Team UT Knoxville

UTK
iGEM

MOTIVATION

In 2015, worldwide processing of crude oil averaged over 96 million gallons/day [1]. One of the largest byproducts of this industry is a mixture of toluene and its derivatives, and as a result roughly 21 million tons of toluene alone are produced each year [2]. These waste products are major environmental hazards. They are substantial contaminants of soil, groundwater, and air. Once these toxins reach soil and groundwater, they do not readily degrade and contaminate effected areas for extended periods of time [3]. Because they are produced on such a massive scale and in excess, almost all major toluene derivatives are both cheap and easily accessible. Our project focuses on degradation of these toxins and conversion to safer and industrially useful aromatic aldehydes by engineered E. coli.

Figure 1: Outline of our project's advantages

Aromatic aldehydes have a wide range of industrial uses, most prominently in the flavor and fragrance industry. Artificial almond, vanilla, cherry, peach, spearmint, and many other flavors or scents all make use of aromatic aldehydes. They are also used in plastics manufacturing, as easily degradable insecticides or fungicides, and as pharmaceutical precursors. Industrial aromatic aldehyde synthesis takes advantage of the low price and high availability of toluene derivatives as a substrate for production. Although this makes good use of the toxic precursors, the industrial processes used today present their own set of challenges, primarily that the most common catalysts are known environmental hazards themselves [4]. With this in mind, our project's goal is to construct a safe and renewable platform for synthesis of aromatic aldehydes from a cheap toluene derivative feedstock.

Economy

In addition to the main advantage of bioremediation, this platform provides another benefit in its economic value. All substrates used in our experiments are of low value due to the fact that they are waste products (Table 1), but producing each aldehyde provides an increase in value of up to 25 fold. In cases where the alcohol intermediate is more valuable than the aldehyde (notably 3-methylanisole and 3-chlorotoluene), that chemical can be produced using inly the XO module (part BBa_K1966000) instead of the composite part (BBa_K1966003) we have submitted, which will be discussed in further detail. The ability to modulate production between the expression of XO alone or XO with BADH gives us a specificity that is not contained within any one current manufacturing method today.

Table 1: Summary of values of precursors, intermediates, and aldehydes that will be investigated.

Pseudomonas Putida TOL Pathway

The soil microbe Pseudomonas putida is known to break down multiple toluene derivatives through a well-characterized metabolism [5]. P. putida uses a xylene monooxygenase consisting of two subunits, xylA and xylM, to oxidize a methyl substituent on a derivative to an alcohol group. Following this, the native benzyl alcohol dehydrogenase (xylB) reduces the alcohol group to an aldehyde, generating an aromatic aldehyde. The most important aspect of this synthesis is that no group other than a methyl group located at the 1-prime carbon will be affected. This means that any groups at the meta or para positions of the ring remain untouched by the pathway, allowing for the generation of a robust library of aromatics. These enzymes are both limited by an inability to act on compounds with groups in the ortho position, but other similar enzymes exist in hydrocarbon-degrading bacteria that are active for such substrates (these are similarly not active for groups in the meta and para positions). It has been demonstrated that multiple derivatives can undergo successful uptake and metabolism by P. putida and that derivatives with other groups such as halogens or carbonyls can be altered by the xylene monooxygenase [6], [7]. The activity of benzyl alcohol dehydrogenase on groups other than alcohols and carbon chains has not been explored.

Figure 2: Basic function of P. putida TOL pathway.

E. coli Platform: A Novel Approach for Synthesis of Aromatic Aldehydes

Our group engineered the genes xylA, xylB, and xylM into Escherichia coli, and the modified organism now functions as the biocatalyst for aromatic aldehyde synthesis. E. coli was selected as an expression host because it is a well understood model organism, and it has been previously indicated in literature that aspects of our platform are functional in this host [7].

Constructs

The constructs listed below were all submitted by the UKT iGEM team for this year's project. BBa_K1966000 contains xylA and xylM, the two subunits of P. putida xylene monooxygenase (XO). Both subunits must be expressed in order to produce functional XO. BBa_K1966001 contains xylB, the P. putida benzyl alcohol dehydrogenase (BADH). Part BBa_K1966003 is a composite part built from BBa_K196600 and BBa_K1966001 via digestion/ligation in the Biobrick cloning platform. Finally, BBa_K1966004 contains P. putida xylR, a regulatory protein and its associated promoter, Pu. The RFP in this part is amplified from registry part BBa_J04450. All constructs are under the control of a Lac promoter (part BBa_R0011) and contain the RBS BBa_B0034 and terminator BBa_B0010 as needed. Confirmed sequences of these parts are available in the registry.

