About our Research

My research group focuses on understanding the evolution, genetics and molecular biology of industrial yeasts, specifically the lager yeasts Saccharomyces pastorianus. We use adaptive evolution, synthetic biology and metabolic engineering approaches to expand the physiological and metabolicproperties of yeasts, to improve the fermentation process, generate novel taste profiles in beer and enable the single celled microorganism to carry out new tasks such as making bioethanol from biomass.

1. Lager yeasts

Yeasts have been used in the fermentation of alcoholic beverages for several millennia, with the earliest recordings of beer production dating back as early as 7000 B.C. The yeast, Saccharomyces pastorianus, used in the production of lagers is new to the scene, having arisen just 500-600 years ago as a result of a hybridisation event between two unrelated yeast species, Saccharomyces cerevisiae and Saccharomyces eubayanus. The happenstance of the interspecies hybridisation event between the mesophilic S. cerevisiae, with high fermentative capacity and the cryotolerant S. eubayanus, in a beer vat in Central Europe, created an ideal new yeast capable of adapting to the new conditions of brewing in the Middle Ages.

My research group focuses on understanding the complex genetic make-up of the Saccharomyces pastorinaus. We are addressing questions such as:

What is the genome structure and genetic composition of Saccharomyces pastorianus ?

S. pastorinaus strains can be divided into two broad groups (I and II) based on gene content and structure. The parent genomes have recombined at specific chromosomal locations to create a unique set of hybrid chromosomes. We identified a common sequence motif at the recombination epicentres, indicative of a common molecular mechanism controlling these recombination events. My research group also discovered that recombination at these sites is induced in response to environmental stress. Thus, stresses encountered during industrial fermentations play an important role in the evolution of these yeasts.

Recombination epicentres in lager yeasts

Recombination epicentres in lager yeasts. A hybrid lager gene showing the percentage identity to the S. cerevisiae strain S288C (red) and S. eubayanus strain FM1318 (blue). The recombination epicentre is identified from the region where the percentage sequence identities to the individual parental species intersect. B. Common motifs at recombination epicentres in lager yeasts.

Origins of the parental species of S. pastorianus?

We are interested in finding the origins of the parental species that gave rise to the lager yeasts S. pastorianus. The S. eubayanus parent is not indigenous to Europe but was discovered in Patagonia in South America. Subsequently, S. eubayanus isolates have been discovered in China and Tibet. How did the species arrive in Europe for the brief encounter with the S. cerevisiae parent? Current hypotheses suggest that S. eubayanus may have arrived in Europe from China via the Silk Road. What of the origins of the S. cerevisiae parent? By analysing the genomes of hundreds of yeast species, we are showed that S. pastorianus contain genetic information related to modern day

Ale and Stout yeasts, leading us to hypothesise that the two types of S. pastorianus arose as a result of sequential rounds of hybridisation, firstly between S. eubayanus and an Ale-like yeast, followed by a second hybridisation of this progenitor strain with a Stout yeast to give rise to Group I and II yeasts respectively. This seminal discovery was recently highlighted in several articles published in The Irish Times in 2014 and 2015.

Proposed evolutionary path of lager yeasts.

Unique genetic characteristics of S. pastorianus.

We have identified several genes that are unique to the genomes of S. pastorianus. These include hybrid genes emerging from recombination between parental chromosomes as well as genes located at the tips of chromosomes, which have been lost in most other yeast species but preferentially retained in S. pastorianus. One such hybrid gene is XRN1, encoding for a 5’ to 3’ exonuclease that plays a central role in RNA metabolism in yeast cells. Through its role in degrading cellular RNAs, XRN1 contributes to maintaining the steady state pool of cellular RNAs and thus has a major influence in the final proteome of the cell. Lager yeasts contain multiple hybrid forms of XRN1 that contain part S. eubayanus and part S. cerevisiae sequences. We are currently examining how co-expression of hybrid genes affects the RNA landscape of the lager yeast cell.

Another interesting gene is HYPO, named for the fact that it is a gene encoding for a hypothetical open reading frame. Originally identified a “lager-specific” gene we have recently shown that HYPO is also found in the genomes of Ale and Stout yeasts but absent in wine and laboratory strains of yeasts, suggesting that it’s function is important for beer fermentations. Using a GFP-tagged gene, we showed that HYPO locates to the plasma membrane in a unique punctate pattern. The presence of HYPO increases maltose uptake by cells prompting us to rename this gene FMU, Facilitator of Maltose Uptake.

Through analysing the effects of these unique gene products on the biochemistry and physiology of the cell, we aim to decipher the complex algorithm of gene expression that results in the unique fermentation and physiological characteristics of S. pastorianus.

2. Improving yeast strains to expand to biological capabilities of yeast cells

Accelerated adaptive evolution generates copy number changes in lager yeast strains.

Generating new improved strains of yeasts through adaptive evolution.

We use adaptive evolution approaches to generate novel yeast strains with improved physiological characteristics to expand the repertoire of yeast strains available for industrial applications. Using a novel accelerated evolution method, we are generating non-GMO yeast strains with new characteristics such as tolerance to high ethanol levels, improved growth on high sugar concentrations and strains with improved aroma and taste profiles.

