Development of molecular and genomic tools for the low pH production host Saccharomyces bulderi (aka Kazachstania bulderi)

点击翻译按钮获取中文摘要

Bio-refinery has been proposed as a solution to replace oil-derived products with sustainable biotechnologies, which is to produce value-added chemicals from renewable feedstocks. Biomass conversion processes are hampered by the high costs linked to product purification and recovery, which in many cases can be as high as 50%-80% of the total production cost. The primary reason that product recovery cost is so high is because organic acid fermentation needs to be controlled at neutral pH to ensure that the fermentation microorganisms is at its optimal performance condition. When product, organic acid is produced and gradually accumulates in the fermenter, broth pH decreases and drifts immediately. Base will then be added to adjust pH, which results in formation of the organic acid salt. Given that pKa values for most organic acids of commercial interests are between 3 and 5, the use of production hosts that can produce organic acids efficiently below pH 4.0, will decrease or eliminate the formation of organic acid salts. There is therefore, a need to develop new production hosts that have an optimum pH below 4.0.Several species of filamentous fungi can naturally produce high levels of organic acids, however they are difficult to work with because of their filamentous growth, lack of genetic versatility, and the risk of potential harmful by-products such as aflatoxins. Varieties of yeast strains are known for their capability of growth under acidic conditions, and are more amenable to genetic manipulation. Saccharomyces bulderi (aka Kazachstania bulderi), isolated in anaerobic maize silage, is a Saccharomyces sensu lato yeast species with novel physiological characteristics, able to sustain efficient growth rate over a wide range of pHs between 5.0 and 2.5. Such growth characteristics are the results of specific physiological adaptations occurred in this species, making K. bulderi an excellent candidate to be developed as a new production host for low pH fermentation. The genus Kazachstania has around 63 associated species, and despite the fact is closely related to Saccharomyces only limited genetic studies and molecular tools are available. This genus is quite diversified in term of phenotypes, morphologies, genome sizes and chromosome numbers, compared to the genus Saccharomyces. Here, we propose to fully characterise the three known species of K. bulderi at genetic and genomic level. We intent to carry out whole genome sequencing, assemble the genomes into chromosomes, determine polymorphisms, ploidy and chromosomal rearrangements. This knowledge will give us the molecular starting point to understand this species and to create an array of genetic tools for its swift manipulation. Specifically we will engineer the strains to produce a proxy organic acid (i.e. lactic acid) as a proof of concept level. Data on global gene expression collected for these strains grown at high and low pH will help us to identify the key players responsible for the specific physiological adaptations to acidic environments. Hybridisation between yeast species occurs readily in natural and domesticated environments, bringing together different traits in the same genetic background. Hybrids can be resilient to specific conditions and therefore perform better in some harsh industrial environments. We intend to cross different strains and species of Kazachstania genus and assess the resulting hybrids for genome stability and mitochondria DNA inheritance (since different type of mitochondria can affect phenotype). Hybrids with improved biomass at low pH will be selected.The ultimate goal is to be able to evaluate K. bulderi as a new production host for the production of organic acids by fermentation.