Genomic analysis of Kazachstania aerobia and Kazachstania servazzii reveals duplication of genes related to acetate ester production

Kazachstania aerobia and Kazachstania servazzii can affect wine aroma by increasing acetate ester concentrations, most remarkably phenylethyl acetate and isoamyl acetate. The genetic basis of this is unknown, there being little to no sequence data available on the genome architecture. We report for the first time the near-complete genome sequence of the two species using long-read (PacBio) sequencing (K. aerobia 20 contigs, one scaffold; and K. servazzii 22 contigs, one scaffold). The annotated genomes of K. aerobia (12.5Mb) and K. servazzii (12.3Mb) were compared to Saccharomyces cerevisiae genomes (laboratory strain S288C and wine strain EC1118). Whilst a comparison of the two Kazachstania spp. genomes revealed few differences between them, divergence was evident in relation to the genes involved in ester biosynthesis, for which gene duplications or absences were apparent. The annotations of these genomes are valuable resources for future research into the evolutionary biology of Kazachstania and other yeast species (comparative genomics) as well as understanding the metabolic processes associated with alcoholic fermentation and the production of secondary ‘aromatic’ metabolites (transcriptomics, proteomics and metabolomics).Mandy Man-Hsi Lin, Michelle E. Walker, Vladimir Jiranek, and Krista M. Sumb

Measures to improve wine malolactic fermentation

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.Krista M. Sumby, Louise Bartle, Paul R. Grbin, Vladimir Jirane

Characterisation of intracellular esterases from Oenococcus oeni and Lactobacillus hillgardii and their potential for application in wine

Poster 53Oenococcus oeni is the lactic acid bacterium (LAB) most typically used in winemaking to carry out malolactic fermentation (MLF). Alongside this, LAB can metabolise precursors present in wine during the MLF and as a consequence alter its chemical composition and quality. Aroma compounds such as esters and the quantities in which they occur can play a particularly important role in determining wine quality (Sumby et al. 2010). Esters are primarily formed during fermentation by yeast after which LAB are capable of modifying ester concentration during MLF. Ester hydrolysis and synthesis can be catalysed by esterases, which have greatest specificity for water-soluble short-chain esters. With a view towards understanding and enhancing the role of LAB in ester profile modifications during MLF, we report the cloning, heterologous expression, partial purification, and biochemical characterisation of EstA2, EstB28, and EstCOo8 esterases from O. oeni and EstC34 esterase from Lactobacillus hillgardii. Enzyme function under the harsh physicochemical conditions frequently encountered in wine was examined to evaluate their potential applicability in this context. The influence of pH (3.0 to 8.0), temperature (10 to 60°C) and ethanol (2 to 22% (vol/vol)) on esterase activity was determined. All retain at least partial activity under wine like conditions of pH, temperature and ethanol (Sumby et al, 2009; 2012, 2013a). Substrate specificity trials were conducted in which eight different pNP-linked ester substrates. This activity was also confirmed using natural substrates (Sumby et al, 2013b). Substrate specificity of the enzymes was shown to vary, thereby suggesting possible applications in wine for targeted ester removal (Figure 1). Having established that EstA2 and EstB28 should retain at least partial activity in wine, they were assayed for activity in two separate wines by using SPME-GCMS to monitor the appearance or disappearance of esters. EstB28 and EstA2 demonstrated duel hydrolysis and synthesis activity in wine and are the first LAB esterases demonstrated to retain activity in wine (Table 1). Once the esterase enzymes were characterised the basis for previously observed strain specific differences in ester hydrolysis by whole cells was investigated through wine MLF trials. Ester concentration changes in wine were dependent on the strain conducting the MLF. Further investigations will allow for a more informed choice of MLF strain

Ethanol-tolerant lactic acid bacteria strains as a basis for efficient malolactic fermentation in wine: evaluation of experimentally evolved lactic acid bacteria and winery isolates

