Unconventional Yeast Research Articles

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.

2016: This Year in JoVE

Welcome to JoVE's Year in Review, where we highlight some of the most interesting video protocols of the past year. In January 2016 JoVE started out with a bang...or rather, it didn't need to...as JoVE Engineering gave us a protocol for the production of synthetic nuclear melt glass. This is a valuable tool for forensic study of post-detonation, without the boom. February was a treasure of a month for JoVE - not only did we publish our 6000th article, but in JoVE Chemistry we showcased research on gold nanoparticles from the laboratory of a Nobel Prize winner. In March, JoVE Biology described RNA interference in the malaria vector Mosquito Anopheles gambiae, through direct pupal injection. This promising research may pave the way for the next generation of mosquito-borne disease control. April heralded the launch of a new Science Education collection - Chemistry. Here, we unveiled protocols covering the fundamentals of general, organic, and Inorganic chemistry - giving students the chance to explore lab techniques, common equipment, and core theories of Chemistry. Shhh! In May, JoVE Environment profiled a novel technique for tracking and identifying individual cheetahs from just their footprints...allowing researchers to better monitor and protect Africa's most endangered large Felid. June gave us the new Science Education Clinical Skills collections, providing a foundation for performing physical examinations of patients including the assessment of vital signs, and basic external and internal evaluations. In July, JoVE Neuroscience was buzzing with cool science. We showcased research into the taste perception of honeybees to different nutrients and toxins and how this influences the feeding behavior of these critical pollinators. JoVE Medicine got to the heart of the matter in August...profiling a new method to help investigate angina and identify constrictions in coronary arteries. September launched three new JoVE Journal sections covering Genetics, Cancer Research, and Biochemistry.These new collections include groundbreaking research into topics as diverse as genetic engineering of unconventional yeast strains for biofuels...mass spectrometry for meat authentication...and mixed cell culture models to mimic tumor microenvironments. In October, JoVE Immunology and Infection took us behind the scenes in a Biosafety Level 4 facility. In a four article series, we showed how researchers operate aerobiology chambers...use safety suits and biosafety cabinets...and perform medical imaging in an environment containing life threatening diseases like the Ebola virus. November's JoVE Behavior explored...um...oh yes...memory.Our authors showed that turtles actively use memory and recall to find their seasonal habitats, and not simply environmental cues. Finally, in December, JoVE Bioengineering helped us to see things more clearly, with a new technique for performing 3D super-resolution microscopy of individual cell structures and components. This Year in Review was just a sampling of more than 1100 video-articles that JoVE published in 2016. Browse the JoVE archives for thousands of other videos, and come back each week to see brand-new material in JoVE: The Journal of Visualized Experiments

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

September 2016-This Month in JoVE: Introducing JoVE Genetics, JoVE Biochemistry, and JoVE Cancer Research

This month we are pleased to introduce three new sections of the JoVE family: Genetics, Biochemistry, and Cancer Research. JoVE Genetics contains methodologies for exploring all aspects of genes and heredity-from human genetics to model organisms, epigenetics to evolutionary genetics, and gene editing to gene therapy. This section features a method for genotyping pufferfish species by liquid chromatography/mass spectrometry, described by Miyaguchi. This technique can aid in the appropriate identification and differentiation of toxic species, which is not only important for public health but also for forensics and investigations of food fraud. Also in JoVE Genetics, Yu et al. report methods for genetically engineering the unconventional yeast Yarrowia lipolytica with improved gene deletion efficiency. The engineered Y. lipolytica strains have potential applications in biofuel and biochemical production. JoVE Biochemistry comprises methods that advance our understanding of biomolecule structure and function, as well as their interactions and transformations during biological processes. This month, Gunning et al. present a method of meat authentication using multiple reaction monitoring (MRM) mass spectrometry, which identifies peptides and gives relative quantitation for detecting adulterant species in meat mixtures. This method is sensitive enough to detect 1% horsemeat in beef products. Also in JoVE Biochemistry, Head and Liu describe a method for identifying small molecule-binding proteins using photoaffinity labeling. The target proteins are bound and covalently labeled within the live cellular environment, which helps preserve native protein structure and binding conditions. JoVE Cancer Research encompasses a broad range of techniques used to advance the understanding and treatment of cancer. This includes methodologies for studying carcinogenesis, developing innovative diagnostics and therapeutics, and uncovering the mechanisms of drug resistance. This month in JoVE Cancer Research, Ansari et al. report a method of targeted cell isolation via glass surface functionalization. This method can identify biomarkers of resistance or susceptibility to anti-angiogenic therapies. Also in this section, Domogauer et al. present a mixed cell culture model that mimics the tumor microenvironment. With this model, the intercellular communication within the tumor microenvironment can be studied under various conditions. You've just had a sneak peek of the articles in the newest sections of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6% for the wild-type strain and 94.1% for the ku70/ku80 double-deficient strain, within 4days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3%, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.

