Precise control of SCRaMbLE in synthetic haploid and diploid yeast

Michael Shen,Juan Wang,Ying-Jin Yuan,Duo Liu,Leslie A Mitchell,Yi Wu,Hao Qi,Hao-Ran Zhang,Ying-Xiu Cao,Bin Jia,Bing-Zhi Li,Xiao Zhou,Hong Liu,Xia Li,Shuo Pan,Bo Li,Jef D Boeke,Ze-Xiong Xie,Nan Jia

doi:10.1038/s41467-018-03084-4

Abstract

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

Highlights

The strategy of engineering heterologous pathways within host microorganisms has been used to improve the production of high-added-value biomolecules such as pharmaceuticals[1] and biofuels[2,3]
This increase may be because the Gal[1] promoter was highly induced with galactose and produced large amounts of Cre-estrogen-binding domain (EBD), small amounts of which escaped Hsp[90] binding and entered the nucleus. When both galactose and estradiol were added to the media, the doubling time (DT) of the synV yeast was increased to 10 h, 1.5-fold higher than that of the control
High stability and reliability enable the iterative SCRaMbLEing of synthetic chromosomes without removing the Cre plasmid

Summary

Introduction

The strategy of engineering heterologous pathways within host microorganisms has been used to improve the production of high-added-value biomolecules such as pharmaceuticals[1] and biofuels[2,3]. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity that can be followed by screening for advantageous organisms. The synthetic yeast chromosomes synII, synIII, synV, synVI, synIXR, synX, and synXII have been fully synthesized and incorporated into Saccharomyces cerevisiae without major fitness defects[4,5,7,8,9,10,11,12,13], offering an opportunity to generate tremendously diverse host yeast strains that can be screened for the production of high-added-value biomolecules[14]. We construct a galactose-driven Cre-EBD as an AND gate in a genetic switch for the precise control of SCRaMbLEing synthetic haploid and diploid yeast. We analyze SCRaMbLEd derivatives with improved carotenoid production by high-throughput sequencing and long-read sequencing, and we figure out the high-production phenotypes to the new genotypes

Methods

Results

Conclusion