Figure 3: Parts submitted into registry

Substrates

m-xylene and p-xylene

m-xylene and p-xylene are both major components of BTX mixtures. Because XO is sterically prevented from acting upon toluene derivatives with functional groups in the ortho position, we have only investigated m-xylene and p-xylene here. Conversion of both xylenes to their respective aromatic aldehydes generates a more valuable, more useful, and less toxic molecule. The table below shows the increases in value and uses of each product. m-tolualdehyde is used as an artificial flavor and fragrance additive for many sweet fruit flavors, such as peach or banana, and is most often used in conjunction with other flavoring agents. p-tolualdehyde is a widely used source of artificial cherry. As shown in Table 1, m-tolualdehyde is nearly 12 fold more valuable per gram than its precursor.

Figure 4

Toluene and 3-Chlorotoluene

Toluene is the main component of BTX waste. It is produced at such a large scale and in such excess worldwide that it is the cheapest of all components of BTX (Table 1). Toluene's corresponding aldehyde product benzaldehyde is used as the most common source of almond flavoring and fragrance across the globe, and is therefore much more valuable than toluene. This substrate has previously been demonstrated at length, as it is the main target of the P. putida TOL pathway.

3-chlorotoluene is not found naturally in BTX waste. However, it is still a highly toxic compound and our platform provides a method of transformation to 3-chlorotolualdehyde. This compound's most important use in in research as a protease inhibitor. This compound was chosen more to show novel functions of P. putida BADH than for its practical application; this is the first ever demonstration that the enzyme acts upon halogenated toluene derivatives. The alcohol intermediate generated during synthesis of 3-chlorotolualdehyde is far more valuable (approx. 15x) than the aldehyde product (Table 1). Instead of the composite part BBa_K196003, part BBa_K196000 (containing only the XO module) can be used to produce the alcohol instead. This is the case with all substrates.

Figure 5

3-methyl Anisole and 4-methyl Anisole

3-methyl anisole and 4-methyl anisole are less common components of oil waste than toluene and xylenes, but are still highly toxic. Only the XO module has been shown in literature to function with both of these substrates, making us the first group to observe the BADH function for each. These are 2 of the 3 novel functions we have found for P. putida BADH. The corresponding aldehydes for the 3 and 4-methyl compounds are m and p-anisaldehyde, respectively.

m-anisaldehyde and p-anisaldehyde have the most diverse range of applications of our target products. They are primarily used in drug synthesis for antiasthmatics, antihistamines, bronchodilators, contraceptives, and many others. p-anisaldehyde also finds uses as a flavor and fragrance additive, which, when used either alone or with other chemicals, can create a taste or scent of flowers, aniseed, fennel, and licorice. It is commonly found in detergent and laundry soap scents, and is sometimes also used for specialized TLC conditions in protein separations. Given all its potential applications, p-anisaldehyde is nearly 25 times more valuable than the respective substrate (Table 1).

Figure 6

P. putida xylene monooxygenase (XO) has been shown in literature to have a wide substrate range specificity, including all 6 substrates above (8, wide range substrate paper). However, benzyl alcohol dehydrogrenase (BADH) has not been demonstrated to act on 3-choloro benzyl alcohol, 3-methoxy benzyl alcohol, or 4-methoxy benzyl alcohol. One of the significant goals of this project is to characterize BADH for new functionality on these chemicals.

RESULTS

Our platform was successful in converting the majority of toluene, m-xylene, and p-xylene to their respective aldehyde products. As shown in Figure 8, toluene, m-xylene, and p-xylene were all remediated at high rates. Our parts are successful in converting toluene, m-xylene, and p-xylene into their respective alcohols and aldehydes.