Exploring medium-chain-length polyhydroxyalkanoates production in the engineered yeast Yarrowia lipolytica.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or β-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, we constructed a genetically modified strain of Y. lipolytica by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 with a PTS1 peroxisomal signal. When in single copy, the codon optimized PhaC1 allowed the synthesis of 0.205 % DCW of PHA after 72 h cultivation in YNBD medium containing 0.1 % oleic acid. By using a multi-copy integration strategy, PHA content increased to 2.84 % DCW when the concentration of oleic acid in YNBD was 1.0 %. Furthermore, when the recombinant yeast was grown in the medium containing triolein, PHA accumulated up to 5.0 % DCW with as high as 21.9 g/L DCW, which represented 1.11 g/L in the culture. Our results demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for PHA production using food waste, which contains lipids and other essential nutrients.

One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21%, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.

Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential

‘Wild’ and spontaneously fermented beers are growing in popularity in the craft beer industry. Most of these beers are fermented by the use of either pure cultures of unconventional yeast and bacteria or spontaneous fermentation using mixed local microflora. This study examined the potential of using pure strains of new isolates of wild yeast in the fermentation of a unique beer. The microbial communities from the fruit of pindo palm, loquat, hackberry and blackberry were collected in liquid culture, then plated for isolation. Ten isolates were selected for further analysis. Strains were identified by restriction fragment length polymorphism (RFLP) analysis and analysed for growth in a simple liquid media, fermentation in a complex media, alcohol tolerance and acid tolerance. Despite identification of some strains as the same species, they displayed a wide range of physiological properties. All strains were tolerant of pH values as low as 2.4, but none were tolerant of pH 1.9. Alcohol tolerance of different strains varied from 6 to 12%. Several strains had properties that suggest potential as primary fermenters, including the alcohol fermentation of a beer wort. Organoleptic properties of beers fermented with several of the strains demonstrated potential for commercial brewing. Copyright © 2014 The Institute of Brewing & Distilling

Functional integration of multiple genes into the genome of the oleaginous yeastRhodosporidium toruloides

The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R.toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R.toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.

Phytase production by the unconventional yeast Pichia anomala in fed batch and cyclic fed batch fermentations

-1 ) in comparison with that in batch (11569 UL -1 ) and fixed volume fed batch (36911 UL -1 ) fermentations in cane molasses medium. Phytase productivity was sustainable in cyclic fed batch fermentation over seven days, and therefore, this mode of fed batch fermentation appears to be a better approach for producing phytase in a cost-effective manner.

Rethinking the cane sugar mill by using selective fermentation of reducing sugars by Saccharomyces dairenensis, prior to sugar crystallization

Abstract High yield sugarcane is expected to resolve the competition between food and fuel regarding farmland and biomass resources. However, its higher composition of reducing sugars (i.e., glucose and fructose), which inhibit sucrose crystallization, hinders the production of sugar from high yield sugarcane. Under the conventional integrated sugar–ethanol manufacturing system, high biomass yield causes only the increase of ethanol production because of the increase in unrecovered sugar after extraction, which represents a failure in resolving the competition. The technology presented here is the world's first to solve this problem via selective ethanol fermentation using Saccharomyces dairenensis, an unconventional yeast that ferments only reducing sugars and leaves sucrose untouched. A laboratory-scale test using sugarcane juices with a high composition of reducing sugars (100 g kg−1) resulted in a sucrose crystal yield increase from a single extraction, from 16.2 to 65.1%, by introducing selective fermentation. The second extraction, from the molasses, which was enabled by the lowered residual reducing sugar composition, further enhanced the total sugar crystal yield (up to 83.4%). A simulation of the application of this technology in the U.S.A. revealed that both sugar and ethanol production were enhanced, whereas sugar production declined by the mere adoption of high yield cultivar, even with the increase in sugarcane yield.

