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 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 tool 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 Gluconobacter oxydans and Gluconacetobacter xylinus, we attempted to conjugate 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, there is potential for further genetic modification to create a designer beverage.

Recapitulation

One of the primary focuses of our project is the nature of the symbiotic environment of fermenting kombucha. Recapitulation refers to the reformation of kombucha from individual isolates of microbes taken from the beverage. We learned which microbes were essential for the brewing of kombucha and whether or not the beverage could be recreated from its constituent microbes after they had been genetically modified.

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.¹ 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 the 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.

References

1. Mamlouk, D., and Gullo, M. (2013) Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation. Indian Journal of Microbiology 53, 377–384.

Brazzein

One of the potential methods to create designer kombucha is to add a brazzein gene into the bacterial strains. 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. Commercial production of brazzein is limited, however, 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, including kombucha. By genetically engineering the brazzein gene into the bacteria in kombucha, the drink could be sweetened without adding excessive calories.

pH Sensors

Many of the microorganisms involved in the fermentation of kombucha produce acidic metabolites that lower the pH of the culture. Using pH-sensitive promoter regions to control the expression of reporter chromoproteins in the bacteria in kombucha would allow visualization of the pH change in the kombucha's liquid portion and SCOBY. The promoters Cpx, P-atp2, and Cadc would be used to indicate pH in the neutral, basic, and acidic ranges, respectively. These constructs could be transformed into Escherichia coli to sense pH changes in a variety of products, such as kombucha or milk. Modification of Gluconobacter oxydans was also explored as an alternative to E. coli to avoid disturbing the kombucha microbiome. Three endogenous upstream regions of loci that were reported to show increased mRNA synthesis as pH decreased were obtained. 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 pH-sensitive promoters with different reporters and transforming into multiple organisms, the visualization of the microbes and their location in kombucha would be possible. This would serve as a stepping stone into the transformation of multiple kombucha organisms with these different reporter constructs, meaning organism concentration at a specific time during the brewing process could be visualized.