Biotechnology of non-Saccharomyces yeasts—the basidiomycetes

Yeasts are the major producer of biotechnology products worldwide, exceeding production in capacity and economic revenues of other groups of industrial microorganisms. Yeasts have wide-ranging fundamental and industrial importance in scientific, food, medical, and agricultural disciplines (Fig. 1). Saccharomyces is the most important genus of yeast from fundamental and applied perspectives and has been expansively studied. Non-Saccharomyces yeasts (non-conventional yeasts) including members of the Ascomycetes and Basidiomycetes also have substantial current utility and potential applicability in biotechnology. In an earlier mini-review, “Biotechnology of non-Saccharomyces yeasts—the ascomycetes” (Johnson Appl Microb Biotechnol 97: 503–517, 2013), the extensive biotechnological utility and potential of ascomycetous yeasts are described. Ascomycetous yeasts are particularly important in food and ethanol formation, production of single-cell protein, feeds and fodder, heterologous production of proteins and enzymes, and as model and fundamental organisms for the delineation of genes and their function in mammalian and human metabolism and disease processes. In contrast, the roles of basidiomycetous yeasts in biotechnology have mainly been evaluated only in the past few decades and compared to the ascomycetous yeasts and currently have limited industrial utility. From a biotechnology perspective, the basidiomycetous yeasts are known mainly for the production of enzymes used in pharmaceutical and chemical synthesis, for production of certain classes of primary and secondary metabolites such as terpenoids and carotenoids, for aerobic catabolism of complex carbon sources, and for bioremediation of environmental pollutants and xenotoxicants. Notwithstanding, the basidiomycetous yeasts appear to have considerable potential in biotechnology owing to their catabolic utilities, formation of enzymes acting on recalcitrant substrates, and through the production of unique primary and secondary metabolites. This and the earlier mini-review (Johnson Appl Microb Biotechnol 97:503–517, 2013) were motivated during the preparation and publication of the landmark three-volume set of “The yeasts: a taxonomic study, 5th edition” (Kurtzman et al. 2011a, b).     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

Biotechnology of ornamental plants: when beauty joins science—preface from the editors

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Biotechnology and molecular biology of the α-glucosidase inhibitor acarbose

The -glucosidase inhibitor acarbose, O-{4,6-dideoxy-4[1s-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino--d-glucopyranosyl}-(14)-O--d-glucopyranosyl-(14)-d-glucopyranose, is produced in large-scale fermentation by the use of strains derived from Actinoplanes sp. SE50. It has been used since 1990 in many countries in the therapy of diabetes type II, in order to enable patients to better control blood sugar contents while living with starch-containing diets. Thus, it is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market. Cultures of Actinoplanes sp. also produce various other acarbose-like components, of which component C is hard to separate during downstream processing, which is one of the most modern work-up processes developed to date. The physiology, genetics and enzymology of acarbose biosynthesis and metabolism in the producer have been studied to some extent, leading to the proposal of a new pathway and metabolic cycle, the carbophore. These data could give clues for further biotechnological developments, such as the suppression of side-products, enzymological or biocombinatorial production of new metabolites and the engineering of production rates via genetic regulation in future.     

Molecular polymorphism of β-fructosidase SUC genes in the Saccharomyces yeasts

The molecular polymorphism of SUC genes that encode β-fructosidase has been investigated in the yeast genus Saccharomyces. We have determined the nucleotide sequences of subtelomeric SUC3, SUC5, SUC7, SUC8, SUC9, and SUC10 genes of S. cerevisiae and the SUCa gene of S. arboricola. Comparisons of the nucleotide sequences of all known SUC genes revealed the predominance of C → T transitions in the third codon position, which were silent. The amino acid sequences of β-fructosidases studied have identity of 88–100%. SUCa (S. arboricola) and SUCb (S. bayanus) proteins, which had amino acid identity with other SUC proteins of less than 92%, were the most divergent. It was determined that accumulation of the polymeric SUC genes takes place in industrial populations of S. cerevisiae, while the other Saccharomyces species (S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, and S. paradoxus) each harbor only one SUC gene. Subtelomeric repeats of β-fructosidase SUC genes could appear in the genome of S. cerevisiae under the effect of selection in the course of their domestication.     

Biotechnology of β-adrenergic receptors

The emergence of Biotechnology has provided pharmacologists with a variety of methods for investigating the structure, the function, and the regulation of membrane-bound receptors with a precision that was not imagined even five years ago. These new tools have been developed and used to analyze the known catecholamine β₁- and β₂ receptors and to discover and study a new subtype, the β-adrenergic receptor. We review here the salient features of each of these three receptors, compare their structural and functional properties, and propose models to explain their differential regulation in time and space. A whole family of proteins has now been found to share with the β-adrenergic receptors their most prominent features, including seven transmembrane domains and coupling with GTP-binding “G” proteins. We therefore propose that the biotechnology-based procedures developed for the β-adrenergic receptors will be well applicable to the other members of this “R₇G” family of receptors.     