Background and Aims: Reliable malolactic fermentation (MLF) is essential for process efficiency and spoilage prevention in wine. This study extends previous research in our laboratory, aimed at the development and selection of new bacterial strains for reliable MLF in wine, focusing on ethanol‐tolerant lactic acid bacteria (LAB) strains. Sensory differences of seven LAB strains were assessed, including two commercial strains, two ethanol‐tolerant strains derived from directed evolution and three isolates from a high ethanol Grenache. Methods and Results: In this study, the performance of 30 LAB strains was first assessed in fermented chemically defined grape juice media. Seven of the best performing strains were then tested in small‐scale (5 L) fermentations in Shiraz and Shiraz‐Grenache blend wines. All wines were evaluated with a sensory panel using free choice profiling. Conclusions: Despite significantly different MLF kinetics between the strains there were no strain‐specific differences on the final wines. The choice of LAB strain did not adversely change the sensory properties of either wine. Significance of the Study: These findings provide reassurance that the efficient LAB strains (G71 and G55) and the modified directed evolution strains do not compromise the sensory properties of wines despite their marked MLF benefits.K.M. Sumby, J. Niimi, A.L. Betteridge, V. Jirane

Yeast bioprospecting versus synthetic biology–which is better for innovative beverage fermentation?

Producers often utilise some of the many available yeast species and strains in the making of fermented alcoholic beverages in order to augment flavours, aromas, acids and textural properties. But still, the demand remains for more yeasts with novel phenotypes that not only impact sensory characteristics but also offer process and engineering advantages. Two strategies for finding such yeasts are (i) bioprospecting for novel strains and species and (ii) genetic modification of known yeasts. The latter enjoys the promise of the emerging field of synthetic biology, which, in principle, would enable scientists to create yeasts with the exact phenotype desired for a given fermentation. In this mini review, we compare and contrast advances in bioprospecting and in synthetic biology as they relate to alcoholic fermentation in brewing and wine making. We explore recent advances in fermentation-relevant recombinant technologies and synthetic biology including the Yeast 2.0 Consortium, use of environmental yeasts, challenges, constraints of law and consumer acceptance.Lucien Alperstein, Jennifer M Gardner, Joanna F Sundstrom, Krista M Sumby, Vladimir Jirane

Influence of Kazachstania spp. on the chemical and sensory profile of red wines

We report the fermentative traits of two Kazachstania species (K. aerobia and K. servazzii) in non-sterile red wine and the resulting chemical and sensory properties. This builds on our previous work which revealed that Kazachstania spp. increased acetate esters in sterilised white wine. In this study Kazachstania spp. were initially evaluated in laboratory-scale fermentations (500 mL) in Merlot must to assess whether similar increases in chemical/volatile compounds would occur. The impact of malolactic fermentation (MLF) by Oenococcus oeni (VP41) on aroma composition was considered and found to reduce ester profiles in Merlot wines. The sensory implications of sequential inoculation with Kazachstania spp., followed by Saccharomyces cerevisiae, were then evaluated in small-lot fermentations (7 kg) of Shiraz must. Fungal diversity was monitored during early fermentation stages and was influenced by the early implantation of Kazachstania spp., followed by the dominance of S. cerevisiae. The effect of MLF in Shiraz wines was inconclusive due to high ethanol levels providing an inhospitable environment for lactic acid bacteria. When compared to S. cerevisiae alone, Kazachstania spp. significantly increased acetate esters, particularly phenylethyl acetate and isoamyl acetate, in both Merlot and Shiraz. The Shiraz wines fermented with Kazachstania spp. had higher jammy and red fruit aroma/flavour compared to S. cerevisiae (monoculture) wines. No influence was observed on colour one-year post-bottling. Results from this study show the contribution of Kazachstania spp. to the aroma profile of red wines and demonstrate their potential as starter cultures for improving the aromatic complexity of wines.Mandy Man-Hsi Lin, Paul K. Boss, Michelle E. Walker, Krista M. Sumby, Vladimir Jirane