Characterization on 3-methyl anisole, 4-methyl anisole, and 3-chlorotoluene was attempted but unsuccessful (Figure 8). It has been previously explained in literature that though xylene monooxygenase can act on these substrates [7] it does so with far lower specificity than to its primary targets (toluene, m-xylene, and p-xylene). Due to the fact that characterization was carried out under limited oxygen conditions to combat evaporation, we hypothesize that the combination of limited oxygen availability and low enzyme substrate specificity inhibited significant production of the desired products. Further investigation of BADH with corresponding alcohol intermediates for these substrates is described below.

Figure 7: Successful production of corresponding alcohols and aldehydes for toluene, m-xylene, and p-xylene.

Figure 8: Extent of conversion of toluene, m-xylene, and p-xylene to either alcohol or aldehyde.

GC-MS Results

Figures 9-11 are GC-MS chromatograms for the 3 substrates that were successfully able to be converted to their related alcohols and aldehydes by XO and BADH, respectively. These samples were collected after 8 hours of incubation with 5 mM of associated substrate. For more information on characterization and GC-MS methods, click here.

Figure 9: Chromatogram for m-xylene sample

Figure 10: Chromatogram for p-xylene sample

Figure 11: Chromatogram for toluene sample

As shown in Figure 8, XO was not shown to be active as expected from literature [7] for 3-chlorotoluene, m-anisaldehyde, and p-anisaldehyde. This is expected to be due to low enzyme activity and extremely limited oxygen availability at experimental conditions as mentioned above. However, P. putida BADH is NADH-dependent and is expected to function without issue in an anaerobic environment. Though BADH has been previously demonstrated to be functional with benzyl alcohol, 3-methylbenzyl alcohol, and 4-methylbenzyl alcohol [5], it has not been tested with the alcohol products that were originally expected from XO: 3-chlorobenzyl alcohol, 3-methoxybenzyl alcohol, and 4-methoxybenzyl alcohol. Using the same experimental setup as in XO+BADH characterization, cells with only BADH were cultured and tested with these 3 novel substrates. BADH was shown to convert both 3-chlorotoluene and 3-methoxybenzyl alcohol to 3-chlorobenzaldehyde and m-anisaldehyde. It was not functional for 4-methoxybenzyl alcohol (Figure 12).

Figure 12: Activity of BADH on 3 novel substrates. BADH was not able to convert 4-methoxybenzyl alcohol to p- anisaldehyde.

Figure 13: Aldehyde production from alcohol substrates

Though it was not shown that P. putida BADH was able to produce p-anisaldehyde, the iGEM team was still able to identify two previously unknown activates of the enzyme. BADH's activity on substrates with both halogen and methoxy functional groups shows that it is a reasonably wide substrate-range enzyme and is a suitable choice for generation of a library of aromatic aldehydes from aromatic alcohols.

Indigo Production

The final aspect of our product is indigo production. This activity was unexpected and was only realized once characterization of xylene monooxygenase on potential substrates. Xylene monooxygenase is one of many oxygenases reported in literature to have the activity described below, and produces indigo at a significant titer.

Along with its oxygenase activity on methyl groups, XO can act on naturally generated indole from E. coli and synthesize indigo dye with the mechanism shown below in Figure 14. This allows us to synthesize alcohols, aldehydes, and indigo crystals all at once with the same method, yielding over 2 g/L indigo while simultaneously achieving our main project goals. It should be noted that indigo production could compete for enzyme activity and limit productivity of aldehydes and alcohols, but we do not have a method at this time of preventing competition given that indigo production will occur any time the enzymes are active and indole is present.

Figure 14: Mechanism of indigo synthesis.

In order to confirm that indigo was the compound synthesized, samples of extracted indigo in chloroform were kept and a UV-Vis spectra was checked for each (Figure 15). Spectra results indicated that indigo is in fact the compound produced.

Figure 15: The spectrum on the left is the expected spectra for indigo (15, UTA chem), and spectra on the right apply to equal volume cultures grown with and without BADH as labeled. BADH+XO gives a much stronger signal than XO alone.

Once indigo synthesis was observed, indigo production was enhanced with induction of indole into cultures in stationary phase. These conditions are only intended for indigo synthesis and not aromatic aldehyde or alcohol generation.