Distribution of aquatic yeasts — Effect of incubation temperature and chloramphenicol concentration on isolation

Fresh (river), estuarine, and marine waters in and along the coastline of Connecticut were cultured by the membrane filter technique at 20 and 37°C on a complex medium containing 0–1000 mg/L of chloramphenicol. Using counts on medium with 500 mg/L antibiotic as a base, ratios of total and pink yeast counts were recorded for other chloramphenicol concentrations at both temperatures for the waters sampled. Variable results were obtained; in general, both total and pink yeast counts decreased with increasing antibiotic levels, being most apparent at > 400 mg/L chloramphenicol. Medium without antibiotic and with 100 mg/L always produced baterial overgrowth. A total of 209 white yeasts were isolated from all platings; the genera Torulopsis, particularly T. Candida, and Candida were dominant with lesser numbers of Cryptococcus, Trichosporon, sporogenous genera, and Kloeckera. Most species isolated were found on media at all chloramphenicol levels. Comparisons were made of yeast distributions in these temperate waters with reports from other areas.Contribution No. 101 from the University of Connecticut Marine Research Laboratory.     

Biotechnology: the language of multiple views in Māori communities

In Aotearoa (New Zealand), the government funded studies on communicating biotechnology to different sectors in the community from 2003 to 2006. Subsequently, a researcher covering the Māori sector performed a content analysis of data gathered in the community. Qualitative analysis methods included examining text from participant interviews, focus groups, government documents, newspapers, Internet sites, and current literature. Content was coded by identifying common themes in the English and the Māori language. Words like genetic modification (GM), genetic engineering (GE), and biotechnology were explained to provide a basic understanding between the communities and researcher. The terminology applied in the research was essential to achieve communication between the researcher and the community. The resultant themes represented seven views to interpret the communities association with biotechnology: purist Māori, religious Māori, anti Māori, pro Māori, no Māori, uncertain Māori, and middle Māori views. The themes are taken from the analysis of data compiled after 3 years of completing different stages of a research project. The views indicate that a common understanding can be achieved in the diverse range of Māori tribal communities providing those communicating biotechnology can identify the view and interpretations communities associate with biotechnology. This knowledge is essential for government agencies, researchers, community practitioners, scientist, and businesses that desire to dialogue with Māori communities in the language of biotechnology.     

A possible relationship between the fatty acid composition of yeasts and the ‘petite’ mutation

A number of Crabtree-positive and Crabtree-negative yeasts were tested for their ability to yield respiratory-deficient (petite) mutants on treatment with acriflavine. Crabtree-positive species produced petite mutants but did not contain the polyunsaturated linoleic (C 18.2) and linolenic (C 18.3) fatty acids. Crabtree-negative species contained these fatty acids and were resistant to acriflavine. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Anamylopsoraceae — a new family of lichenized ascomycetes with stipitate apothecia (Lecanorales: Agyriineae)

The anatomy, chemistry and developmental morphology ofAnamylopsora pulcherrima is investigated. Some characters, including the ascus structure, suggest a close affinity with theAgyriaceae. However, the chemistry and the pycnidial structure differ as well as the ascoma ontogeny.Anamylopsora has a gymnocarpous ascoma development and the ascogonia are produced in stipes.Trapelia coarctata, as a typical member of theAgyriaceae, shows a hemiangiocarpous ascoma ontogeny. The anatomical, chemical and ontogenetical characters of several families are compared withAnamylopsora and it is shown that the genus is best placed in a monotypic familyAnamylopsoraceae Lumbsch & Lunke, fam. nova, which is placed in theAgyriineae (Lecanorales).This paper is dedicated to Prof. DrAino Henssen (Marburg) on the occasion of the 70th birthday.     

Should we hail the Red King? Evolutionary consequences of a mutualistic lifestyle in genomes of lichenized ascomycetes

The Red Queen dynamic is often brought into play for antagonistic relationships. However, the coevolutionary effects of mutualistic interactions, which predict slower evolution for interacting organisms (Red King), have been investigated to a lesser extent. Lichens are a stable, mutualistic relationship of fungi and cyanobacteria and/or algae, which originated several times independently during the evolution of fungi. Therefore, they represent a suitable system to investigate the coevolutionary effect of mutualism on the fungal genome. We measured substitution rates and selective pressure of about 2000 protein‐coding genes (plus the rDNA region) in two different classes of Ascomycota, each consisting of closely related lineages of lichenized and non‐lichenized fungi. Our results show that independent lichenized clades are characterized by significantly slower rates for both synonymous and non‐synonymous substitutions. We hypothesize that this evolutionary pattern is connected to the lichen life cycle (longer generation time of lichenized fungi) rather than a result of different selection strengths, which is described as the main driver for the Red Kind dynamic. This first empirical evidence of slower evolution in lichens provides an important insight on how biotic cooperative interactions are able to shape the evolution of symbiotic organisms.     

