Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness

Genome and Transcriptome Analysis of the Food-Yeast Candida utilis

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis

New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for <i>Saccharomyces cerevisiae</i> Regulated by 2,4-Diacetylphloroglucinol and Other Ligands

Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of <i>Escherichia coli</i> TetR, including the transcriptional regulator PhlF from <i>Pseudomonas</i> and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; <i>i.e.</i>, DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the <i>p</i>-cumate switch based on CymR from <i>Pseudomonas</i>, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (<i>p</i>-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of “On” and “Off” switches for yeast biotechnology

Transcription results of 2 putative hexose transporters of <i>C. utilis</i>.

<p>Transcription results of 2 putative hexose transporters of <i>C. utilis</i>.</p

Phylogenetic tree of nitrate assimilation related genes.

<p>This tree was built on concatenated homologous sequences of all three genes (nitrate/nitrite transporter, nitrate reductases, and nitrite reductase) in Ascomycota and Basidiomycota from the subkingdom Dikarya (Materials and Methods) by the ML method.</p

<i>C. utilis</i> ADH and ALD genes, and transcription results.

*<p>Protein localization site was predicted by WoLF PSORT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037226#pone.0037226-Horton1" target="_blank">[34]</a>.</p><p>Cyto.: Cytoplasm, Mito.: Mitochondrion.</p

Antisense transcripts of <i>C. utilis ADH1</i> (cut01g0000110) differentially expressed between log- and stationary-phases.

<p>Expressions at the <i>ADH1</i> locus was visualized using the Genaris integrated Next-Generation Sequencing Data Analysis Platform (GiNeS) (Genaris, Inc., Kanagawa, Japan).</p

Genome sequencing overview.

<p>Genome sequencing overview.</p

Yeast Golden Gate (yGG) for the Efficient Assembly of <i>S. cerevisiae</i> Transcription Units

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae. (Figure Presented)