Another unexpected result related to indigo production was that the presence of benzyl alcohol dehydrogenase (BADH, BBa_K1966001) increases indigo production. No previous literature has reported BADH or any dehydrogenase activity related to indigo. However, production levels with BADH under these conditions showed over 8-fold increase over XO alone; conditions designed for enhanced indigo production was 1.3-fold greater (Figure 17). The spectra above (Figure 15) also shows a significantly greater amount of indigo was present in cultures with BADH expressed. Extractions from XO+BADH vs. XO-only are visibly more colorful, and pelleted cells are far darker (Figure 16).

Figure 16: 1) Pelleted cultures of BL21 DE3+XO (A-B) vs pelleted cultures of BL21 DE3+XO+BADH (C-D). 2)Indigo extracted from cultures of BL21 DE3+XO+BADH (F) and BL21 DE3+XO (E). 3)Crystallized isolated indigo.

Figure 17: Production of indigo under varied conditions.

We hypothesize that the BADH promotes the conversion of indoxyl to 3-indoxole, which is necessary to produce indigo (Figure 14). This dehydrogenase is active on a wide range of aromatic alcohols, and given that indoxyl is an aromatic alcohol it likely is a substrate that BADH has specificity towards. Alcohols on 5-membered aromatic rings have been shown in literature as functional substrates for XO but not with BADH. This is the first report of use of dehydrogenase to enhance indigo production.

Pu Promoter

The Pu promoter of P. putida is activated in response to the presence of toluene, m-xylene, and p-xylene when the activator protein xylR is also present in conjunction with IHF and RNAP Ïƒ54 (both of which are naturally found in E. coli) [16]. This mechanism is depicted in Figure 18. Our part BBa_K1966004 contains all the necessary parts for regulation of the Pu promoter in E. coli.

Figure 18: Regulation of the P. putida Pu promoter.

Figure 19: Constructed and proposed constructs for Pu promoter-regulated expression.

When constructed as in part BBa_K196600x and grown with IPTG to induce the lac promoter, the Pu promoter is intended to sense and respond to toluene derivative substrates in media and subsequently express genes for their remediation. Part BBa_K1996004 is designed to express RFP under the same conditions.

In order to prove functionality of the Pu promoter, the team first characterized its responsiveness in RFU via RFP expression levels in BBa_K1966004. The construct showed no leaky expression of RFP without substrate induction, but upon introduction of substrate no fluorescence was observed (Figure 20).

Sequencing indicates that the construct is properly constructed despite the lack of response. The team has submitted BBa_K1966004 for potential future use and in order to make the piece available to any other teams that are interested in its use. The UTK iGEM team is continuing to troubleshoot the construct and registry information for the part will be updated if it is shown to be functional.

Figure 20: Characterization of part BBa_K1966004.

Real-World Application

The process of bioremediation occurs in complex environments, therefore organisms developed to serve as biocatalysts for waste disposal will need to be robust enough to survive and perform their function under multiple chemical and environmental stresses. To show the efficacy of our approach to bioremediation, we subjected our organism to complex chemical mixtures that simulate those it would be exposed to in the field as a bioremediator and in industry as a production module. We chose the complex chemical mixtures of gasoline and simulated BTX (toluene and the three xylene isomers) to serve as stand-ins, as they would provide complex substrates from which to measure the concentration of our terminal products, benzyl alcohol and benzaldehyde. Showcasing our organism's ability to function under real-world conditions will allow for optimization of the strain and its potential application to degrading waste streams such as gasoline run-off and the production of valuable chemical intermediates.

We used two scenarios to show real-world applications of our remediation platform: remediation of gasoline and a simulated BTX mixture. Gasoline, which contains significant amounts of toluene (5-7%, added to improve octane rating [17]), was added to cultures with BL21 DE3+BBa_K1966003 (XO+BADH) at a concentration of 5% v/v. The chromatogram below shows GC-MS results of gasoline alone and gasoline after 8 hours of induction with cells able remediate it (Figure 21). Although little benzaldehyde was produced, a significant amount of toluene was converted to the more environmentally friendly benzyl alcohol. It is likely that at a higher gasoline concentration more benzyl alcohol and benzaldehyde would be detected, but because toluene was only present at approximately 0.25%v/v in the culture substrate availability is limited. The concentration of gasoline was kept low due to safety concerns in the lab.

Figure 21: Conversion of toluene to benzyl alcohol and trace amounts of benzaldehyde in 0.5% v/v gasoline. Toluene concentration is decreased noticeably.

