Description

Kombucha is a beverage made when a symbiotic community of bacteria and yeast ferments sugared tea. Though kombucha has been consumed for thousands of years in the East, the drink has enjoyed a recent resurgence in popularity. Several kombucha breweries operate in Austin, Texas, our team’s hometown. The role microbes play in the production of the beverage led our team to wonder if synthetic biology could allow us to create “designer kombucha” with enhanced properties, such as more appealing flavors or additional nutrients. In order to do so, our team attempted to isolate the strains responsible for the fermentation of kombucha, identify them, genetically modify them, and finally add the individual strains into tea media to recreate the drink. We additionally considered potential applications of the ability to genetically modify the microbial population of kombucha, such as reducing the ethanol content of the beverage and improving taste with brazzein, a sweet-tasting protein.

Kombucha Strains

One of the earliest goals of our project was to analyze the microbes responsible for the production of kombucha. First, we needed to identify them. Samples of store-bought kombucha were plated onto a variety of media with various dilutions to isolate microbes. Then, morphologically different colonies were selected and saved as a frozen glycerol stock for further use. Once we obtained a collection of microbial isolates, each microbe was sequenced and identified by using Polymerase Chain Reaction to amplify a particular ribosomal RNA gene. The 16S gene was selected for with bacterial strains, and the ITS gene was amplified for the fungal samples. After the samples were sequenced, we utilized the Ribosomal Database Project (RDP) SeqMatch to identify our isolated species of bacteria and yeast. By identifying these kombucha strains, we were able to use our own experimentally-isolated strains for processes like recapitulation and conjugation to obtain more revealing data.

Conjugation

In order to demonstrate that genetic engineering is possible with the organisms Gluconobacter oxydans and Gluconacetobacter xylinus, we conjugated(????????????) GFP into the microbes using a DAP (Diaminopimelic Acid) auxotroph strain of E. coli. The plasmid, pBTK520, contains GFP and a spectiomycin resistance gene. By proving that conjugation is possible with these microbes, this opens the door for further genetic modification in order to create a designer beverage.

Recapitulation

Kombucha is a beverage that exists due to a symbiotic environment. This symbiosis, which the microbes of kombucha thrive in, is the main study of the recapitulation project. Recapitulation refers to the reformation of kombucha from individual isolates of microbes taken from the beverage. In the beginning of the project, it was determined what the key microbes in kombucha were, and the process of recapitulation is to determine, that if after being taken apart and genetically modified, can these microbes still function in symbiosis while maintaining their new genetic information,

Ethanol

During the fermentation process, yeast in kombucha produce ethanol, the type of alcohol present in beer, wine, and other alcoholic beverages. This presents a challenge to kombucha brewers who wish to market their product as a non-alcoholic beverage. If the alcohol content of a manufacturer’s kombucha exceeds 0.5% at any point during production, the manufacturer may not market their beverage as non-alcoholic and must be regulated as a producer of alcoholic beverages. One way to tackle this problem with synthetic biology is to ferment with yeast that produce less ethanol. However, this is impractical. Some bacteria in the SCOBY oxidize ethanol produced by the yeast to produce acetic acid, which is a major component of the beverage’s distinctive, tart flavor.

Another approach is to increase the rate at which the bacteria convert the ethanol to acetic acid. Two enzymes are responsible for this process: an alcohol dehydrogenase and an aldehyde dehydrogenase (Mamlouk and Gullo, 2013). Using Golden Gate assembly, we plan to assemble a construct containing the coding sequences for these genes and insert the construct into Gluconacetobacter hansenii, an acetic acid bacterium similar to those found in kombucha. Then, we plan to recapitulate kombucha with both transformed and control Ga. hansenii to evaluate the ethanol content over the course of the fermentation with gas chromatography-mass spectrometry. We also plan to determine whether increasing the acetic acid production will lead to a pH change that could affect the flavor of the beverage by testing the pH and observing the cultures for visible differences. If we are able to produce the Ga. hansenii that cause the kombucha to have a lower ethanol content over the course of the fermentation, kombucha brewers could use the modified bacterium to help ensure the ethanol content of their product stays below the legal limit.

Brazzein

One of the projects that we are working on involves adding a brazzein gene into the bacteria found in Kombucha. Brazzein, a protein found in the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is an extremely sweet substance. It is 2,000 times sweeter than sucrose by weight. This makes it a healthy and economical alternative to sugar. However commercial production of brazzein is limited because it comes from a tropical plant. If it could be more easily harvested, it could be used to improve the flavor of various foods and drinks. If it is added to Kombucha, the drink could be sweetened without adding excessive calories.

pH Sensors

Kombucha is a living beverage with many different microorganisms contributing toward its formation during the brewing process. However, these many organisms change their environment with the products of the reactions being performed, such as the lowering of pH. If pH sensitive promoter regions could be identified, and used within the organisms in kombucha, then the visualization of pH changes in the environment could be seen in both the liquid portion and the SCOBY. Three such regulatory mechanisms have been identified, the Cpx, P-atp2, and Cadc promoter regions that are used in promoter-reporter constructs to detect pHs in the neutral, basic, and acidic ranges respectively. These constructs could be used in Escherichia coli to sense pH changes in a variety of products, such as kombucha or milk. Alternatively, to not disturb the organism balance that exists in kombucha, the modification of the abundant Gluconobacter oxydans was explored. Three endogenous upstream regions of loci that were reported to show increased mRNA synthesis as pH dropped were acquired, and using Golden Gate assembly, these putative promoters will be placed on a plasmid with a specific reporter sequence (Hanke et al, 2012). By placing these variety of pH sensitive promoters with different reporters and transforming multiple organisms, then the visualization of the organisms in kombucha and where they reside would be possible.