Non-conventional Yeast Research Articles

Identification of genes involved in xylose metabolism of Meyerozyma guilliermondii and their genetic engineering for increased xylitol production

Meyerozyma guilliermondii, a non-conventional yeast that naturally assimilates xylose, is considered as a candidate for biotechnological production of the sugar alternative xylitol. Because the genes of the xylose metabolism were yet unknown, all efforts published so far to increase the xylitol yield of this yeast are limited to fermentation optimization. Hence, this study aimed to genetically engineer this organism for the first time with the objective to increase xylitol production. Therefore, the previously uncharacterized genes of M. guilliermondii ATCC 6260 encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) were identified by pathway investigations and sequence similarity analysis. Cloning and overexpression of the putative XR as well as knockout of the putative XDH genes generated strains with about threefold increased xylitol yield. Strains that combined both genetic modifications displayed fivefold increase in overall xylitol yield. Enzymatic activity assays with lysates of XR overexpressing and XDH knockout strains underlined the presumed functions of the respective genes. Furthermore, growth evaluation of the engineered strains on xylose as sole carbon source provides insights into xylose metabolism and its utilization for cell growth.

A differential evolution approach to estimate parameters in a temperature-dependent kinetic model for second generation ethanol production under high cell density with Spathaspora passalidarum

An unstructured–unsegregated temperature-dependent kinetic model was developed and validated to describe ethanol fermentation in a mixture of xylose and glucose for the nonconventional yeast Spathaspora passalidarum NRRL Y-27907 under high cell density in the temperature range of 26–32 °C. The kinetic model consisted of 13 equations and 16 kinetic parameters, describing cell growth, individual xylose and glucose uptake and product formation. Global parametric estimation was executed through a differential evolution algorithm. Temperature-dependent parameters were identified and adjusted to Arrhenius-type equations as a function of temperature. The overall r2 of the process calibration and validation were 0.972 and 0.959, respectively, indicating that the model satisfactorily described the process in the temperature range investigated. Furthermore, using a 3D projection of the model to simulate ethanol production, it was concluded that temperatures between 30 and 32 °C resulted in the highest productivities.

Sugar Alcohols and Organic Acids Synthesis in Yarrowia lipolytica: Where Are We?

Sugar alcohols and organic acids that derive from the metabolism of certain microorganisms have a panoply of applications in agro-food, chemical and pharmaceutical industries. The main challenge in their production is to reach a productivity threshold that allow the process to be profitable. This relies on the construction of efficient cell factories by metabolic engineering and on the development of low-cost production processes by using industrial wastes or cheap and widely available raw materials as feedstock. The non-conventional yeast Yarrowia lipolytica has emerged recently as a potential producer of such metabolites owing its low nutritive requirements, its ability to grow at high cell densities in a bioreactor and ease of genome edition. This review will focus on current knowledge on the synthesis of the most important sugar alcohols and organic acids in Y. lipolytica.

Exploiting new biorefinery models using non-conventional yeasts and their implications for sustainability

Feasible bioprocessing of lignocellulosic biomass requires the use of microbial strains with tolerance to inhibitor compounds and osmotic pressure, able to provide high product yield and productivity. In this sense, this study evaluated the potential of two non-conventional yeasts, Hansenula polymorpha CBS 4732 and Debaryomyces hansenii CBS 767, for use on biomass conversion in a biorefinery perspective. The ability of the strains to consume pentose and hexose sugars, to resist the toxic compounds present in hydrolysates, as well as to produce sugar alcohols and ethanol, was investigated. H. polymorpha showed highlighted resistance to toxic compounds and relevant ability to consume xylose and produce xylitol and ethanol under these conditions, at 37 °C. D. hansenii was a great producer of arabitol from glucose. The implications for sustainability due to the use of these yeasts in biorefineries was discussed. These results open up new perspectives for the development of the biorefinery sector.

