SCRaMbLE generates evolved yeasts with increased alkali tolerance

Lu Ma,Yi Wu,Yunxiang Li,Ying-Jin Yuan,Xinyu Chen,Mingzhu Ding

doi:10.1186/s12934-019-1102-4

Abstract

BackgroundStrains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes.ResultsHaploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae.ConclusionsSCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.

Highlights

Strains with increased alkali tolerance have a broad application in industrial, especially for bioremedia‐ tion, biodegradation, biocontrol and production of bio-based chemicals
We further validated that the deletion of YER161C (SPT2) in this region could lead to the enhanced phenotype under alkaline stress
Cre recombinase fused with estrogen-binding domain (EBD) has been used to control the SCRaMbLE system in previous studies [19, 22]

Summary

Introduction

Strains with increased alkali tolerance have a broad application in industrial, especially for bioremedia‐ tion, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombina‐ tion and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. Ma et al Microb Cell Fact (2019) 18:52 recombinase in vivo, the synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system could drive generation of a yeast library with massive structural variations. The transcriptional response to alkaline stress in S. cerevisiae was studied by a short-term exposure in alkaline pH [33] These methods are non-rational designed and make it complicated to uncover phenotype-genotype correlations. The SCRaMbLE method provides an efficient way to generate evolved strains with increased alkali tolerance and a straightforward way to dissect the correlation of phenotype and genotype (Fig. 1)

Methods

Results

Conclusion