Genome sequencing of 15 acid-tolerant yeasts.

We report draft genome sequences for 15 non-conventional Saccharomycotina yeast strains obtained from public culture repositories. Included in our collection are eight strains of Pichia with broad tolerance to dicarboxylic acids. The genome sequences of these strains will enable comparative genomics of acid-tolerant phenotypes and strain engineering of non-conventional hosts.

Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid.

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids.

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L-1, a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

An Engineered Aro1 Protein Degradation Approach for Increased cis,cis-Muconic Acid Biosynthesis in Saccharomyces cerevisiae.

Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the Saccharomyces cerevisiae yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in an aro3Δ aro4Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar.IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.

Assessment of bioavailable B vitamin content in food using in vitro digestibility assay and LC-MS SIDA.

Standardized analytical methods, where each B vitamin is extracted from a given sample individually using separate procedures, typically ensure that the extraction conditions provide the maximum recovery of each vitamin. However, in the human gastrointestinal tract (GIT), the extraction conditions are the same for all vitamins. Here, we present an analytically feasible extraction protocol that simulates conditions in the GIT and provides a measure of the content of bioavailable vitamins using LC-MS stable isotope dilution assay. The results show that the activities of both human gastric and duodenal juices were insufficient to liberate absorbable vitamers (AV) from pure cofactors. The use of an intestinal brush border membrane (IBBM) fraction derived from the mucosal tissue of porcine small intestine ensured at least 70% AV recovery. The rate of AV liberation, however, was strongly dependent on the cofactor, e.g., in the case of NADH, it was magnitudes higher than in the case of thiamine diphosphate. For some vitamins in some food matrices, the use of the IBBM fraction assay resulted in lower values for the content of AV than conventional vitamin determination methods. Conventional methods likely overestimate the actual bioavailability of some vitamins in these cases. Graphical abstract Assessment of bioavailable B vitamin content in food.

Utilization of (15)N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids.

Lactococcus lactis subsp. lactis IL1403 was grown in medium containing unlabelled free amino acids and (15)N-labelled yeast hydrolysate to gain insight into the role of peptides as a source of amino acids under conditions where free amino acids are abundant. A mathematical model was composed to estimate the fluxes of free and peptide-derived amino acids into and out of the intracellular amino acid pool. We observed co-consumption of peptides and free amino acids and a considerable efflux of most free amino acids during growth. We did not observe significant differences between the peptide consumption patterns of essential and non-essential amino acids, which suggests that the incorporation of a particular amino acid is more dependent on its availability in a readily assimilated form than the organism's auxotrophy for it. For most amino acids the contribution of peptide-bound forms to the formation of biomass was initially between 30 and 60 % with the remainder originating from free amino acids. During the later stages of fermentation we observed a decrease in the utilization of peptide-bound amino acids, thus indicating that the more readily assimilated peptides are gradually exhausted from the medium during growth.

YAP1 over-expression in Saccharomyces cerevisiae enhances glutathione accumulation at its biosynthesis and substrate availability levels.

Microbiological production of glutathione using genetically engineered yeast strains has a potential to satisfy the increasing industrial demand of this tripeptide. In the present work accumulation of glutathione in response to YAP1 over-expression in Saccharomyces cerevisiae was studied. The over-expression resulted in intracellular glutathione level over two times higher than in the parent strain. Transcript analyses revealed that, in addition to the genes encoding enzymes in the glutathione biosynthesis pathway (GSH1 and GSH2), the expression levels of the genes in the cysteine biosynthesis pathway (CYS3 and CYS4) were also significantly higher in the YAP1 over-expressed strain. This suggests that YAP1 over-expression affects glutathione accumulation at both its biosynthesis and substrate availability levels.

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants.

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.

Glutathione accumulation in ethanol-stat fed-batch culture of Saccharomyces cerevisiae with a switch to cysteine feeding.

Shot-wise supplementation of cysteine to a yeast culture is a common means of promoting glutathione (GSH) production. In the present work, we study the accumulation kinetics of cysteine, gamma-glutamylcysteine, and GSH and the expression of genes involved in GSH and sulfur metabolism in ethanol-stat fed-batch cultures as a result of switching to a medium enriched with cysteine and glycine. Supplementation in this fashion resulted in a rapid but short-term increase in the rate of GSH synthesis, while the expression of GSH1 decreased. Expression of GSH1 and GSH synthesis rate were observed to revert close to the base level after a few hours. These results indicate that, under such conditions, the control of GSH synthesis at higher concentrations occurred at the enzymatic, rather than the transcriptional level. The incorporation of cysteine into GSH was limited to approximately 40% of the theoretical yield, due to its requirement as a source of sulfur for protein synthesis under conditions whereby the sulfate assimilation pathway is down-regulated. This was supported by the expression profiles of genes involved in cysteine and homocysteine interconversion.

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in environmental stress monitored by expression of the stress response protein Hsp12p.

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in different environmental stress conditions during both respiratory growth and aerobic fermentative growth in the presence of excess glucose was investigated by monitoring the level and rate of expression of the stress response protein Hsp12p using the fluorescent fusion construct Hsp12p-Gfp2p. The initial expression level and the rate of Hsp12p synthesis was significantly greater under glucose-limited conditions in the chemostat (D<0.14 h(-1)) compared with when excess glucose was present in the auxostat. Decreasing the dilution rate and the glucose concentration further in the A-stat resulted in increased Hsp12p expression, which was more marked when a rapid rather than a gradual change was affected. Common stress factors such as NaCl, ethanol and elevated temperature caused stress responses in both D-stat and auxo-accelerostat culture. The magnitude of the stress response depended on the stress factor, cultivation conditions as well as the rate of change of the stress factor. The rate of Hsp12p synthesis increased due to all applied stresses, with the observed increase between 2 and 20 times lower when the stress was applied gradually rather than rapidly. The results suggested that the Hsp12p expression rate is a good indicator of applied stress in S. cerevisiae.