Industrial Yeast Research Articles

Functional diversity and plasticity in the sugar preferences of Saccharomyces MALT transporters in domesticated yeasts.

Maltose and maltotriose, together with glucose, are the major carbohydrates found in malts. Thus, brewing yeasts grown in malt-based brewing processes with serial re-pitching have likely increased their ability to uptake these sugars during domestication by modulating the expression and copy number of maltose transporter genes (MALT, also known as Malx1). However, the molecular basis for and structural insights into the sugar preferences of MALT proteins remain to be elucidated. Here we report the functional evaluation of two novel Saccharomyces cerevisiae MALT proteins, ScMalt#2p and ScMalt#5p, from industrial brewing yeasts, focusing on their maltose and maltotriose preferences. Structural models of the MALT proteins generated by AlphaFold2 and functional analyses of substitution mutants revealed that a very small number of amino acid residues in two spatially adjacent transmembrane helixes, TMH7 and TMH11, appear to be crucial for sugar preference. Thus, subtle conformational alterations conferred by a small number of amino acid polymorphisms within MALTs would contribute to the adaptation of domesticated brewing yeasts to the constrained carbohydrate environment of industrial wort during beer brewing.

Metabolic Engineering and Adaptive Evolution for Efficient Production of l-Lactic Acid in Saccharomyces cerevisiae.

l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.

OPTIMISATION OF TECHNOLOGICAL PARAMETERS OF FERMENTATION OF HIGHLY CONCENTRATED WORT FROM GRAIN RAW MATERIALS FOR BIOETHANOL PRODUCTION

Bioethanol is one of the most promising alternatives to fossil fuels and can be produced from various renewable sources rich in carbohydrates. A priority direction of biotechnological research is the development of new resource-saving technologies of bioethanol production. For effective bioconversion of carbohydrate-containing raw materials into bioethanol, it is advisable to optimise the technological parameters of fermenting highly concentrated wort obtained from grain raw materials. Laboratory experimental studies of the samples according to the newly developed technology have helped to substuntiate the technological parameters of fermenting highly concentrated wort and establishing the regular patterns in how the ethanol concentration in fermented washes changes depending on the initial concentration of dry matter in the wort, the industrial yeast concentration, and the fermentation temperature. Grain wort was fermented with the osmophilic thermotolerant race of distiller’s yeast Saccharomyces cerevisiae DО–16 (IMB Y-5099) at 32–37°C. The initial concentration of dry matter was 26–30%. To optimise the technological parameters of the process of fermenting highly concentrated grain wort, an experiment was designed, which resulted in building mathematical models. To obtain the second-order regression equations in natural form, the basic level of each technological parameter of the process and its variability interval were determined. The mathematical model developed makes it possible to calculate the ethanol concentration depending on the initial concentration of the wort, the concentration of industrial yeast, and the fermentation temperature. Fermentation of highly concentrated wort for bioethanol production was optimised according to the equations of the mathematical model. It has been established that to synthesise the maximum alcohol concentration in washes using the highly concentrated wort fermentation technology, the wort concentration should be 30% of dry matter, the concentration of industrial yeast 40 million/cm³, and the temperature of the main fermentation 35°C.

Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.

Construction of a novel plasmid for an industrial yeast Candida glycerinogenes by dual-autonomously replicating sequence strategy

Due to the lack of available episomal plasmid, the improvement of many industrial strains, especially exogenous gene expression, is severely restricted. The failure of autonomous replication or low copy number of episomal plasmids is the main reason for the failure of many episomal plasmids construction. In this paper, Candida glycerinogenes, an industrial strain lacking episomal plasmids, was employed as the topic. A series of GFP-based plasmids containing autonomously replicating sequence (ARS) from different strain sources were constructed and analyzed for performance, and it was found that only the panARS from Kluyveromyces lactis compared with other nine low capacity ARSs proved to have the best performance and could be used to construct episomal plasmid. Further, the dual-ARS strategy was used to optimize the episomal plasmid, and the results indicated that only the dual-ARS plasmid+PPARS2 with double different ARSs, not the dual-ARS plasmid+panARS with double same ARSs, showed an improvement in all properties, with an increase in transformation efficiency of about 36% and a synchronous trend of fluorescence intensity and copy number, both by about 40%. In addition, constructed episomal plasmids were used to express the exogenous gene CrGES, and the fact that geraniol was found proved the versatility of the plasmids. The successful construction of episomal plasmids will also substantially facilitate genetic engineering research and industrial use of C.glycerinogenes in the future, as well as providing a feasible approach to create episomal plasmids for industrial strains.