Metabolic engineering of Pichia pastoris for production of isopentanol (3-Methyl-1-butanol)

In recent years, the increasingly serious and clear effects of climate change have increased interest in renewable fuels and platform chemicals. Microbial platforms that can produce these compounds in an economically efficient way have emerged as an attractive alternative to the traditional production approaches. Here, we engineered the industrially-relevant yeast Pichia pastoris to produce the platform chemical 3-methyl-1-butanol (3M1B, isopentanol) directly from the renewable carbon source glucose. Specifically, we overexpressed the endogenous valine and leucine biosynthetic pathways to increase the production of the key pathway intermediate, 2-ketoisocaproate (2-KIC). Overexpression of the artificial keto-acid degradation pathway converted 2-KIC into 3M1B. Down-regulation of the side-product ethanol production using the CRISPR/Cas9 system led to a strain that is able to produce 3M1B at a titer of 191.0 ± 9.6 mg/L, the highest titer reported to date in a non-conventional yeast. We envision that our yeast system will pave the way for an efficient production system for this important class of platform compounds.

Microbiological Production of Isocitric Acid from Biodiesel Waste and Its Effect on Spatial Memory.

Within this work, the microbial synthesis of (2R,3S)-isocitric acid (ICA), a metabolite of the nonconventional yeast Yarrowia lipolytica, from biodiesel waste, has been studied. The selected strain Y. lipolytica VKM Y-2373 synthesized ICA with citric acid (CA) as a byproduct. This process can be regulated by changing cultivation conditions. The maximal production of ICA with the minimal formation of the byproduct was provided by the use of a concentration of (NH4)2SO4 (6 g/L); the addition of biodiesel waste to cultivation medium in 20–60 g/L portions; maintaining the pH of the cultivation medium at 6, and degree of aeration between 25% and 60% of saturation. Itaconic acid at a concentration of 15 mM favorably influenced the production of ICA by the selected strain. The optimization of cultivation conditions allowed us to increase the concentration of ICA in the culture liquid from 58.32 to 90.2 g/L, the product yield (Y) by 40%, and the ICA/CA ratio from 1.1:1 to 3:1. Research on laboratory animals indicated that ICA counteracted the negative effect of ammonium molybdate (10−5 M) and lead diacetate (10−7 M) on the learning and spatial memory of rats, including those exposed to emotional stress.

The Xylose Metabolizing Yeast Spathaspora passalidarum is a Promising Genetic Treasure for Improving Bioethanol Production

Currently, the fermentation technology for recycling agriculture waste for generation of alternative renewable biofuels is getting more and more attention because of the environmental merits of biofuels for decreasing the rapid rise of greenhouse gas effects compared to petrochemical, keeping in mind the increase of petrol cost and the exhaustion of limited petroleum resources. One of widely used biofuels is bioethanol, and the use of yeasts for commercial fermentation of cellulosic and hemicellulosic agricultural biomasses is one of the growing biotechnological trends for bioethanol production. Effective fermentation and assimilation of xylose, the major pentose sugar element of plant cell walls and the second most abundant carbohydrate, is a bottleneck step towards a robust biofuel production from agricultural waste materials. Hence, several attempts were implemented to engineer the conventional Saccharomyces cerevisiae yeast to transport and ferment xylose because naturally it does not use xylose, using genetic materials of Pichia stipitis, the pioneer native xylose fermenting yeast. Recently, the nonconventional yeast Spathaspora passalidarum appeared as a founder member of a new small group of yeasts that, like Pichia stipitis, can utilize and ferment xylose. Therefore, the understanding of the molecular mechanisms regulating the xylose assimilation in such pentose fermenting yeasts will enable us to eliminate the obstacles in the biofuels pipeline, and to develop industrial strains by means of genetic engineering to increase the availability of renewable biofuel products from agricultural biomass. In this review, we will highlight the recent advances in the field of native xylose metabolizing yeasts, with special emphasis on S. passalidarum for improving bioethanol production.

Evaluation of the non-conventional yeast strain Wickerhamomyces anomalus (Pichia anomala) X19 for enhanced bioethanol production using date palm sap as renewable feedstock

In the present study the non-conventional yeast Wickerhamomyces anomalus X19, was evaluated as biocatalyst for direct, low cost bioethanol production, using natural date palm sap (DPS) as substrate. Initial experiments were conducted with synthetic media in order to characterize the strain in terms of its nitrogen requirements, the effect of initial substrate concentration on the ethanol yields and the main kinetic constants during alcoholic fermentation. Subsequently, the effect of aerobic or anaerobic conditions was assessed with DPS as substrate, in batch mode and the possible improvement of ethanol yields was further investigated via fed batch experiments. It was shown that W. anomalus X19 was not inhibited by low pH (<3) and high initial sugar concentrations (up to 160 g/L), leading in all cases to ethanol yields that exceeded 80% of the theoretically estimated values. The highest ethanol concentration from DPS fermentation was achieved in fed batch experiments, reaching 73.11 g/L and the ethanol yield was 0.46 g/g sugars, which corresponded to more than 90% of the theoretical maximum.