Preventing bacterial infections in yeast fermentations.

Bacterial infections is a common problem encountered in industrial fermentations that can result in economic and/or reputation loss to the industry if the alcoholic beverage has to be recalled. The most common contaminations are caused by Lactobacillus species. We set about to identify antimicrobials that might prevent bacterial contaminations in beer fermentations.

We discovered that small amphipathic peptides such as defensins effectively destroy Lactobacilli in beer fermentations. We engineered S. pastorianus strains to produce the human defensin-3 peptide (HBD3) in situ through heterologous gene expression of a defensin gene cassette. Expression of HBD3 prevented proliferation of Lactobacillus brevis in pilot-scale fermentations indicating that defensins can provide prophylactic protection against beer-spoiling bacteria.

Using HBD3 as a gold standard, we searched for plant-derived defensin-like peptides that might substitute for HBD3 as an additive to beer fermentations. Several plant peptides, such as cp-thionin 2 and fabatin 2, were shown to be as effective as HBD3. Using a synthetic rational design approach, we used the known physiochemical properties of active peptides to design synthetic de novo peptides with a greater antimicrobial potency than HBD3. These novel peptides will provide the fermentation industries with tangible solutions to antibacterial infections in large-scale production plants.

Identification of plant peptides with antimicrobial activity against beer-spoiling bacteria.

Identification of plant peptides with antimicrobial activity against beer-spoiling bacteria.

Yeasts as cell factories for generating energy from biomass.

As the world searches for alternative sources of renewable energy, yeasts offer a unique solution to generate bioethanol from renewable resources due to their inherent ability to convert sugars into ethanol. Lignocellulose biomass, derived from dedicated energy crops or from industrial or agricultural waste, provides an alternative renewable source of green energy for bioethanol production. The extraction of useable sugars from lignocellulose biomass requires a suite of enzymes for the utilization of both cellulose and xylose, the two major components of lignocellulose biomass, however yeasts do not have the necessary genes to encode these enzymes. Using a novel gene cassette development strategy, we generated strains of yeasts expressing the genes required to metabolise the 5-carbon sugar, xylose, and the complex carbohydrate, cellulose. The engineered yeast strain is capable of co-utilising xylose and cellulose, generating an alcohol yield of 82% from the available sugars.

Engineering yeasts for cellulose degradation. The enzymes CBH1/2, EG1 and BGL1 can be tethered to the cell wall or secreted into the medium.

Engineering yeasts for xylose utilisation. The enzymes XR/XDH or XI can be expressed in yeast.

Other relevant publications from Bond Lab

'Molecular Mimics of the Tumour Antigen MUC1', PLOS One, 7, 11 (2012), doi: 10.1371/journal.pone.0049728 [James, T.C. and U, Bond]

'Phage display biopanning identifies the translation initiation and elongation factors (IF1- a and eIF-3) as components of Hsp70-peptide complexes in breast tumour cells.', Cell Stress Chaperones, 17, 2 (2012), 145-56 [Siebke C, James TC, Cummins R, O'Grady T, Kay E, Bond U.]

'The PolyA tail length of yeast histone mRNAs varies during the cell cycle and is influenced by Sen1p and Rrp6p', Nucleic Acids Research, 40, 6 (2012), 2700-11 [Suzanne Beggs, Tharappel C. James and Ursula Bond]

'Deletion of the nuclear exosome component RRP6 leads to continued accumulation of the histone mRNA HTB1 in S-phase of the cell cycle in Saccharomyces cerevisiae.', Nucleic Acids Research, 35, 18 (2007), 6268-79 [Canavan, R and U. Bond]

'Stressed Out! The effects of environmental stress on mRNA metabolism.', FEMS Yeast Research, 6 (2006), 160-170 [U. Bond]

'A Novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.', Journal of Immune Based Therapies and Vaccines, 4, 12 (2006), 2-13 [Arnaiz B, Madrigal-Estebas L, Todryk S, James TC, Doherty DG, Bond U.]

'Structural mimics of heat shock protein 70- associated peptides from breast tumour cells can prime T-cells to respond to tumour antigens', European Journal of Cancer, 2 (2004), 79 [Arnaiz, B., L. Madrigal, D. Doherty, T.C.James and U. Bond]

'A sequence element downstream of the yeast HTB1 gene connects mRNA'3 end processing, transcription termination and cell cycle regulation of a histone gene', Mol. Cell Biol., 22 (2002), 8415-25 [Campbell, S., M. li del Olmo, Beglan, P. and U. Bond]

'Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells', RNA, 5 (1999), 1586-96[Bracken A. and U. Bond]

Microbiology

About our Research

1. Lager yeasts

What is the genome structure and genetic composition of Saccharomyces pastorianus ?

Read more about this

Origins of the parental species of S. pastorianus?

Read more about this

Unique genetic characteristics of S. pastorianus.

Read more about this

2. Improving yeast strains to expand to biological capabilities of yeast cells

Generating new improved strains of yeasts through adaptive evolution.

Read more about this

Preventing bacterial infections in yeast fermentations.

Read more about this

Yeasts as cell factories for generating energy from biomass.

Read more about this

Other relevant publications from Bond Lab