Performing Department
(N/A)
Non Technical Summary
Tropical fruit flavors are highly desirable in the fermented beverage market. The popularity of these flavors is especially evident in the beer industry, where sales of beers made with tropical flavoring hops, primarily grown in the American Pacific Northwest and in Australia/New Zealand has skyrocketed in the last decade. While demand for these beers and the tropical flavoring-hops used to make them has increased in recent years, extreme weather events like droughts and high winds have simultaneously threatened, and in some cases damaged U.S. flavoring-hop harvests. As increasingly dry and adverse weather events are predicted to become more frequent due to climate change, U.S. hop harvests, hop farmers, and the production of in-demand beer styles will become increasingly threatened in coming years. While efforts are underway to support the $500M/yr American hop industry through the breeding of more resilient and drought tolerant hop cultivars, current drought-tolerant plants lack the pungent, fruity flavor profiles that brewers and beer drinkers demand.In this SBIR application, we propose to develop genetically engineered strains of brewers yeast that will produce strong, tropical fruit flavors during beer fermentation, using non-aromatic precursor molecules already existing in drought-tolerant hops as substrates. Specifically, we will engineer yeast for production of three volatile thiol molecules, 3MH (guava flavor), 3MHA (passionfruit), and 4MMP (black currant), each of which is a major contributor to the tropical flavor notes of currently popular hops varieties. As differing ratios of 3MH, 3MHA, and 4MMP molecules impart distinctive flavors in beer, we will construct a set of brewing yeast strains in which each strain produces distinct quantities of these three volatile thiols. This set of strains will provide brewers with an easily scalable toolkit that produces a diversity of tropical fruit flavored beers, using American-grown non-aromatic drought tolerant hops as substrates.This work will build upon our prior success engineering brewing yeast strains for enhanced 3MH biosynthesis during beer fermentation. In our proposed Phase I research, we will develop yeast strains that produce varying ratios of 3MH to 3MHA during fermentation, then test the consumer acceptance and commercial potential of this technology. In Phase II work, we will develop strains that additionally produce 4MMP during fermentation, then increase production of all three thiols so as to be comparable to the concentrations achieved through addition of popular flavoring hop varieties. The end result of Phase II work will be a set of strains that produce distinct, marketable, concentrations of 3MH, 3MHA, and 4MMP during beer fermentation. This technology will provide valuable tools that help the beer and hop industries meet consumer demand while insulating them from the threat of droughts and severely reduced hop harvests.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The overall goal of Phase I SBIR research is to improve beer quality by developing yeast strains that produce tropical-flavored thiols without off-flavors. The objectives of Phase I research that will allow us to reach this goal are to 1) identify an acyltransferase that converts 3MH to 3MHA without ethyl acetate production, and 2) vary acyltransferase expression to produce beer with distinct 3MH to 3MHA ratios. Once we achieve these objectives, we will evaluate successful achievement of improved beer quality by performing proof-of-principle brewing trials with chemical analytics and quanititative food science methods.
