Team:USP-Brazil/Project

Detecthol

The Chassis

Looking for a chassis that would resist the presence of ethanol, and specially of methanol, the simplest solution was to use yeast cells, largely employed in the production of biofuels, such as Saccharomyces cerevisae [6]. The best choice turned out to be a methylotrophic organism, that is, one that can use methanol as a carbon source [7]. Pichia pastoris is a species of methylotrophic yeast with its genome sequenced [11]that is commonly used in the production of recombinant proteins [8], mainly due to its populational characteristics, such as growth rate and cell density, which make cell suspensions paste-dense [9], and to its methanol-responsive promoters, that could be part of a genetic circuit that would respond to the presence of methanol. In addition, P.pastoris cultures are able to grow in media with up to 10% of ethanol [10], which makes it a perfect candidate four our chassis.

Imagem da Pichia pastoris, the methylotrophic yeast.

Molecular detection

Overview

In order to detect methanol in alcoholic drinks, we searched for P.pastoris promoters inducible by methanol, and preferably known to have well-established genetic recombination techniques.

Table 1: LEGENDA DA TABELA LEGENDA DA TABELA LEGENDA DA TABELA

Gene name	Gene product	Regulation	Expression level
AOX1	Alcohol oxidase 1	Induced by methanol	Strong (naturally ∼5% of mRNA and ∼30% of total protein)
AOX2	Alcohol oxidase 2	Induced by methanol	∼5%–10% of PAOX1
DAS	Dihydroxyacetone synthase	Induced by methanol	Strong (similar to PAOX1)
FLD1	Formaldehyde dehydrogenase	Induced by methanol and methylamine	Strong (similar to PAOX1)

The three best choices (see table above) were PAOX1, PFLD1 and PDAS. Since we did not find much information on PDAS, while PAOX1 and PFLD1 are well characterized, both of them having commercial plasmids for genomic integration [13][14], we chose the latter two as our molecular detectors. That, in a simple genetic construction, could work as a proof of concept. A priori, our output system would be monomeric RFP (mRFP1, BBa_E1010), being used for fluorescence tests and for efficiently characterizing the relative promoter strength. We also expect to be able to see the production of RFP with the naked eye, once in E.coli it is shown to be possible.

Figure 1: Fluorescent proteins expressed in an E. coli suspension. Respectively, amilCP BBa_K592009 (blue), amilGFP BBa_K592010 (yellow) and RFP BBa_E1010 (red).

On the final design of a biodetector, we believe the best choice would be the one built by the 2010 iGEM team of Imperial College [15], where the origin of the colorimetric output would be cell lysis of the bioengineered organisms, in a time scale of minutes. Our team chose to focus on the detection system via methanol-responsive promoters, and to characterize them well as BioBricks.

P_AOX1

PAOX1 is a strong promoter which can be controlled by simple changes in its carbon source [16], and it is the most common choice for expression of heterologous proteins in P. pastoris, having a naturally elevated expression rate, of circa 5% of the RNA and 30% of total protein production [12].

The challenge in using P_AOX1 is its regulation. This promoter is prone to a strong catabolic repression by hexoses and ethanol [17] — the main component of alcoholic beverages. Fortunately, ethanol is also involved in the degradation of peroxisomes, cellular compartments where P. pastoris realizes the metabolism of methanol; this aspect is interesting for our application, since it means methanol will not be degraded as fast as it would in the absence of ethanol. Therefore, the degradation of peroxisomes would enhance the activation of P_AOX1, by allowing methanol to stay for longer in the cell.

In order to develop this biosensor, it is necessary to evaluate the rates at which the promoter P_AOX1 is activated via methanol or inhibited via ethanol. In addition, we decided to study a modification on Pichia pastoris' Mxr1p transcription factor [18] that should alter its interaction with PAOX1 by turning ethanol into an activator of the promoter [19]. If the rate of activation by ethanol stays below the rate of activation by methanol, the latter should be identifiable when the drink is diluted. It would then be possible to create a color guide that would help one differ pure and contaminated beverages (For more information, see the Modeling section).