Biological characteristics of the heterotrophic marine microalgae Thraustochytrium pachydermum TSL10 isolated from the sea area of Truong Sa Archipelago, Vietnam

The sea area of the Truong Sa Archipelago, Vietnam - outpost island region of the country has a special geographical location and plays an important role in economic development as well as security and defense of Vietnam. Here, the first time for isolation of heterotrophic marine microalgae is precious. In this paper, the results on isolation, identification, and selection of suitable culture conditions of the strain Thraustochytrium pachydermum TSL10 isolated from the sea area of Spratly Island, Truong Sa Archipelago, Vietnam in 2021 were presented. The optimal culture conditions for the growth of T. pachydermum TSL10 in the flask were M1 medium, 3% glucose, 1% yeast extract and 25 - 28 °C temperature. Dry biomass and lipid content reached the highest levels of 12.54 ± 1.23 g/L and 28.65 ± 1.11 % dry cell weight (DCW), respectively. In a 30 L fermentor, T. pachydermum TSL10 grew well in a medium containing 9% glucose and 1% industrial extract yeast. After 120 hours of cultivation, dry biomass, lipid, protein, carbohydrate, carotenoids, and astaxanthin reached the highest levels of 30.76 ± 1.32 g/L; 22.95 ± 0.28% DCW; 6.65 ± 0.19% DCW, 15.96 ± 0.021% DCW, 178.00 ± 2.43 µg/L, and 8.30 ± 0.28 µg/L, respectively. The fatty acid composition contains mainly omega - 6 and omega - 9 fatty acids, accounting for 21.69% of total fatty acids (TFA) and 34.56% of TFA. The fatty acid profile was mainly dominated by linoleic acid and oleic acid, accounting for 21.54% of TFA and 34.23% of TFA, respectively. The results showed that TSL10 biomass was suitable raw material for screening bioactive compounds.

Development of an industrial yeast strain for efficient production of 2,3-butanediol

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

Increasing Ethanol Tolerance and Ethanol Production in an Industrial Fuel Ethanol Saccharomyces cerevisiae Strain

The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae.

Copy number variants impact phenotype-genotype relationships for adaptation of industrial yeast Saccharomyces cerevisiae.

The industrial yeast Saccharomyces cerevisiae possesses a plastic genome enabling its adaptation to varied environment conditions. A more robust ethanologenic industrial yeast strain NRRL Y-50049 was obtained through laboratory adaptation that is resistant to 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF), a major class of toxic chemicals associated with lignocellulose-to-biofuel conversion. A significant amount of knowledge has been achieved in characterizing its tolerant phenotypes and molecular mechanisms of the resistance. Recent findings on a limited number of nonsynonymous SNP (single nucleotide polymorphism) detected in NRRL Y-50049 compared with its progenitor NRRL Y-12632 raised doubt of SNP roles in the tolerance adaptation. The genotype-phenotype relationship for yeast adaptation to the toxic chemicals is yet unclear. Here, we examine copy number variant (CNV) of the adapted strain NRRL Y-50049 to address phenotype-genotype relationships. As a background information, CNV of model strain S288C of the reference genome was also examined versus the industrial-type strain NRRL Y-12632. More than 200 CNVs, mostly duplication events, were detected in NRRL Y-12632 compared with the laboratory model strain S288C. Such enriched genetic background supports its more diversified phenotype response for the industrial yeastthan the laboratory strainS288C. Comparing the two industrial strains, we found extra nine CNVs in the mitochondrial genome and 28 CNVs in the nuclear genome of NRRL Y-50049 versus its progenitor NRRL Y-12632. Continued DNA recombination event and high rate of CNV observed in NRRL Y-50049 versus its progenitor suggests that CNV is more impactful than SNP in association with phenotype-genotype relationships of yeast adaptation to the toxic chemical stress. COX1 and COB loci were defined as DNA recombination hotspots in the mitochondrial genome for the industrial yeast based on the high frequency of CNVs observed in these loci. KEY POINTS: • COX1 and COB loci are identified as DNA recombination hotspots for the industrial yeast. • The industrial yeast type strain NRRL Y-12632 possesses more CNVs vs the reference genome S288C. • CNV is more important than SNP on phenotype-genotype relationships for yeast adaptation.

