Enzymatic glycosylation of aloesone performed by plant UDP-dependent glycosyltransferases.

Aloesone is a bioactive natural product and biosynthetic precursor of rare glucosides found in rhubarb and some aloe plants including Aloe vera. This study aimed to investigate biocatalytic aloesone glycosylation and more than 400 uridine diphosphate-dependent glycosyltransferase (UGT) candidates, including multifunctional and promiscuous enzymes from a variety of plant species were assayed. As a result, 137 selective aloesone UGTs were discovered, including four from the natural producer rhubarb. Rhubarb UGT72B49 was further studied and its catalytic constants (kcat = 0.00092 ± 0.00003 s-1, KM = 30 ± 2.5 µM) as well as temperature and pH optima (50 °C and pH 7, respectively) were determined. We further aimed to find an efficient aloesone glycosylating enzyme with potential application for biocatalytic production of the glucoside. We discovered UGT71C1 from Arabidopsis thaliana as an efficient aloesone UGT showing a 167-fold higher catalytic efficiency compared to that of UGT72B49. Interestingly, sequence analysis of all the 137 newly identified aloesone UGTs showed that they belong to different phylogenetic groups, with the highest representation in groups B, D, E, F and L. Finally, our study indicates that aloesone C-glycosylation is highly specific and rare, since it was not possible to achieve in an efficient manner with any of the 422 UGTs assayed, including multifunctional GTs and 28 known C-UGTs.

Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed been used by humans since the previous centuries for biotransformations. Among them, yeasts combine the advantage of a unicellular state with a eukaryotic organization. Moreover, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycete budding yeast, is widely known for its peculiar tolerance to different stresses, among which are organic acids. Moreover, the recent reclassification of the species, including diverse hybrids, is further expanding both fundamental and applied interests. It is therefore reasonable that despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here we describe in detail the methods for determining chromosome number, size, and aneuploidy, transformation, classical target gene disruption or gene integration, and designing of episomal expression plasmids helpful for engineering the yeast Z. bailii .

Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate.

BACKGROUND: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars-in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. RESULTS: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. CONCLUSIONS: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.

Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations.

BACKGROUND: The selection of bioengineering platform strains and engineering strategies to improve the stress resistance of Saccharomyces cerevisiae remains a pressing need in bio-based chemical production. Thus, a systematic effort to exploit genotypic and phenotypic diversity to boost yeast's industrial value is still urgently needed. RESULTS: We analyzed 5,400 growth curves obtained from 36 S. cerevisiae strains and comprehensively profiled their resistances against 13 industrially relevant stresses. We observed that bioethanol and brewing strains exhibit higher resistance against acidic conditions; however, plant isolates tend to have a wider range of resistance, which may be associated with their metabolome and fluxome signatures in the tricarboxylic acid cycle and fatty acid metabolism. By deep genomic sequencing, we found that industrial strains have more genomic duplications especially affecting transcription factors, showing that they result from disparate evolutionary paths in comparison with the environmental strains, which have more indels, gene deletions, and strain-specific genes. Genome-wide association studies coupled with protein-protein interaction networks uncovered novel genetic determinants of stress resistances. CONCLUSIONS: These resistance-related engineering targets and strain rankings provide a valuable source for engineering significantly improved industrial platform strains.

Changes in SAM2 expression affect lactic acid tolerance and lactic acid production in Saccharomyces cerevisiae.

BACKGROUND: The great interest in the production of highly pure lactic acid enantiomers comes from the application of polylactic acid (PLA) for the production of biodegradable plastics. Yeasts can be considered as alternative cell factories to lactic acid bacteria for lactic acid production, despite not being natural producers, since they can better tolerate acidic environments. We have previously described metabolically engineered Saccharomyces cerevisiae strains producing high amounts of L-lactic acid (>60 g/L) at low pH. The high product concentration represents the major limiting step of the process, mainly because of its toxic effects. Therefore, our goal was the identification of novel targets for strain improvement possibly involved in the yeast response to lactic acid stress. RESULTS: The enzyme S-adenosylmethionine (SAM) synthetase catalyses the only known reaction leading to the biosynthesis of SAM, an important cellular cofactor. SAM is involved in phospholipid biosynthesis and hence in membrane remodelling during acid stress. Since only the enzyme isoform 2 seems to be responsive to membrane related signals (e.g. myo-inositol), Sam2p was tagged with GFP to analyse its abundance and cellular localization under different stress conditions. Western blot analyses showed that lactic acid exposure correlates with an increase in protein levels. The SAM2 gene was then overexpressed and deleted in laboratory strains. Remarkably, in the BY4741 strain its deletion conferred higher resistance to lactic acid, while its overexpression was detrimental. Therefore, SAM2 was deleted in a strain previously engineered and evolved for industrial lactic acid production and tolerance, resulting in higher production. CONCLUSIONS: Here we demonstrated that the modulation of SAM2 can have different outcomes, from clear effects to no significant phenotypic responses, upon lactic acid stress in different genetic backgrounds, and that at least in one genetic background SAM2 deletion led to an industrially relevant increase in lactic acid production. Further work is needed to elucidate the molecular basis of these observations, which underline once more that strain robustness relies on complex cellular mechanisms, involving regulatory genes and proteins. Our data confirm cofactor engineering as an important tool for cell factory improvement.

Molecular tools and protocols for engineering the acid-tolerant yeast Zygosaccharomyces bailii as a potential cell factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

Recombinant protein production in yeasts.

Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins.

The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

BACKGROUND: Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. RESULTS: The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. CONCLUSIONS: The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant.

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.

Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype.

BACKGROUND: Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a beta-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. RESULTS: The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Deltagas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of approximately 400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Deltagas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1beta secretion disruption had no relevant effect. CONCLUSIONS: The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Investigating the multibudded and binucleate phenotype of the yeast Zygosaccharomyces bailii growing on minimal medium.

The yeast Zygosaccharomyces bailii, known to have peculiar resistance to several environmental constraints, is very little known with respect to its genetics and life cycle. In addition to molecular and biochemical studies, cytofluorimetric and morphological analyses can also add information necessary to shed light on its interesting features. In the present study, the DNA and protein content as well as the cellular morphology of Z. bailii populations growing in minimal medium supplemented with different carbon sources and with the addition of different organic acids were investigated. The results show the occurrence of a multibudded phenotype and of a low, but significant percentage of binucleate cells occurring in the early-stationary phase. These traits appear to be different in comparison with the better-known laboratory yeast Saccharomyces cerevisiae. Experiments and speculations about these features and possible implications with Z. bailii main characteristics are discussed.