Biocatalytic exploitation of enoate reductase from unconventional yeasts

Biocatalytic exploitation of enoate reductase from unconventional yeasts

Ethanol production from olive prunings by autohydrolysis and fermentation with Candida tropicalis

Hydrolysates from olive prunings (a renewable, low-cost, easily available, agricultural residue) were fermented with the unconventional yeast Candida tropicalis NBRC 0618 to produce not only ethanol fuel but also xylitol as a by-product, which adds value to the economic viability of the bioprocess. Autohydrolysis took place at 200 °C in a stirred stainless-steel tank reactor. The influence of the solid/liquid ratio in the reactor was studied. Fermentation experiments were conducted in a batch-culture reactor at a temperature of 30 °C, a stirring rate of 500 rpm and pH values of between 5.0 and 6.5. Under the operating conditions tested the highest yields of ethanol and xylitol were obtained with the hydrolysate fermented at pH 5.0 and solely the airflow that entered via the stirring vortex. Under these conditions, the instantaneous ethanol yield was 0.44 g g −1 and the overall xylitol yield 0.13 g g −1.

Pichia Power: India's Biotech Industry Puts Unconventional Yeast to Work

As a platform for making recombinant proteins, E. coli is hard to beat. This universal gut bacterium grows quickly and eagerly, and—with simple genetic tinkering—will make virtually any foreign protein. However, E. coli, a prokaryote, can't handle posttranslational modifications; the proteins it makes are often misfolded and insoluble, requiring expensive processing steps to make them usable. Mammalian cells, in contrast, fold and modify proteins with ease but are much harder to culture. One organism that potentially combines the advantages of bacterial and mammalian expression systems is Pichia pastoris, a harmless species of yeast that feeds on methanol.

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha in a model wastewater system

In search of the optimal way to reduce the hazards of environmental contamination by formaldehyde (FD) and methanol the use of unconventional yeasts is proposed as exemplified by the methylotrophic yeast Hansenula polymorpha. In a very simplified environment of a model wastewater solution, H. polymorpha cells were able to grow on, and metabolize formaldehyde and methanol, applied as sole carbon sources, at concentrations typical for wastewaters of the chemical industry. Several experimental conditions were tested for cell growth and biodegradation kinetics. It was found that the yeast culture inoculated at low cell density was able to grow on initial FD levels up to 400 mg/l and the biomass yield was dependent on both, the amount of total carbon added and the physiological state of the cells. When high density of preadapted cell culture was used, the methylotrophs were fully viable and able to degrade formaldehyde present at initial concentrations up to 700 mg/l. The maximum limiting FD consumption rate was determined as approx. 400 mg/l per hour. Methanol, at concentrations up to 2%, was easily utilized and did not have a negative effect on cell growth and respiration. It is suggested that in real wastewaters the eukaryotic microorganisms — in contrast to bacteria — might reveal greater adaptation potential to toxic levels of formaldehyde as well as to other wastewater constituents.

Nonconventional Yeasts: Their Genetics and Biotechnological Applications

To date, more than 500 species of yeasts have been described. Most of the genetic and biochemical studies have, however, been carried out with Saccharomyces cerevisiae. Although a considerable amount of knowledge has been accumulated on fundamental processes and biotechnological applications of this industrially important yeast, the large variety of other yeast genera and species may offer various advantages for experimental study as well as for product formation in biotechnology. The genetic investigation of these so-called unconventional yeasts is poorly developed and information about corresponding data is dispersed. It is the aim of this review to summarize and discuss the main results of genetic studies and biotechnological applications of unconventional yeasts and to serve as a guide for scientists who wish to enter this field or are interested in only some aspects of these yeasts.

Nutrition An Interdisciplinary Survey

AbstractWith a critical world food situation and an increasing protein gap between the rich and the poor, scientific and technological solutions exist, at least in principle, to extend food production and thus avoid world wide famine. Human food requirement, malnutrition, the world markets of supply and food habits are reviewed. Conventional developments of food production consist in expanding arable land, the Green Revolution more fertilizers and irrigation, plant protection and the accumulation of reserves. But the efficient processing of animal and plant proteins is equally important and so is the production of new concentrated proteins. Amongst the more unconventional developments, algae, fungi, yeast and bacteria as single cell proteins, are only the first steps in the biotechnological production of natural substances for human consumption to remove the food crisis.