Recent Advances in the Metabolic Engineering of Yeasts for Ginsenoside Biosynthesis

Ginsenosides are a group of glycosylated triterpenes isolated from Panax species. Ginsenosides are promising candidates for the prevention and treatment of cancer as well as food additives. However, owing to a lack of efficient approaches for ginsenoside production from plants and chemical synthesis, ginsenosides may not yet have reached their full potential as medicinal resources. In recent years, an alternative approach for ginsenoside production has been developed using the model yeast Saccharomyces cerevisiae and non-conventional yeasts such as Yarrowia lipolytica and Pichia pastoris. In this review, various metabolic engineering strategies, including heterologous gene expression, balancing, and increasing metabolic flux, and enzyme engineering, have been described as recent advanced engineering techniques for improving ginsenoside production. Furthermore, the usefulness of a systems approach and fermentation strategy has been presented. Finally, the present challenges and future research direction for industrial cell factories have been discussed.

Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones

Thirteen Non-Conventional Yeasts (NCYs) have been investigated for their ability to reduce activated C=C bonds of chalcones to obtain the corresponding dihydrochalcones. A possible correlation between bioreducing capacity of the NCYs and the substrate structure was estimated. Generally, whole-cells of the NCYs were able to hydrogenate the C=C double bond occurring in (E)-1,3-diphenylprop-2-en-1-one, while worthy bioconversion yields were obtained when the substrate exhibited the presence of a deactivating electron-withdrawing Cl substituent on the B-ring. On the contrary, no conversion was generally found, with a few exceptions, in the presence of an activating electron-donating substituent OH. The bioreduction aptitude of the NCYs was apparently correlated to the logP value: Compounds characterized by a higher logP exhibited a superior aptitude to be reduced by the NCYs than compounds with a lower logP value.

Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production

Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids.Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ​± ​5 ​mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ​± ​17.7 ​mg/L in shake flasks, and 1232 ​± ​64 ​mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.

Multinuclear Yeast Magnusiomyces (Dipodascus, Endomyces) magnusii is a Promising Isobutanol Producer.

Higher alcohol isobutanol is a promising liquid fuel. During alcoholic fermentation, Saccharomyces cerevisiae produces only trace amounts of isobutanol. Screening the collection of nonconventional yeasts show that Magnusiomyces magnusii accumulates 440mg of isobutanol per L in rich YPD medium. Here, the transformation protocol for M. magnusii is adapted based on the use of the dominant markers conferring resistance to nourseothricin or zeocin; the strong constitutive promoter TEF1 is cloned and a reporter system based on LAC4 gene from Kluyveromyces lactis coding for β-galactosidase is constructed. In order to increase isobutanol production in M. magnusii, the heterologous gene ILV2 from S. cerevisiae is expressed in M. magnusii under control of the TEF1 promoter. The best stabilized transformants produce 620mg of isobutanol per L in YPD medium and 760mg L-1 in the medium with 2-oxoisovalerate. This suggests that M. magnusii is a promising organism for further development of a robust isobutanol producer.

Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

The large-scale production of recombinant proteins (rProt) is becoming increasingly economically important. Among the different hosts used for rProt production, yeasts are gaining popularity. The so-called non-conventional yeasts, such as the methylotrophic Pichia pastoris and the dimorphic Yarrowia lipolytica, are popular choices due to their favorable characteristics and well-established expression systems. Nevertheless, a direct comparison of the two systems for rProt production and secretion was lacking. This study therefore aimed to directly compare Y. lipolytica and P. pastoris for the production and secretion of lipase CalB in bioreactor. Y. lipolytica produced more than double the biomass and more than 5-fold higher extracellular lipase than P. pastoris. Furthermore, maximal CalB production levels were reached by Y. lipolytica in half the cultivation time required for maximal production by P. pastoris. Conversely, P. pastoris was found to express 7-fold higher levels of CalB mRNA. Secreted enhanced green fluorescent protein –in isolation and fused to CalB– and protease inhibitor MG-132 were used in P. pastoris to further investigate the reasons behind such discrepancy. The most likely explanation was ultimately found to be protein degradation by endoplasmic reticulum-associated protein degradation preceding successful secretion. This study highlighted the multifaceted nature of rProt production, prompting a global outlook for selection of rProt production systems.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

