Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts.

BACKGROUND

The thermotolerant methylotrophic yeast 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.

RESULTS

In 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 . Using the CMGE-ML integration method, three genes from , from and from of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in . Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette -- -- - 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 using CMGE-MC to integrate genes and , respectively. In addition, the CMGE-MC method was successfully developed in .

CONCLUSIONS

An 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 and other yeast species.