Team:Austin UTexas/Demonstrate
Demonstrate
Click on one of the images below to learn more about our results!
Kombucha Strains
Conjugation
Recapitulation
Ethanol
pH
Kombucha Strains
- Successfully isolated microbes from various samples of kombucha.
- Identified strains of bacteria and yeast using rRNA gene sequencing.
- Characterized each of the isolated microbes to facilitate further experimentation.
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Conjugation
- Attempted conjugation with G. oxydans.
- Performed minimum inhibitory concentration experiments between G. oxydans and spectinomycin, carbenicillin and kanamycin.
- Determined that G. oxydans is resistant to spectinomycin and carbenicillin.
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Recapitulation
- In a process called "recapitulation," we successfully created a kombucha-like culture by adding individual strains of microbes instead of a living culture containing the entire kombucha microbiome.
- Determined that the microbe Ga. hansenii is essential for the fermentation of kombucha.
- Determined that two distinct strains of the yeast Lachancea fermentati are necessary for the fermentation of kombucha, including one that appears to produce high quantities of C02.
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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.1 One way to tackle this problem with synthetic biology is to ferment with yeast that produce less ethanol. However, this may be 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.2 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-producing 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 create a microbial community that results in a lower ethanol content within the kombucha during fermentation, kombucha brewers could use the modified bacterium to help ensure the ethanol content of their product stays below the legal limit.
References
- Alcohol and Tobacco Tax and Trade Bureau. Kombucha Information and Resources. 2016. https://www.ttb.gov/kombucha/kombucha-general.shtml
- 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 substance1. 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 sugar or excessive calories. While still being a GMO product, this beverage would be low in sugar and could appeal to a health-conscious consumer.
References
- Yan, Sen et al. “Expression of Plant Sweet Protein Brazzein in the Milk of Transgenic Mice.” Ed. Xiao-Jiang Li. PLoS ONE 8.10 (2013): e76769.
- Brazzein protein structure acquired from European Bioinformatics Institute
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 promoters to control the expression of reporter proteins, such as GFP or a chromoprotein, would allow visualization of the pH change. The promoters Cpx, P-atp2, and Cadc were selected as pH sensors to indicate pH in the neutral, basic, and acidic ranges, respectively.1,3,5,6 These constructs have been or will be transformed into Escherichia coli to confirm pH sensitivity prior to introduction to kombucha and to see if these constructs could be utilized as sensors in mediums besides kombucha.
Modification of Gluconobacter oxydans, a bacterium in kombucha, is also planned 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.2 Golden Gate assembly is currently being used to quickly assemble these promoters upstream of Venus (pYTK033).4 Once successful, these pH-sensitive promoters with different reporters will be used to visualize the different members of the kombucha microbiome overtime.
References
- BIT-China-2015
- Hanke, T., Richhardt, J., Polen, T., Sahm, H., Bringer, S., and Bott, M. (2012) Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology. Journal of Biotechnology 157, 359–372.
- Kuper, C., and Jung, K. (2005) CadC-mediated activation of the cadBA promoter in Escherichia coli. Journal of Molecular and Microbiological Biotechnology 1, 26–39.
- Lee ME, DeLoache, WC A, Cervantes B, Dueber, JE. (2015) A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. ACS Synthetic Biology 4 975-986
- Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528.
- Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069.
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