Delft University of Technology Maltose and maltotriose metabolism in brewing-related Saccharomyces yeasts Brickwedde, Anja DOI 10.4233/uuid:0c847298-0007-4922-aff4-00beb248d664 Publication date 2019 Document Version Final published version Citation (APA) Brickwedde, A. (2019). Maltose and maltotriose metabolism in brewing-related Saccharomyces yeasts. https://doi.org/10.4233/uuid:0c847298-0007-4922-aff4-00beb248d664 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. Maltose and maltotriose metabolism in brewing-related Saccharomyces yeasts Dissertation for the purpose of obtaining the degree of doctor at Delft University of Technology by the authority of the Rector Magnificus Prof. dr. ir. T.H.J.J. van der Hagen, chair of the Board for Doctorates, to be defended publicly on Wednesday 6 March 2019 at 15:00 o’clock by Anja BRICKWEDDE Master of Science in Environmental and Industrial Biology (ISTAB), University of Applied Sciences Bremen, Germany, born in Nordenham, Germany This dissertation has been approved by the promotors. Composition of the doctoral committee: Rector Magnificus, chairperson Prof. dr. J.T. Pronk, Delft University of Technology, promotor Dr. J-M.G. Daran, Delft University of Technology, promotor Independent members: Dr. K. Voordeckers, Catholic University of Leuven (KU Leuven) Dr. J.P. Morrissey, University College Cork Prof. dr. T. Boekhout, University of Amsterdam Prof. dr. E.J. Smid, Wageningen University & Research Prof. dr. P. Osseweijer, Delft University of Technology Substitute member: Prof. dr. W.R. Hagen, Delft University of Technology The research presented in this thesis was performed at the Industrial Microbiology Section, Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, the Netherlands. The project was financed by the Seventh Framework Programme of the European Union in the frame of the SP3 people support for training and career development of researchers (Marie Curie), Networks for Initial Training (PITN-GA-2013 ITN-2013-606795) YeastCell. Contents Summary ............................................................................................................ 1 Samenvatting ...................................................................................................... 5 Chapter 1: Introduction......................................................................................... 9 Chapter 2: Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS 12357T .............................................................. 31 Chapter 3: S. cerevisiae x S. eubayanus interspecific hybrid, the best of both worlds and beyond ............................................................................................................. 75 Chapter 4: Evolutionary engineering in chemostat cultures for improved maltotriose fermentation kinetics in Saccharomyces pastorianus lager brewing yeast ................. 103 Chapter 5: Outlook ........................................................................................... 127 References ...................................................................................................... 133 Acknowledgements .......................................................................................... 161 Curriculum vitae .............................................................................................. 165 List of publications ........................................................................................... 167 Summary Summary Grain-based fermented beverages have been produced since, several millennia BCE, human societies developed a settled lifestyle based on agriculture. In the 15th century CE, lager type beers similar to the present-day ones first emerged in Central Europe. Lager beer is produced from barley, hops, and water. Conversion of carbohydrates from grains to ethanol and CO2 is catalysed by Saccharomyces pastorianus yeasts. Those yeasts are hybrids of S. cerevisiae and S. eubayanus that, through their ability to efficiently ferment the predominant wort sugars at low temperatures, combine traits from both parents. Remarkably, S. pastorianus strains have exclusively been isolated from man-made environments related to lager beer brewing, and not from natural ecosystems, suggesting that they have arisen and evolved in breweries. Maltose and maltotriose are taken up by S. pastorianus via a set of proton symporters, the structural genes for which are derived from each of its parents and subsequently evolved throughout the process of brewing yeast domestication. In contrast to the well-studied maltose uptake and hydrolysis in S. cerevisiae, knowledge on transport and metabolism of this α-glucoside by S. eubayanus is limited. The study described in Chapter 2 of this thesis systematically investigates the functionality of all annotated genes encoding potential maltose transporters present in the S. eubayanus type strain CBS12357, namely MALT1, MALT2, MALT3, and MALT4. First, using Oxford Nanopore Technology’s MinION long-read sequencing platform, a near-complete genome sequence of this strain was assembled, which provided, amongst other new genomic features, complete sequence information of the four MAL loci. All S. eubayanus MALT genes were then separately expressed in a maltose-transport-negative S. cerevisiae strain background. The four genes were shown to support growth on synthetic medium with maltose as sole carbon source, thereby confirming their ability to encode functional maltose transport proteins. In addition to the heterologous expression of single transporters in S. cerevisiae, MALT genes were systematically deleted in S. eubayanus CBS12357 using CRISPR-Cas9 based genome editing to explore their contribution to maltose utilization in their natural genetic context. Deletion of MALT2 and MALT4, which share 99.7% sequence identity, led to a strain which did not grow on maltose. Conversely, strains carrying deletions in both MALT1 and MALT3 showed a similar physiology as the initial strain. The conclusion that Malt2 and Malt4 are the only maltose transporters active in CBS12357 was further supported by increased expression levels of MALT2 and MALT4 in cultures grown on maltose. In contrast, MALT1 and MALT3 showed negligible expression levels in maltose-grown cultures. In Chapter 3 of this thesis, two major characteristics of lager yeasts that are relevant for the success of the brewing process, cryotolerance and the ability to ferment maltose and maltotriose, are investigated in an interspecies hybrid that was constructed via mass mating of a haploid derivative of the S. eubayanus type strain CBS12357 and a 1 Summary haploid S. cerevisiae laboratory strain of the CEN.PK family. The performance of the constructed hybrid, strain IMS0408, was then compared to those of its parents in anaerobic batch cultures grown on different media and at different temperatures. While S. eubayanus displayed significantly higher growth rates than S. cerevisiae in anaerobic batch cultures below 25 °C, the laboratory hybrid IMS0408 performed as well as the best parent or even better at most tested temperatures. In contrast to its S. eubayanus parent, the hybrid strain was further able to consume maltotriose, the second most abundant sugar in wort, in cultures grown on sugar mixtures. This observation showed how acquisition of the S. cerevisiae genome contributed an important brewing related characteristic of the hybrid. The hybrid strain IMS0408 showed a best parent heterosis in two major characteristics that are relevant in the brewing environment. This heterosis illustrates how an early, spontaneous S. pastorianus lager brewing hybrid might have outcompeted other Saccharomyces species, including its parental ones, under the low- temperature, high-maltotriose conditions of lager fermentation processes. While maltose is fermented rapidly by most S. pastorianus strains, maltotriose is often taken up much slower than maltose and in many cases not completely fermented. Such suboptimal maltotriose fermentation kinetics compromise the stability and economics of brewing processes. Chapter 4 explores whether evolutionary engineering can be applied to improve the kinetics of maltotriose fermentation and, in particular, of the transport of this oligosaccharide across the yeast plasma membrane. Evolutionary engineering was chosen over targeted genetic modification because of the genetic complexity of maltose and maltotriose metabolism, which made it difficult to a priori identify targets for genetic modification. Moreover, in view of limited customer acceptance for beverages made with genetically modified organisms (GMOs), it is attractive for industrial applications that strains obtained by evolutionary engineering approaches are not considered to be GMOs. The lager brewing