpha
Summary
The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. One attractive property of O. polymorpha is able to integrate up to 100 copies of target gene into the genome mediated by non-homologous end joining (NHEJ), which can be used to highly express heterologous genes and synthesize various biotechnology products [1, 4, 7, 8]. It can synthesize glycoproteins with human compatible oligosaccharides [9, 10]. Several prophylactic HBV vaccines have been marketed [1, 4, 7, 8]
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