A simulated BTX was generated by adding a mixture of o-xylene, m-xylene, p-xylene, and toluene at 1.25 mM each to cultures for a total of 5 mM BTX mixture. The chromatogram in Figure 22 shows only toluene, benzyl alcohol, and benzaldehyde as o-xylene, m-xylene, and p-xylene cannot be reasonably differentiated with available GC-MS methods, so chromatograms for these compounds would be inconclusive. However, it is demonstrated in Figure 22 below that in the simulated BTX mixture benzaldehyde and benzyl alcohol are both generated from toluene.

Figure 22: Conversion of toluene in simulated BTX mixture.

Conclusions

The genes XylA, XylM, and XylB from the P. putida TOL pathway were successfully integrated into E. coli to form a system for bioremediation of and manufacturing using toluene derivatives.
Toluene, m-xylene, and p-xylene were converted to their respective alcohols and aldehydes using high cell density cultures.
Toluene from gasoline was successfully converted to benzyl alcohol which simulated a bioremediation scenario.
Toluene in a culture with mixed xylenes was successfully converted to benzyl alcohol and aldehyde which simulated a BTX waste stream bioremediation scenario.
Indigo dye was produced both in toluene derivative cultures, and in cultures with added indole.
Indigo production was increased when the host contained XylB.

Sources

Short Term Energy Outlook, March 2016. U.S. Energy Information Administration. www.eia.gov
Clark, J. H. & Deswarte, F. Introduction to chemicals from biomass. (Wiley.com, 2016).
Ukpaka, C. P. Modelling the BTX Contaminants Degradation in Soil Environments Using Bioremediation Techniques. International Journal of Current
Aluminum chloride, anhydrous MSDS. Sigma-Aldrich. Version 3.10. Accessed 4.04.2016
Harayama, S. et al. Characterization of Five Genes in the Upper-Pathway Operon of TOL Plasmid pWW0 from Pseudomonas putida and Identification of Gene Products. 1989. Journal of Bacteriology, Vol. 171, No. 9.
Kunz, D. and Chapman, P. Catabolism of Pseudocumene and 3-Ethyltoluene by Pseudomonas putida mt-2: Evidence for New Functions of the TOL Plasmid. April 2001. Journal of Bacteriology, Vol. 146, No. 1.
Wubbolts, M. et al. TOL plasmid-specified xylene oxygenase is a wide-range monooxygenase capable of olefin epoxidation. July 1994. Enzyme Microbiology Technology, Vol. 16.
Suzuki, Masahiko et al. Primary Structure of Xylene Monooxygenase: Similarities to and Differences from the Alkane Hydroxylation System. March 1991. Journal of Bacteriology, Vol. 173, No. 5.
Meta-Tolualdehyde. The Good Scents Company. 2016.
National Center for Biotechnology Information. PubChem Compound Database; CID=7725, https://pubchem.ncbi.nlm.nih.gov/compound/7725 (accessed Oct. 17, 2016).
Oâ€™Connor, Kevin E. and Hartmans, Sybe. Indigo Formation by Aromatic Hydrocarbon-Degrading Bacteria. March 1998. Biotechnology Letters, Vol. 20, No. 3.
Ensley, Burt. D et al. Expression of Naphthalene Oxidation Genes in E. coli Results in Biosynthesis of Indigo. October 1993. Science, Vol. 222, No. 4620.
McClay, Kevin et al. Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments. September 2005. Applied and Environmental Microbiology, Vol. 71, No. 9.
Oâ€™Connor, Kevin E et al. Indigo Formation by Microorganisms Expressing Styrene Monooxygenase Activity. November 1997. Applied and Environmental Microbiology, Vol. 63, No. 11.
Chemistry of Fibers, Textiles, and Garments-Dyeing of Garments. University of West Indies-Mona Department of Chemistry. 2015.
Kim, Mi Na et al. Construction and comparison of E. coli whole-cell biosensors capable of detecting aromatic compounds. 2005. Journal of Microbiological Methods, Vol 60.
Environmental Fact Sheet, Toluene Health Information Summary ARD-EHP-4. New Hampshire Department of Environmental Services. 2005.

Team:UT-Knoxville/Project