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

生物炼油是以可持续生物技术替代石油产品的一种解决方案，即从可再生原料中生产高附加值的化学品。生物质转化过程受到与产品纯化和回收相关的高成本的阻碍，在许多情况下，其可高达总生产成本的50%-80%。产品回收成本如此之高的主要原因是因为有机酸发酵需要控制在中性pH，以确保发酵微生物处于其最佳性能条件。当产品有机酸产生并逐渐积累在发酵罐中时，肉汤pH值立即降低并漂移。然后添加碱以调节pH值，从而形成有机酸盐。考虑到商业利益的大多数有机酸的pKa值在3和5之间，使用可以在低于pH 4.0下有效地产生有机酸的生产主体将减少或消除有机酸盐的形成。因此，需要开发具有低于4.0的最佳pH的新生产宿主。几种丝状真菌可以天然地产生高水平的有机酸，但是由于它们的丝状生长、缺乏遗传多样性以及潜在有害副产物如黄曲霉毒素的风险，它们难以工作。已知各种酵母菌株在酸性条件下生长的能力，并且更易于遗传操作。保加利亚酵母（Saccharomyces bulderi，又名Kazachstania bulderi）是一种从厌氧玉米青贮饲料中分离出来的酵母菌，具有新颖的生理特性，能够在5.0至2.5的pH范围内维持有效的生长速率。这种生长特性是该种特有的生理适应的结果，使K. bulderi是一个很好的候选人，被开发为新的生产宿主低pH发酵。哈萨克斯坦属有大约63个相关物种，尽管与酵母菌密切相关，但只有有限的遗传研究和分子工具可用。与酵母属相比，该属在表型、形态、基因组大小和染色体数目方面相当多样化。在这里，我们建议完全覆盖三个已知的K。在遗传和基因组水平上。我们打算进行全基因组测序，将基因组组装成染色体，确定多态性，倍性和染色体重排。这一知识将为我们提供分子起点，以了解这一物种，并创造一系列遗传工具，以快速操纵它。具体而言，我们将工程菌株产生代理有机酸（即乳酸）作为概念验证水平。收集的这些菌株在高pH和低pH下生长的全球基因表达数据将有助于我们确定负责酸性环境特定生理适应的关键参与者。酵母物种之间的杂交在自然和驯化环境中很容易发生，在相同的遗传背景下将不同的性状结合在一起。混合动力车可以适应特定的条件，因此在一些恶劣的工业环境中表现更好。我们打算交叉不同的菌株和物种的哈萨克斯坦属，并评估所得的杂种的基因组稳定性和线粒体DNA遗传（因为不同类型的线粒体可以影响表型）。将选择在低pH下生物量提高的杂交种。bulderi作为通过发酵生产有机酸的新生产宿主。

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.