Fit-for-purpose yeast and bacteria via directed evolution

This project sought to generate ‘fit-for-purpose’ yeast and lactic acid bacterial (LAB) strains better suited to problematic wine and juice conditions via Directed Evolution (DE). DE involves growing an organism in a stressful environment where it can mutate and adapt over time. Screening of DE populations and LAB isolates from un-inoculated fermentations has generated 5 yeast and 8 LAB improved strains. The molecular basis behind the observed improved performance during fermentation was also investigated.

The ability of a range of MLF bacteria to grow and undertake malolactic fermentation in wines with combinations of low pH, high alcohol and the presence of SO2 and medium chain fatty acids was assessed. A number of strains were isolated with increased tolerance to SO2 and lower pH, and in some cases, can still undertake malolactic fermentation in the presence of 18% (v/v) ethanol. Depending on the conditions, these tolerances resulted in a reduction of malolactic fermentation by up to 50%, or completion of MLF whilst the parent strains did not (i.e. became stuck).

Twelve wine yeast were chosen as source strains for improvement and these were subjected to two conditions of directed evolution (i) exposure to the combination of high sugar and ethanol and low pH and (ii) high fructose and ethanol content (i.e. as per a late-stage fermentation). Five evolved yeast cultures were found to have superior fermentation performance, as measured by total fermentation duration i.e. the time taken to utilize all sugars. For instance strain RM7-71 fermente all available sugars 23 hours before the commercial yeast Uvaferm 43 (well known to be capable of rapid fermentation).

Whole genome sequencing was performed on 18 lactic acid bacteria including 12 O. oeni and 5 Lb. plantarum, and 6 wine yeast. This forms foundational data and work is ongoing to capitalise on the information contained in this data. For example further studies will investigate how modification of particular genes could confer robust and efficient malolactic fermentation. For the more efficient yeast, the fermentation efficiency of particular strains is likely conferred by multiple genes fine-tuning multiple processes.

The yeast ‘Fermentome’, is defined as the genes and/or processes required by yeast to sense and respond to the multiple stresses encountered during growth in grape juice to allow complete and efficient fermentation. By using 'gene deletion' mutants (using a lab strain of yeast where just one of each individual gene was deleted) 93 lab-strain yeast mutants struggled to ferment, indicating the genes that were missing in these were important for robustness. Using a library of yeast previously prepared by AWRI where just one of each individual gene was deleted, a range of the genes most likely to confer the yeast capacity for efficient fermentation were identified.

During this project 8 PhD students and 4 Masters of Viticulture and Oenology students have been trained. Eight have been awarded their degrees, and 5 have continued careers in research.

The use of specifically selected yeast and bacteria to complete both primary and secondary fermentation still remains the most commonly used and arguably most reliable method to ensure desirable fermentation outcomes in new-world industrial winemaking. Many commercial strains are currently available, however feedback from industry reports that even with these, problem fermentations still exist. We expect this is also exacerbated by the potential for increased variability in the composition of grape musts, impacted by factors such as climate change as well as viticultural practices targeting particular wine styles. This project has generated ‘fit-for-purpose’ yeast and lactic acid bacterial strains better suited to problematic juice conditions, as reported by industry (high sugar, low nitrogen, low pH) and the presence of microbial inhibitors such as sulfur dioxide and medium chain fatty acids. Reliable fermentation outcomes will translate into further improvements to sustainable and economical wine production with enhanced or consistent wine quality.

Objectives

Develop non-GM yeast and bacterial strains for commercial release with better stress tolerance and performance, including one or more of the following properties

• greater tolerance of low nutrient availability, cold or inhibitory compounds
• enhanced flocculation and metabolism of malic acid, and reduced H₂S and SO₂ formation
• tailored performance in ‘extreme’ juices e.g. from overripe or Botrytis affected grapes

Approach

The approach used in this project to generate the new strains is directed evolution guided by an understanding of the genes and physiochemical stresses involved. The utility of the new strains will be evaluated through laboratory and pilot scale fermentations and sector trials together with evaluation of experimental wines.

Sector benefits

The knowledge and the new strains generated by this project will give winemakers increased ability to manage fermentation efficiency and wine composition.