Exploring the diversity of bacteriophage specific to Oenococcus oeni and Lactobacillus spp and their role in wine production

The widespread existence of bacteriophage has been of great interest to the biological research community and ongoing investigations continue to explore their diversity and role. They have also attracted attention and in-depth research in connection to fermented food processing, in particular from the dairy and wine industries. Bacteriophage, mostly oenophage, may in fact be a 'double edged sword' for winemakers: whilst they have been implicated as a causal agent of difficulties with malolactic fermentation (although not proven), they are also beginning to be considered as alternatives to using sulphur dioxide to prevent wine spoilage. Investigation and characterisation of oenophage of Oenococcus oeni, the main species used in winemaking, are still limited compared to lactococcal bacteriophage of Lactococcus lactis and Lactiplantibacillus plantarum (formally Lactobacillus plantarum), the drivers of most fermented dairy products. Interestingly, these strains are also being used or considered for use in winemaking. In this review, the genetic diversity and life cycle of phage, together with the debate on the consequent impact of phage predation in wine, and potential control strategies are discussed. KEY POINTS: • Bacteriophage detected in wine are diverse. • Many lysogenic bacteriophage are found in wine bacteria. • Phage impact on winemaking can depend on the stage of the winemaking process. • Bacteriophage as potential antimicrobial agents against spoilage organisms.Zhecun Xu, Michelle E. Walker, Jin Zhang, Jennifer M. Gardner, Krista M. Sumby, Vladimir Jirane

Apilactobacillus apisilvae sp. nov., Nicolia spurrieriana gen. nov. sp. nov., Bombilactobacillus folatiphilus sp. nov. and Bombilactobacillus thymidiniphilus sp. nov., four new lactic acid bacterial isolates from stingless bees Tetragonula carbonaria and Austroplebeia australis

Four strains, SG5_A10T , SGEP1_A5T , SG4_D2T , and SG4_A1T , were isolated from the honey or homogenate of Australian stingless bee species Tetragonula carbonaria and Austroplebeia australis. Based on 16S rRNA gene phylogeny, core gene phylogenetics, whole genome analyses such as determination of amino acid identity (AAI), cAAI of conserved genes, average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH), chemotaxonomic analyses, and the novel isolation sources and unique geography, we propose three new species and one genus with the names Apilactobacillus apisilvae sp. nov. (SG5_A10T = LMG 32133T = NBRC 114991T ), Bombilactobacillus thymidiniphilus sp. nov. (SG4_A1T = LMG 32125T = NBRC 114984T ), Bombilactobacillus folatiphilus sp. nov. (SG4_D2T = LMG 32126T = NBRC 115004T ) and Nicolia spurrieriana sp. nov. (SGEP1_A5T = LMG 32134T = NBRC 114992T ). Three out of the four strains were found to be fructophilic, where SG5_A10T and SGEP1_A5T belong to obligately fructophilic lactic acid bacteria, and SG4_D2T representing a new type denoted here as kinetically fructophilic. This study represents the first published lactic acid bacterial species associated with the unique niche of Australian stingless bees.Scott A. Oliphant, Nathan S. Watson-Haigh, Krista M. Sumby, Jennifer Gardner, Scott Groom, and Vladimir Jirane

Evaluation of indigenous non-Saccharomyces yeasts isolated from a South Australian vineyard for their potential as wine starter cultures

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.Mandy Man-Hsi Lin, Paul K. Boss, Michelle E. Walker, Krista M. Sumby, Paul R. Grbin, Vladimir Jirane

Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p and a low affinity transporter, Soa1p. Sulfate contributes to H2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2 S are highly desirable. We report on the use of toxic analogs of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2 S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K, E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology, and shown to reduce accumulation of H2 S and not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2 S production as well as the potential use of these strains for winemaking and in yeast breeding programs.Michelle E. Walker, Jin Zhang, Krista M. Sumby, Andrea Lee, Anne Houlès, Sijing Li, Vladimir Jirane