Application of direct and indirect strain engineering approaches to unlock the potential of the yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus for bio-based processes

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Jayaprakash, Pooja
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University College Cork
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The efficient implementation of biorefineries to produce bio-based chemicals and fuels requires a sustainable source of feedstock and robust microbial factories. Among others, lignocellulosic biomass represents cheap and sugar-enriched feedstock. The conversion of lignocellulosic biomass into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for the production of natural and recombinant products, including bulk chemicals such as organic acids. In bio-based industrial processes, microorganisms are subjected to different kinds of stresses associated with process conditions. These stressors are known to inhibit cellular metabolism and compromise the performances of a fermentative process, being an important limitation to an effective marketability of biobased microbial products. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop viable and competitive bioprocesses. In the chapter 1 of this thesis we reviewed the link between LCBs composition, choice of enzymatic cocktail and selection of yeast species and strains that need to be considered in an integrated fashion to enable the development of an efficient bio-based process. We discussed the pivotal role of enzymatic cocktail optimization to unlock the potential of non-Conventional yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstock and products of biorefineries. The aim of this PhD work was to expand the industrial potential of two non-conventional yeasts, Zygosaccharomyces parabailii and Kluyveromyces marxianus, by applying direct and indirect strain engineering approaches. These yeasts possess desirable characteristics. K. marxianus has broad specificity for both hexose and pentose sugars as carbon and energy source. Apart from this, its thermotolerance, fast growth and the ability to thrive at pH below 3, make it ideal for industrial use. However, the lack of tolerance of this yeast to inhibitory compounds, particularly weak organic acid produced during LCB pretreatment, hinders its use when this biomass is used as substrates. Although the use of synthetic biology techniques has started to be employed to understand the robustness of K. marxianus and for the production of various chemicals, the mechanisms related to organic acid tolerance are yet to be deciphered. To match this goal, we used Adaptive Laboratory Evolution (ALE), an indirect strain engineering approach, alternative and often complementary to direct engineering. In chapter 2, we aimed to improve the tolerance of K. marxianus to sugar beet pulp (SBP) hydrolysate at pH 3.0 at two different temperatures, 30 oC and 40 oC. Using the ALE approach, we selected K. marxianus evolved isolates with robust phenotype compared to the parental strains, at 30 oC. Differently to K. marxianus, the hybrid yeast Z. parabailii exhibits resistance to weak organic acids (WOA) also at low pH. Understanding the mechanism involved in tolerance to WOA can be used for avoiding the growth of this yeast in food production pipelines as well as for promoting its use as a cell factory for the production of organic acids and other bio-products. In chapter 3 of this study, our aim was to understand the phenotype-genotype correlation involved in the WOA tolerance trait. Using direct engineering method, we constructed and characterised single and double Z. parabailii pdr12 mutants. This study revealed that Pdr12p is involved in tolerance to acetic and butyric acids and not in tolerance towards sorbic and benzoic acids. Furthermore, analysis of the Pdr12p sequence provided insights in the amino acids differences. The pdr12 mutants were constructed by the classical tool of exploiting deletion cassettes. Advances in metabolic engineering and synthetic biology have increased the need for creating techniques such as CRISPR-Cas9 for faster and more efficient genome editing. In chapter 4 of this study our aim was to develop a CRISPR-Cas9 system for simultaneous disruption or deletion of two alleles of a gene in Z. parabailii. We evaluated the use of four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements. The functionality of the gRNA systems was tested by analyzing the inactivation of the ADE2 gene in the wild type strain and the most efficient gRNA system was used to successfully construct a Z. parabailii dnl4 mutant. This mutant exhibited improved homologous recombination in the deletion of both ADE2 alleles. Analysis of mutations in the gRNA target regions of both ADE2 and DNL4 genes suggested inter-allelic rearrangements between the two gene loci, as well as absence of large regions of chromosomes. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories.
Biorefinery , Lignocellulosic biomass , Yeast
Jayaprakash, P. 2022. Application of direct and indirect strain engineering approaches to unlock the potential of the yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus for bio-based processes. PhD Thesis, University College Cork.