Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as ‘global transcription machinery engineering’ (gTME), to induce genetic variation, providing a new source of yeast genetic diversity.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/1574-6976.12073

Improving industrial yeast strains: exploiting natural and artificial diversity

Chitin is the second most abundant organic and renewable source in nature, after cellulose. Chitinases are chitin-degrading enzymes. Chitinases have important biophysiological functions and immense potential applications. In recent years, researches on fungal chitinases have made fast progress, especially in molecular levels. Therefore, the present review will focus on recent advances of fungal chitinases, containing their nomenclature and assays, purification and characterization, molecular cloning and expression, family and structure, regulation, and function and application.

Review of fungal chitinases.

A 1.1 kb rice genomic DNA fragment, containing a chitinase gene under the control of the CaMV 35S promoter, was cloned into the rice transformation vector pGL2. After transformation of Indica rice protoplasts in the presence of polyethyleneglycol, plants were regenerated. The presence of the chimeric chitinase gene in T0 and T1 transgenic rice plants was detected by Southern blot analysis. Western blot analysis of transgenic plants and their progeny revealed the presence of two proteins with apparent molecular weights of 30 and 35 kDa that reacted with the chitinase antibody. Progeny from the chitinase-positive plants were tested for their resistance to the sheath blight pathogen, Rhizoctonia solani. The degree of resistance displayed by the transgenic plants to this pathogen correlated with the level of chitinase expression.

Genetic engineering of rice for resistance to sheath blight and other agronomic characters

Fungal plant diseases are one of the major concerns to agricultural food production world wide. Soil borne pathogenic fungi such as Pythium, Fusarium, Rhizoctonia and Phytopthora attack most of the economically important crop plants (either through seed root before germination or seedling after germination) resulting in loss of billions of dollars. Moreover, the management of chitinous waste is also pressing need today. Mycolytic enzymes (chitinases, proteases and glucanase) producing microorganisms may help in solving these problems. These microorganisms have ability to lyse the fungal cell wall and also have the potential to manage the chitinous waste by producing chitinases. Many chitinolytic microorganisms have potential to control fungal plant pathogens but they are not fully successful in all the cases due to different geological and environmental conditions. Thus, bioprospecting to find novel, highly chitinolytic microorganisms which help in developing potential biocontrol agent. Furthermore, to increase the survivability of biocontrol agents, a formulation may also be necessary. This review is focused on the progress of chitinase genes, chitinolytic microorganisms and their diversity as well as formulation of chitinolytic producers which have the potential to control fungal plant pathogens

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Bioprospecting and antifungal potential of chitinolytic microorganisms

Metagenomics has accelerated the process of discovery of novel biocatalysts by enabling scientists to tap directly into the entire diversity of enzymes held within natural microbial populations. Their characterization has revealed a great deal of valuable information about enzymatic activity in terms of factors which influence their stability and activity under a wide range of conditions. Many of the biocatalysts have particular properties making them suitable for biotechnological applications. A diverse array of strategies has been developed to optimize each step of the process of generating and screening metagenomic libraries for novel biocatalysts. This review covers the diversity of metagenome-derived enzymes characterized to date, and the strategies currently being developed to optimize discovery of novel metagenomic biocatalysts.

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Advances in recovery of novel biocatalysts from metagenomes.

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F1 progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.

/pdf/the-saccharomyces-cerevisiae-chitinase-encoded-by-the-cts1-2-4etvc4wr2f.pdf

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco

The excessive use of pesticides to prevent the outbreak of plant diseases holds various disadvantages for the cost-effectiveness of agricultural production prac- tices, the quality of the end products, the environment and the labour force. The mount- ing public resistance to the use of hazardous pesticides on agricultural crops has hastened the search for natural antimicrobial peptides, enzymes and other types of biological con- trol agents. The objective of this study was to investigate the potential of the antifungal activity of a yeast-derived chitinase for possible future agricultural applications. When plants are exposed to fungal pathogens, they produce pathogenesis-related (PR) defence proteins, such as chitinases, in order to degrade the chitin in the cell walls of the attack- ing fungi, thereby inhibiting further fungal growth and the development of hyphae. We have cloned the Saccharomyces cerevisiae chitinase gene (CTS1-2) into a multicopy 2µ-based plasmid and overexpressed it under the control of the phosphoglycerate kinase I gene (PGK1) promoter (PGK1 P ) and terminator (PGK1 T ) sequences. Secretion of the recombinant CTS1-2-encoded chitinases was directed by the native leader peptide, or by the secretion signals of either the yeast mating pheromone α-factor (MFα1 S ) or the Tri- choderma reesei β-xylanase 2 (XYN2 S ). Northern blot analysis confirmed the PGK1 PT - directed expression of CTS1-2 and an indirect enzyme assay was used to compare the efficiency of secretion of the three different recombinant chitinases. These recombinant chitinases were also shown to effectively inhibit spore germination and hyphal growth of Botrytis cinerea, an economically important fungal pathogen of several agricultural crop plants, including grapevine. This study could lead to the development of yeast chitinases and/or chitinolytic yeasts as biocontrol agents to combat plant diseases and reduce the use of chemical pesticides in the agricultural industry.

M. Carstens

Papers

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco

Overexpression, secretion and antifungal activity of the Saccharomyces cerevisiae chitinase