Brazilian industrial yeasts show high fermentative performance in high solids content for corn ethanol process

An imminent change in the world energy matrix makes it necessary to increase the production of renewable fuels. The United States and Brazil are the world's largest producers, but their production methods are very different, using different raw materials, ground corn and sugarcane juice, respectively. In recent years, strong investments have been made to expand the use of corn in Brazilian ethanol production. The combination of the sugar cane and corn ethanol industries has generated innovations in the sector, such as the "flex" mills, which are traditional sugar cane mills adapted to produce corn ethanol in the sugar cane off-season. Brazil has a portfolio of robust industrial yeasts for sugarcane ethanol production, naturally evolved and selected over the past 50 years. In this work, we analyze for the first time the performance of Brazilian industrial strains (BG-1, CAT-1, PE-2 and SA-1, widely used in sugarcane ethanol production) in corn ethanol production using different stress conditions. Ethanol Red yeast, traditionally used in corn ethanol plants around the world, was used as a control. In terms of tolerance to temperature (35 °C), strains BG-1 and SA-1 stood out. In fermentations with high solids concentration (35%), strain BG-1 reached ethanol contents higher than 19% w/v and had a productivity gain of 5.8% compared to fermentation at 30%. This was the first time that these industrial strains were evaluated using the high solids concentration of 35% and the results point to ways to improve the corn ethanol production process.Graphical

Influence of ergosterol and phytosterols on wine alcoholic fermentation with Saccharomyces cerevisiae strains

Sterols are a fraction of the eukaryotic lipidome that is essential for the maintenance of cell membrane integrity and its good functionality. During alcoholic fermentation, they enhance yeast growth, metabolism and viability, as well as resistance to high sugar content and ethanol stress. Grape musts clarified in excess lead to the loss of solid particles rich in sterols, resulting in sluggish and stuck fermentations. Two sterol sources can help Saccharomyces cerevisiae yeasts to adapt to fermentation stress conditions: ergosterol (synthesized by yeast under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts under anaerobiosis). Little is known about the physiological impact of phytosterols assimilation in comparison with ergosterol and the influence of sterol type on fermentation kinetics parameters. Moreover, studies to date have analyzed a limited number of yeast strains. Thus, the aim of this work was to compare the performances of a set of Saccharomyces cerevisiae wine strains that represent the diversity of industrial wine yeast, fermenting with phytosterols or ergosterol under two conditions: sterol limitation (sterol starvation) and high sugar content (the most common stress during fermentation). Results indicated that yeast cell viability was negatively impacted by both stressful conditions, resulting in sluggish and stuck fermentations. This study revealed the huge phenotype diversity of the S. cerevisiae strains tested, in particular in terms of cell viability. Indeed, strains with better viability maintenance completed fermentation earlier. Interestingly, we showed for the first time that sterol type differently affects a wide variety of phenotype, such as viability, biomass, fermentation kinetics parameters and biosynthesis of carbon central metabolism (CCM) metabolites. Ergosterol allowed preserving more viable cells at the end of fermentation and, as a consequence, a better completion of fermentation in both conditions tested, even if phytosterols also enabled the completion of alcoholic fermentation for almost all strains. These results highlighted the essential role of sterols during wine alcoholic fermentation to ensure yeast growth and avoid sluggish or stuck fermentations. Finally, this study emphasizes the importance of taking into account sterol types available during wine fermentation.

Functional Perspective on Sourdough Bread

In recent years, with the awareness of people, the interest in natural and functional foods has increased. Sourdough is a dough piece in which industrial culture yeasts (Saccharomyces cerevisiae), endogenous yeasts, lactic and acetic bacteria from the air and the ingredients used (flour, water, etc.) are active. In sourdough fermentation, yeast and lactic acid bacteria work together to form the natural flora. It has been proven by studies that breads prepared from sourdough have many advantages such as greater volume increase, stronger aroma, better crumb structure and long shelf life. In addition, sourdough fermentation has very important positive effects on human health. Various additives (malt flours, emulsifiers, microbial enzymes, dairy products, soy flour and potato flour) are used to delay the staling of bread. In the production of sourdough bread, high quality and long shelf-life breads can be obtained without the need for these additives. Thus, both natural and additive-free and functional breads are produced. In this review, it is aimed to raise awareness by giving information about the advantages of using sourdough in bread production. In the study, the concept of sourdough was discussed and information was given about the content of bread prepared using sourdough and its benefits on health.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

BackgroundExpansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale.ResultsCo-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry.ConclusionsPichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

Scale-Up to Pilot of a Non-Axenic Culture of Thraustochytrids Using Digestate from Methanization as Nitrogen Source.