Project Methods
Aim 1: Identify an acyltransferase that converts 3MH to 3MHA without off-flavor production Hundreds of acyltransferase sequences from bacterial, fungal, and plant origins have been identified, but only a handful have been functionally characterized. In the cases where functional characterization was done, 3MH acetylating activity was rarely investigated, and as a result, no acyltransferases aside from ATF1 have been previously shown to produce 3MHA. In order to identify acyltransferases that produce 3MHA, but do not produce off-flavors, we will mine publically available sequences and choose a set of twenty candidate acyltransferases to screen for this desired functionality. In determining which twenty acyltransferase sequences to screen, priority will be given to those that have been functionally characterized and are known to 1) produce acetate esters, 2) produce limited ethyl acetate, and 3) show affinity for six-carbon alcohol substrates that are structurally similar to 3MH. For sequences that have not been functionally characterized, we will prioritize genes from tropical fruit and aromatic hops that display sequence similarity to characterized acetate ester-forming acyltransferases. These twenty acyltransferase genes will be synthesized and cloned into drug-selectable yeast centromeric plasmids with expression driven by the strong constitutive yeast promoter, pPGK1. Each of these plasmids will then be transformed into BY502. Transformed strains will be grown in brewing wort media supplemented with 100ng/L Cys-3MH in semi-anaerobic conditions that mimic the brewing environment. After five days of fermentation, culture supernatents will be harvested and concentrations of 3MH, 3MHA, ethyl acetate, and other esters will be determined by Solid-Phase Microextraction (SPME) GC/MS. The three acyltransferase genes that drive the highest 3MHA to 3MH ratio will be chosen for use in Aim 2. Aim 2: Vary acyltransferase expression to produce beer with distinct 3MH to 3MHA ratios We will first choose a set of ten yeast promoter sequences that display a range of expression strengths throughout the beer fermentation process. Each of these promoters will be paired with each of the top three acyltransferases identified in Aim 1, to generate 30 promoter/acyltransferase pairs. Each pair will be cloned into an "integration plasmid", with flanking sequences that mediate site-specific homologous recombination into the yeast genome. Each of the thirty integration plasmids will be linearized by restriction digest and transformed into BY502. Homozygous, stable integration of each promoter/acyltransferase pair at the target locus will be verified by diagnostic PCR. We will next brew beer with each of these thirty acyltransferase-expressing strains in our in- house pilot brewery. Beer fermentations will be supplemented with hops from drought resistant hop plants at day five, as this is typically when flavoring hops are added to beer fermentations. These hop additions will provide substrates for conversion to 3MH and potential subsequent acetylation to 3MHA. At the end of a ten-day fermentation, samples will be taken from each beer and analyzed by SPME GC/MS to quantity 3MH and 3MHA. Beers will also be kegged and carbonated, and an in-house sensory analysis will score each beer for flavor and aroma characteristics. From these combined analytical and sensory data, we will determine a group of five strains that produced beer with a range of 3MH to 3MHA ratios and that scored well during sensory analysis. This in-house sensory analysis will be sufficient to identify a set of five promising strains. To more rigorously assess the effects of our engineering efforts on beer chemistry, flavor, and stability, we will collaborate with the Shellhammer lab at Oregon State University (OSU). Professor Thomas Shellhammer is an internationally recognized expert in beer and hops chemistry, and he studies the contribution of hops to beer flavor, and the sustainability of beer and hops production. His lab has extensive capabilities for producing beer on a state-of-the-art, commercially-comparable 2 hL pilot brewery coupled with instrumental resources for characterizing raw materials and finished beer quality. His lab has worked with a diverse set of industrial partners including large global brewing companies, small, craft breweries, and industry leading companies in the areas of malt, hops, yeast, enzymes and brewery processing equipment. His facilities and expertise are ideally suited to perform more comprehensive brewery trials with our engineered strains. BY will send the Shellhammer lab the five yeast strains chosen on the basis of their distinct 3MH to 3MHA production profiles. The Shellhammer lab will brew 2 hL of beer with each of these strains using its pilot brewery, and will experiment with adding varying quantities and cultivars of drought-tolerant hops during the fermentation. Finished beer will be analyzed for differences in basic beer chemistry (fermentability and ABV) plus specific hop and fermentation components using HPLC and GC techniques. These same beers will be assessed using human sensory descriptive analysis techniques and an in-house trained panel. These two approaches will allow us to determine the magnitude of volatile thiol release and how it modulates the sensory qualities of the resultant beer. Work performed by the Shellhammer lab will allow us to confidently determine whether our engineering efforts succeeded in generating strains that produce flavor-distinct quantities of 3MH and 3MHA without negatively affecting other beer quality parameters. If successful, this work will represent a significant step forward in our ability to modulate the biotransformation process, and will clearly motivate further work in this area.