Emergence of Phenotypically Distinct Subpopulations Is a Factor in Adaptation of Recombinant Saccharomyces cerevisiae under Glucose-Limited Conditions.

Cells cultured in a nutrient-limited environment can undergo adaptation, which confers improved fitness under long-term energy limitation. We have shown previously how a recombinant Saccharomyces cerevisiae strain, producing a heterologous insulin product, under glucose-limited conditions adapts over time at the average population level. Here, we investigated this adaptation at the single-cell level by application of fluorescence-activated cell sorting (FACS) and showed that the following three apparent phenotypes underlie the adaptive response observed at the bulk level: (i) cells that drastically reduced insulin production (23%), (ii) cells with reduced enzymatic capacity in central carbon metabolism (46%), and (iii) cells that exhibited pseudohyphal growth (31%). We speculate that the phenotypic heterogeneity is a result of different mechanisms to increase fitness. Cells with reduced insulin productivity have increased fitness by reducing the burden of the heterologous insulin production, and the populations with reduced enzymatic capacity of the central carbon metabolism and pseudohyphal growth have increased fitness toward the glucose-limited conditions. The results highlight the importance of considering population heterogeneity when studying adaptation and evolution. IMPORTANCE The yeast Saccharomyces cerevisiae is an attractive microbial host for industrial production and is used widely for manufacturing, e.g., pharmaceuticals. Chemostat cultivation mode is an efficient cultivation strategy for industrial production processes as it ensures a constant, well-controlled cultivation environment. Nevertheless, both the production of a heterologous product and the constant cultivation environment in the chemostat impose a selective pressure on the production organism, which may result in adaptation and loss of productivity. The exact mechanisms behind the observed adaptation and loss of performance are often unidentified. We used a recombinant S. cerevisiae strain producing heterologous insulin and investigated the adaptation occurring during chemostat growth at the single-cell level. We showed that three apparent phenotypes underlie the adaptive response observed at the bulk level in the chemostat. These findings highlight the importance of considering population heterogeneity when studying adaptation in industrial bioprocesses.

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.

The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism.

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree-positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid [3HP]) into an evolved PDC-negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (µmax ) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux ( > 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid-producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering.

Improving Reproducibility in Synthetic Biology.

Synthetic biology holds great promise to deliver transformative technologies to the world in the coming years. However, several challenges still remain to be addressed before it can deliver on its promises. One of the most important issues to address is the lack of reproducibility within research of the life sciences. This problem is beginning to be recognised by the community and solutions are being developed to tackle the problem. The recent emergence of automated facilities that are open for use by researchers (such as biofoundries and cloud labs) may be one of the ways that synthetic biologists can improve the quality and reproducibility of their work. In this perspective article, we outline these and some of the other technologies that are currently being developed which we believe may help to transform how synthetic biologists approach their research activities.

EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.