In addition to testing the P_AOX1 that is native in Pichia pastoris, we have synthesized a modified version of this promoter with up to 33% more strength than the wild type promoter [20], according to the hypothesis that a stronger promoter should allow better visualization of the colorimetric output.

P_FLD1

This second promoter has a high transcription rate when activated, just like P_AOX1, but unlike the previous one it activated by methylamine or methanol [12] [21]. Also, it is not repressed by hexoses, like P_AOX1. Since an extensive search in scientific literature did not uncover any data on the regulation of P_FLD1 by ethanol, this promoter represents a great alternative to P_AOX1 if it is confirmed that they do not share the same repression characteristic related to this dicarbonyl alcohol, eliminating an issue in our project.

Preservation mechanism

Overview

In order to allow portability and storing of the device, and to create a robust and resistant and fast-responding methanol detector, some lyophylization tests were realized, aiming to produce results similar to Saccharomyces cerevisae yeast granules. That was made possible by adapting some protocols [29], originally for different yeast species, to Pichia pastoris. Some tests were realized, essentially looking for answers to the questions below:

• How many cells would have to be lyophilized?
• What is the ideal dilution of the drink?
• How long would it take between the contact between yeast and product and the obtaining of results?

These values predicted via mathematical modelling and tested in laboratory, find the necessary conditions for the use of this modified yeast as a methanol biosensor. The purpose was to test the predictions in non-contaminated beverages, and later in artificially contaminated ones, both as a proof of concept and as a way to test the sensibilities of the sensor.

Freeze-dry

Otto preparando conteúdo.

The device

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References

[22] Average price calculated in sep/2013: https://www.scienceexchange.com/services/gas-chromatography-mass-spectrometry-gc-ms?page=1

[7] International Center for Alcohol Policies (ICAP). Producers, Sellers, and Drinkers: Studies of Noncomercial Alcohol in Nine Countries [Monograph]. Washington, DC (2012).

[19] FIGARO TGS 822 - for the detection of Organic Solvent Vapors http://www.figarosensor.com/products/822pdf.pdf

[16] Norm ISO 1388-8:1981 http://www.iso.org/iso/catalogue_detail.htm?csnumber=5950

[23] http://jenslabs.com/2013/06/06/ketosense-an-arduino-based-ketosis-detector/

[1] AJA van Maris et al. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek, vol 90: 391–418 (2006).

[2] CP Hollenberg and G Gellissen. Production of recombinant proteins by methylotrophic yeasts. Current Opinion in Biotechnology, vol. 8: 554-560 (1997).

[3] LM Damasceno, CJ Huang and CA Batt. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, vol 93:31-39 (2012).

[4] JM Cregg et al. Expression in the Yeast Pichia pastoris. Methods in Enzymology, vol. 463, ch. 13 (2009).

[5] F Ganske and UT Bornscheuer. Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the inonic liquids [BMIM][BF4] and [BMIM][PF6] and organic solvents. Biotechnology Letters, vol. 28: 465-469 (2006).

[6] K De Schutter et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology, vol. 27(6) (2009).

[7] T Vogl and A Glieder. Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnology, vol. 30(4): 385-404 (2013).

[8] http://www.lifetechnologies.com/order/catalog/product/V23020?ICID==%253Dsearch-product

[9] http://www.lifetechnologies.com/order/catalog/product/V17520

[10] https://2010.igem.org/Team:Imperial_College_London/Modules/Fast_Response

[11] M Jalic et al. Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastoris. Biotechnol. Prog. vol. 22: 1465-1473 (2006).

[12] M Inan and M Meagher. Non-Repressing Carbon Sources for Alcohol Oxidase Promoter of Pichia pastoris. Journal of Bioscience and Bioengineering, vol 92(6): 585-589 (2001).

[13] GP Lin-Cereghino et al. Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris. Molecular and Cellular Biology, vol. 26(3): 833-897 (2006).

[14] PK Parua et al. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Molecular Microbology, vol. 85(2): 282-298 (2012).

[15] FS Hartner et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Research, vol 36(12) (2008).

[16] S Shen et al. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene, vol. 216: 93-102 (1998).