SCRaMbLE generates evolved yeasts with increased alkali tolerance

Abstract

Background: Strains 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.

Results: Haploid 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.

Conclusions: SCRaMbLE 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.

Keywords: Alkali tolerance; SCRaMbLE; SPT2; Saccharomyces cerevisiae; Synthetic biology.

Fig. 1

The workflow of using SCRaMbLE to improve and analyze alkali tolerance in yeast. Three sections in varied colors indicated three sequential steps of the whole process including construction of inducible SCRaMbLE system, screening for specific phenotype, correlation analysis of phenotype and genotype

Fig. 2

Characterization of SCRaMbLEd strains with increased alkali tolerance. a Characterization of lethality for SCRaMbLE system. Four strains (synV strain carrying pYW079, synV carrying pYW180, synV&X carrying pYW079 and synV&X carrying pYW079) were cultured in SC-Ura medium and exposed in β-estradiol for varied time (2 h, 4 h, 6 h, 8 h, 12 h, 16 h), and then diluted on YPD plates. SynV and synV&X strains containing empty vector pRS416 were used as control strains. b SCRaMbLEd synV&X strains on YPD medium and selective medium (YPD medium at pH 8.0). Strains on YPD medium were cultured 2 days and strains on selective medium were cultured 4 days in 30 °C before photographed. SynV&X strain containing empty vector pRS416 was used as a control strain. c Phenotype verification of SCRaMbLEd strains. SynV and synV&X strains were used as control strains. d PCRTag analysis. PCRTags of genes YER042W, YER161C, YER163C and YER182W were labeled in red, indicating deletions of these regions in strain yML008. None amplification of PCRTags of YEL017W, YER010C, YER081W, YER118C in both synV and yML008 strains was caused by nonspecific primers. SynV strain was used as a control strain. All PCRTag primers were listed in Additional file 1: Table S1

Fig. 3

Analysis of structural variations in SCRaMbLEd strains. a Structural variations in five of the SCRaMbLEd genomes detected by whole genome sequencing. b Statistical analysis of structural variations in SCRaMbLEd genomes. c Detailed information of 14 varied rearrangement events. Two complex structural variations in section <6 and 7> and <12 and 13> were indicated with thick borders

Fig. 4

Deletion of YER161C (SPT2) increases the alkali tolerance in yeast. a Construction of seamless knockout yeast strains. With two rounds of genome integration, transformants were selected on SC-Ura and 5-FOA medium, respectively. b Verification of targeted deletions by junction PCR. All junction primers were listed in Additional file 1: Table S1. c Verification of individual gene deletions. All primers of individual genes were listed in Additional file 1: Table S1. d Characterization of the knockout yeast strains on YPD medium at pH 8.0. Strains on selective medium were cultured 4 days in 30 °C before photographed. e Growth curves of yML094 and BY4741 at pH 8.3. f Characterization of SPT2 knockout strains under various stressful conditions. g Growth fitness of SCRaMbLEd strains and SPT2 knockout strains at pH 8.0 using NaOH as alkali source. h pH changes of yML094 and BY4741 in liquid YPD medium at pH 8.3