The production of non-fish based docosahexaenoic acid (DHA) for feed and food has become a critical need in our global context of over-fishing. The industrial-scale production of DHA–rich Thraustochytrids could be an alternative, if costs turned out to be competitive. In order to reduce production costs, this study addresses the feasibility of the non-axenic (non-sterile) cultivation of Aurantiochytrium mangrovei on industrial substrates (as nitrogen and mineral sources and glucose syrup as carbon and energy sources), and its scale-up from laboratory (250 mL) to 500 L cultures. Pilot-scale reactors were airlift cylinders. Batch and fed-batch cultures were tested. Cultures over 38 to 62 h achieved a dry cell weight productivity of 3.3 to 5.5 g.L−1.day−1, and a substrate to biomass yield of up to 0.3. DHA productivity ranged from 10 to 0.18 mg.L−1.day−1. Biomass productivity appears linearly related to oxygen transfer rate. Bacterial contamination of cultures was low enough to avoid impacts on fatty acid composition of the biomass. A specific work on microbial risks assessment (in supplementary files) showed that the biomass can be securely used as feed. However, to date, there is a law void in EU legislation regarding the recycling of nitrogen from digestate from animal waste for microalgae biomass and its usage in animal feed. Overall, the proposed process appears similar to the industrial yeast production process (non-axenic heterotrophic process, dissolved oxygen supply limiting growth, similar cell size). Such similarity could help in further industrial developments.

Principled, practical, flexible, fast: a new approach to phylogenetic factor analysis.

Biological phenotypes are products of complex evolutionary processes in which selective forces influence multiple biological trait measurements in unknown ways. Phylogenetic comparative methods seek to disentangle these relationships across the evolutionary history of a group of organisms. Unfortunately, most existing methods fail to accommodate high-dimensional data with dozens or even thousands of observations per taxon. Phylogenetic factor analysis offers a solution to the challenge of dimensionality. However, scientists seeking to employ this modeling framework confront numerous modeling and implementation decisions, the details of which pose computational and replicability challenges.We develop new inference techniques that increase both the computational efficiency and modeling flexibility of phylogenetic factor analysis. To facilitate adoption of these new methods, we present a practical analysis plan that guides researchers through the web of complex modeling decisions. We codify this analysis plan in an automated pipeline that distills the potentially overwhelming array of decisions into a small handful of (typically binary) choices.We demonstrate the utility of these methods and analysis plan in four real-world problems of varying scales. Specifically, we study floral phenotype and pollination in columbines, domestication in industrial yeast, life history in mammals, and brain morphology in New World monkeys.General and impactful community employment of these methods requires a data scientific analysis plan that balances flexibility, speed and ease of use, while minimizing model and algorithm tuning. Even in the presence of non-trivial phylogenetic model constraints, we show that one may analytically address latent factor uncertainty in a way that (a) aids model flexibility, (b) accelerates computation (by as much as 500-fold) and (c) decreases required tuning. These efforts coalesce to create an accessible Bayesian approach to high-dimensional phylogenetic comparative methods on large trees.

Genetic mapping of a bioethanol yeast strain reveals new targets for hydroxymethylfurfural- and thermotolerance

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae’s metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097′s HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.

Toward Methanol-Based Biomanufacturing: Emerging Strategies for Engineering Synthetic Methylotrophy in Saccharomyces cerevisiae.