Biotechnology and the improvement of oil crops – genes,dreams and realities

During the past decade, there have been many optimistic claims concerning the potential of novel oil-based products from genetically engineered crops, particularly for the manufacture of a new generation of renewable, carbon-neutral, industrial materials. Such claims have been underpinned by an impressive series of scientific advances that have resulted in the isolation of genes encoding most of the enzymes directly involved in oil biosynthesis. In some cases, these enzymes have even been re-engineered by site-directed or random mutagenesis to allow production of new fatty acid profiles that are not present in any existing organism. This has opened up the prospect of engineering `designer oil crops' to produce novel fatty acids with chain lengths from C8 to C24 and with a wide range of industrially useful functionalities, including hydroxylation, epoxidation, and conjugated and non-conjugated double or triple bonds. However, there remain significant technical challenges before this promise of designer transgenic crops is likely to be translated into large-scale commercial reality. For example, it has proved surprisingly difficult to engineer high levels of novel fatty acids in genetically engineered transgenic plants, although many wild type seeds can readily accumulate 90–95% of a single fatty acid in their storage oil. Another complication is the recent discovery of multiple pathways of triacylglycerol biosynthesis and the difficulty in ensuring that novel fatty acids are only channelled towards storage triacylglycerols and not to membrane or signalling lipids in major target crops like rapeseed. New findings from our lab have suggested that there may also be problems with the tissue specificity of some of the `seed-specific' gene promoters that are commonly used in transgenic crops. There are also considerable and often underestimated challenges associated with the economics, management and public acceptability of all transgenic crops, even for non-food use. In most cases the projections of petroleum reserves over the next few decades make it unlikely that crop-derived commodity products that substitute for petroleum will be competitive. Also the scale of crop production required to generate millions of tonnes of commodity oils, e.g., for biodegradable plastics, is likely to seriously impinge on food production at a time of increasing global populations, and is therefore unlikely to be acceptable. An alternative strategy to transgenic oil crops is to use molecular breeding techniques in order to develop new crops that already synthesise high levels of novel fatty acids of interest. Finally, the most promising market sectors and product ranges for the future development of oil crop biotechnology will be discussed.     

The Second Editorial Board of Chinese Journal of Biotechnology

ADVISORJIAORui Shen (J.S .Chao)　　Professor(InstituteofPlantPhysiologyandEcology,ShanghaiInstitutesforBiologicalSciences,ChineseAcademyofSciences,Shanghai2 0 0 0 32 ,China)EDITOR IN CHIEFYANGSheng Li　　Academician(ShanghaiResearchCenterforBiotechnology ,ChineseAcademyofSciences,Shanghai 2 0 0 2 33,China)VICE EDITOR IN CHIEF (alphabetically)FANGRong Xiang　　Academician(InstituteofMicrobiology ,ChineseAcademyofSciences,Beijing 10 0 0 80 ,China)HEFu Chu　　A…     

The growth of yeasts at 37° C

Summary More than 2,300 strains of 70 species of yeasts have been tested on yeast autolysate agar at 37° C. Of these, all strains of 15 species grew at this elevated temperature while no strains of 13 species grew well. The remaining 42 species, represented by 2 or more strains each, included strains both capable and incapable of growth at 37° C. It is suggested that such species include two groups of strains, one capable of adaptation to growth conditions at elevated temperatures. In sewage-polluted waters such strains may be indicative of fecal pollution.U.S. Department of Health, Education, and Welfare, Bureau of State Services, Public Health Service.     

The influence of different yeasts on the fermentation,composition and sensory quality of cachaça

Summary Cachaça (sugarcane wine) was produced using different yeast strains, six being strains of Saccharomyces cerevisiae and one each of Candida apicola, Hanseniaspora occidentalis, Pichia subpelliculosa and Schizosaccharomyces pombe. The ethanol yields (%) of the non-Saccharomyces strains were similar to those of the Saccharomyces strains. The following determinations were carried out on the cachaça: acetaldehyde, ethyl acetate, propanol, isobutyl alcohol, isoamyl alcohol, volatile acidity. The cachaças showed variations in the levels of secondary compounds, but these variations did not result in differences (P ≤ 0.05) in the sensory attributes of aroma and flavour and overall impression. Of the volatile compounds quantified in the cachaças, only propanol showed a positive correlation (P ≤ 0.05) with the flavour attributes and overall impression. The S. pombe strain was considered inadequate for the production of cachaça. The cachaças were classified into five groups in an exploratory Hierarchical Cluster Analysis as a function of the volatile compounds. Principal Component Analysis showed that 93% of the variation (PC 1) occurred among the samples, and was explained by the individual volatile compounds and the total secondary compounds, with the exception of isoamyl alcohol only 7% (PC 2) was associated with the volatile acidity. The negative correlations shown between the volatile compounds of the cachaças and the ethanol content of the sugarcane wine, with the exception of acetaldehyde, showed that the variation in ethanol content of the sugarcane wine is an important factor for standardization of the ethanol/volatiles ratio and the beverage quality.