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

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.

The problem faced by bio-refinery industries is the high costs related to product purification and recovery. Organic acid fermentation needs to be controlled at neutral pH where most of fermentation microorganisms are at their optimal performance condition. However, when the organic acid is produced, it accumulates in the fermenter and the broth pH decreases, hampering microbial growth. There is therefore a need to develop new production hosts that have an optimum pH below 4.0. Saccharomyces bulderi (aka Kazachstania bulderi) is a Saccharomyces sensu lato yeast able of grow efficiently over a wide range of pHs between 5.0 and 2.5, making this species an excellent candidate to be developed as a new production host. The genus Kazachstania is quite diversified in term of phenotypes, morphologies, and genome sizes, and despite the fact is closely related to Saccharomyces only limited genetic studies and molecular tools are available.
Here, we propose to characterise at molecular level the three known species of K. bulderi. Whole genome sequences, using both short and long read sequencing platforms, will be obtained and full genome assembly and annotation will be carried out. Knowledge on ORFs, genome structure, ploidy, MAT type locus, and specific metabolic genes will inform the construction of molecular tools for this species and allow strain engineering. As proof of concept level we will construct D-lactic acid or L-lactic acid strains by overexpressing the D- or L- lactate dehydrogenase. Stranded RNA-seq data for these strains grown at high and low pH will be obtained to help us to identify the key players responsible for the physiological adaptations to acidic environments. Hybridisation potential between Kazachstania strains and species will be investigated and the resulting hybrids assessed for genome stability and mitochondria DNA inheritance (since different type of mitochondria can affect phenotype), and improved biomass at low pH.

Who will benefit from this research?

We have identified numerous groups of users and beneficiaries outside the academic research community who will benefit from the research in this proposal. These are companies working on production of bio-based chemicals, company developing fermentation processes, primary school children, secondary school children and member of the public.

How will they benefit from this research?

The research in this proposal is predominantly basic science. The knowledge obtained through this project will provide the fundamental tools, protocols and genomic understanding to develop new improved host strains and hybrids of the Saccharomyces sensu lato group for biotechnological purposes. We can impart this new knowledge to our student beneficiaries through the numerous engagement activities we undertake (see Pathways to Impact). In addition, results from this work will provide valuable information, relevant to industries, on which are the molecular players responsible for the unique capacity of K. bulderi to grow efficiently in a wide range of pHs. There are reports that K. bulderi is the second most abundant yeast present in corn silage (18.6%), in together with other Kazachstania species, represent 36% of the yeast microbiome in corn silage. A better knowledge of the physiological characteristics of K. bulderi might help understand the role that this strain plays on the formation and/or spoilage of corn silage, which is one of the most important animal feeds produced around the world.

What will be done to ensure that they have the opportunity to benefit from this research?

The University of Manchester has a variety of school-oriented activities, which we will utilise to disseminate the latest science concepts: i. attending major annual open days at the Manchester Institute of Biotechnology for A-level students where we will exhibit our work; ii. participating at Manchester Science Week, where we will bring our research researchers into contact with members of the public; iii. participating at British Science Week - Schools' Fair, an event in which ~1000 school children (aged 10-14) are invited on campus to experience engaging talks and hands on experimental activities; iv. developing podcasts/web-based material to disseminate our science to a wide audience; v. working closely with MIB Director of Commercialisation, UMI3 and the intellectual property manager of the BP Biosciences Center to investigate commercial options resulting from research projects and patents; vi. accessing the MIB portfolio of funded industry research collaborations that will help to provide foresight and engagement opportunities; vii. promoting exchange of knowledge and experiences between UoM and BP.
All these engagement activities will continue and develop through feedback from the beneficiaries.