A genetic toolbox for metabolic engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

2-Phenylethanol and 2-phenylethylacetate production by nonconventional yeasts using tequila vinasses as a substrate

Vinasses from the tequila industry are wastewaters with highly elevated organic loads. Therefore, to obtain value-added products by yeast fermentations, such as 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEA), could be interesting for industrial applications from tequila vinasses. In this study, four yeasts species (Wickerhamomyces anomalus, Candida glabrata, Candida utilis, and Candida parapsilosis) were evaluated with two different chemically defined media and tequila vinasses. Differences in the aroma compounds production were observed depending on the medium and yeast species used. In tequila vinasses, the highest concentration (65 mg/L) of 2-PEA was reached by C. glabrata, the inhibitory compounds decreased biomass production and synthesis of 2-PEA, and biochemical and chemical oxygen demands were reduced by more than 50 %. Tequila vinasses were suitable for the production of 2-phenylethylacetate by the shikimate pathway. A metabolic network was developed to obtain a guideline to improve 2-PE and 2-PEA production using flux balance analysis (FBA).

Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica.

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine de novo, the oleaginous yeast Yarrowia lipolytica cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain.IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

Evolution of Aromatic Profile of Torulaspora delbrueckii Mixed Fermentation at Microbrewery Plant

Nowadays, consumers require quality beer with peculiar organoleptic characteristics and fermentation management has a fundamental role in the production of aromatic compounds and in the overall beer quality. A strategy to achieve this goal is the use of non-conventional yeasts. In this context, the use of Torulaspora delbrueckii was proposed in the brewing process as a suitable strain to obtain a product with a distinctive aromatic taste. In the present work, Saccharomyces cerevisiae/T. delbrueckii mixed fermentation was investigated at a microbrewery plant monitoring the evolution of the main aromatic compounds. The results indicated a suitable behavior of this non-conventional yeast in a production plant. Indeed, the duration of the process was very closed to that exhibited by S. cerevisiae pure fermentation. Moreover, mixed fermentation showed an increase of some aromatic compounds as ethyl hexanoate, α-terpineol, and β-phenyl ethanol. The enhancement of aromatic compounds was confirmed by the sensory evaluation carried out by trained testers. Indeed, the beers produced by mixed fermentation showed an emphasized note of fruity/citric and fruity/esters notes and did not show aroma defects.

Non-conventional yeasts as superior production platforms for sustainable fermentation based bio-manufacturing processes

Non-conventional yeasts are an excellent option for a number of different industrial bioprocesses. They possess beneficial natural phenotypes, which translates to several fermentation advantages when compared to traditional hosts, like <i>Saccharomyces cerevisiae</i>. The non-conventional yeasts <i>Yarrowia lipolytica</i>, <i>Trichosporon oleaginosus</i>, <i>Kluyveromyces marxianus</i>, <i>Dekkera bruxellensis</i>, <i>Pichia kudriavzevii</i>, <i>Debaryomyces hansenii</i> and <i>Hansenula polymorpha</i>, are considered desirable industrial hosts due to their natural characteristics, including tolerance to several by-products and inhibitors, thermotolerance, salt resistance or osmo- and xerotolerance. Therefore, they are a great alternative for the industrial production of bioethanol, fine chemicals, lipids and recombinant proteins, among others. In this review, we summarize the best natural characteristics of those seven non-conventional yeasts and their use in industrial biotechnology, as well as the molecular/synthetic biology tools available for their genetic modification. Moreover, possible limitations regarding their performance in industrial fermentations and a list of challenges to overcome in the future are also discussed.

Recombination Machinery Engineering for Precise Genome Editing in Methylotrophic Yeast &lt;i&gt;Ogataea Polymorpha&lt;/i&gt;

Methanol biotransformation can expand biorefinary substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR/Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ), increase HR rates from 20-30% to 60-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated systems has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the first production of fatty alcohols in O. polymorpha. These findings will simplify the genetic engineering in non-conventional yeasts, and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.