Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol biosynthesis. Their extensive use in treatment and prevention of cardiovascular diseases placed statins among the best selling drugs. Construction of
Saccharomyces cerevisiae
cell factory for the production of high concentrations of natural statins will require establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. To establish active export of statins from yeast, and thereby detoxification, we integrated a putative efflux pump-encoding gene
mlcE
from the mevastatin-producing
Penicillium citrinum
into the
S. cerevisiae
genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin) and semi-synthetic statin (simvastatin) when compared to the wild type strain. Expression of RFP-tagged
mlcE
showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins.
Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.
Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and the identification of phenotypic maximas would be of great benefit to synthetic biology. Thus, new research tools allowing simple and rapid mapping of sequence to function are required to forward these research fields. Here, we describe CRI-SPA, a method allowing the transfer of a chromosomal genetic feature from a donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion and Selective Ploidy Ablation and is executed within a week. We demonstrate the power of CRI-SPA by transferring four genes responsible for the production of betaxanthin, a yellow biosensor for the morphine precursor L-DOPA, into each strain of the yeast knock-out collection (≈4800 strains), providing a genome-wide overview of the genetic requirements for betaxanthin production. CRI-SPA is fast, highly reproducible, can be massively parallelized with automation and does not require selection for the transferred genetic feature.
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