Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts

TLDR

Ogataea polymorpha is a thermotolerant methylotrophic yeast widely used for basic research and industrial production of chemicals, biofuels, and other bioproducts. This study aimed to develop a CRISPR‑Cas9‑assisted multiplex genome editing (CMGE) system for efficient genetic manipulation of yeasts. CMGE combines multiplex gene knock‑outs, multi‑locus (ML) and multi‑copy (MC) integration methods to perform deletions, insertions, and precise point mutations in O. polymorpha.

Abstract

The 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.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 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.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 O. polymorpha and other yeast species.