The global expansion of biomanufacturing is currently limited by the availability of sugar-based microbial feedstocks, which require farmland for cultivation and therefore cannot support large increases in production without impacting the human food supply. One-carbon feedstocks, such as methanol, present an enticing alternative to sugar because they can be produced independently of arable farmland from organic waste, atmospheric carbon dioxide, and hydrocarbons such as biomethane, natural gas, and coal. The development of efficient industrial microorganisms that can convert one-carbon feedstocks into valuable products is an ongoing challenge. This review discusses progress in the field of synthetic methylotrophy with a focus on how it pertains to the important industrial yeast, Saccharomyces cerevisiae. Recent insights generated from engineering synthetic methylotrophic xylulose- and ribulose-monophosphate cycles, reductive glycine pathways, and adaptive laboratory evolution studies are critically assessed to generate novel strategies for the future engineering of methylotrophy in S.cerevisiae.

Gene Coexpression Connectivity Predicts Gene Targets Underlying High Ionic-Liquid Tolerance in Yarrowia lipolytica.

ABSTRACTMicrobial tolerance to organic solvents such as ionic liquids (ILs) is a robust phenotype beneficial for novel biotransformation. While most microbes become inhibited in 1% to 5% (vol/vol) IL (e.g., 1-ethyl-3-methylimidazolium acetate), we engineered a robust Yarrowia lipolytica strain (YlCW001) that tolerates a record high of 18% (vol/vol) IL via adaptive laboratory evolution. Yet, genotypes conferring high IL tolerance in YlCW001 remain to be discovered. In this study, we shed light on the underlying cellular processes that enable robust Y. lipolytica to thrive in inhibitory ILs. By using dynamic transcriptome sequencing (RNA-Seq) data, we introduced Gene Coexpression Connectivity (GeCCo) as a metric to discover genotypes conferring desirable phenotypes that might not be found by the conventional differential expression (DE) approaches. GeCCo selects genes based on their number of coexpressed genes in a subnetwork of upregulated genes by the target phenotype. We experimentally validated GeCCo by reverse engineering a high-IL-tolerance phenotype in wild-type Y. lipolytica. We found that gene targets selected by both DE and GeCCo exhibited the best statistical chance at increasing IL tolerance when individually overexpressed. Remarkably, the best combination of dual-overexpression genes was genes selected by GeCCo alone. This nonintuitive combination of genes, BRN1 and OYE2, is involved in guiding/regulating mitotic cell division, chromatin segregation/condensation, microtubule and cytoskeletal organization, and Golgi vesicle transport.IMPORTANCE Cellular robustness to cope with stressors is an important phenotype. Y. lipolytica is an industrial robust oleaginous yeast that has recently been discovered to tolerate record high concentrations of ILs, beneficial for novel biotransformation in organic solvents. However, genotypes that link to IL tolerance in Y. lipolytica are largely unknown. Due to the complex IL-tolerant phenotype, conventional gene discovery and validation based on differential gene expression approaches are time-consuming due to a large search space and might encounter a high false-discovery rate. Here, using the developed Gene Coexpression Connectivity (GeCCo) method, we identified and validated a subset of most promising gene targets conferring the IL-tolerant phenotypes and shed light on their potential mechanisms. We anticipate GeCCo being a useful method to discover the genotype-to-phenotype link.

Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris.

Methanol-based biorefinery is a promising strategy to achieve carbon neutrality goals by linking CO2 capture and solar energy storage. As a typical methylotroph, Pichia pastoris shows great potential in methanol biotransformation. However, challenges still remain in engineering methanol metabolism for chemical overproduction. Here, we present the global rewiring of the central metabolism for efficient production of free fatty acids (FFAs; 23.4 g/L) from methanol, with an enhanced supply of precursors and cofactors, as well as decreased accumulation of formaldehyde. Finally, metabolic transforming of the fatty acid cell factory enabled overproduction of fatty alcohols (2.0 g/L) from methanol. This study demonstrated that global metabolic rewiring released the great potential of P. pastoris for methanol biotransformation toward chemical overproduction.

The Influence of Yarrowia lipolytica Glycosylation on the Biochemical Properties and Oligomerization of Heterologous Invertase

Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher KM for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorter chains than hypermannosylated glycans from S. cerevisiae. Furthermore, galactose was detected as the modifying sugar in the glycan structures of invertase expressed in Y. lipolytica. N-glycans did not affect invertase activity but were important for its oligomerization. The expressed enzyme aggregated into dimers, tetramers, hexamers, and octamers, as well as structures of higher molecular mass, which might be decamers, which have not been described so far in the literature.