Taurine is a free aminoacid, it improves heart strength and also works as a cell buffer in order to protect them from harmful effects of UV light and toxic substances. Furthermore, taurine plays an important role in forming bile salts, fat digestion and also is involved in homeostasis regulating intracellular concentrations Na+ and Ca2+ and neurotransmissions equilibriums (Tovar, 2013).
The hypotaurine is the key intermediate for taurine synthesis, it´s an obligatory precursor leading to taurine but also it has been proposed that hypotaurine may act as an antioxidant, although the mechanism of the oxidation of hypotaurine is not fully understood. The enzyme responsible of the transformation of hypotaurine to taurine, hypotaurine dehydrogenase has not been totally identified in some tissues, thus, some researchers suggest that the conversion may be nonenzimatic or either spontaneously.
Brain, lung and sexual tissue contain high levels of hypotaurine, there is evidence to support that it can protect these cells from oxidative damage during hypotaurine quenching of carbon or oxygen-based free radicals can itself react with oxygen, removing dissolved oxygen from the microenvironment undergoing oxidative stress.
Taurine and hypotaurine are closely linked. Taurine, with its special stability, is considered a metabolic end product of methionine and cysteine. Most metabolic pathways give hypotaurine as a precursor to taurine. Hypotaurine differs from taurine only in the oxidation state of the sulfur center and the resulting change in the acidity of the molecule. In general, hypotaurine is much more reactive than taurine. Recent experimental data have shown that hypotaurine reacts rapidly and efficiently with the hydroxyl radical, superoxide radical, and hydrogen peroxide. The ability to scavenge these reactive oxygen species rapidly is a prerequisite for a molecule to act as an antioxidant in vivo.
Tovar, J. (2013). Sistema de aminoácidos neurotransmisores: Taurina. Recuperado el 27 de marzo de 2016, de http://www.javeriana.edu.co/Facultades/Ciencias/neurobioquimica/libros/neurobioquimica/taurina.htm
Team:BIOSINT Mexico/Description
WIKI IGEM 2016
In 2006 were identified four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3 that shared low overall identity and conserved important active-site residues to the enzyme cysteine dioxygenase (CDO) which provides important oxidized metabolites of cysteine such as sulfate and taurine. For this it was used PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs. This work was one of the first and the few in show experimental evidence for the presence of CDO in prokaryotes (Dominy, Simmons, Karplus, Gehring & Stipanuk, 2006).
Conservation of functional residues within eukaryotic and putative bacterial CDOs
Also in 2013, the paper “Discovery of a substrate selectivity motif in amino acid decarboxylases unveils a taurine biosynthesis pathway in prokaryotes” publish that the sequence, structural, and mutational analyses of the structurally and sequentially related hCSAD and human glutamic acid decarboxylase (huGADL) enzymes revealed a three residue substrate recognition motif, within the active site that is responsible for coordinating their respective preferred amino acid substrates.
Mammalian cysteine metabolism and Hypotaurine formation in mitochondria.
However more experiments about the taurine and hypotaurine production and function have been carried out in eukaryotic organisms.
In 2006 a study of in vivo production of taurine, hypotaurine and sulfate following subcutaneous administration of L-cysteinesulfinate (CSA) to rats and mice indicate that liver is the most active tissue for taurine production, followed by kidney, also that external CSA, hypotaurine and taurine are easily taken up by these tissues (Nakamura, Yatsuki & Ubuka, 2006).
In 2008, in an investigation of the hypotaurine production from L-cysteinesulfinate in rat liver mitochondria was indicated the presence of L-cysteinesulfinate decarboxylase activity and the possibility of antioxidant roles of cysteine metabolites like hypotaurine (Ubuka, Okada & Nakamura, 2008).
In 2010, in the publication “Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae”, taurine was used as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio.
Although there are no records of iGEM teams who have worked with the direct bioproduction of taurine or hypotaurine, some teams have used them as precursors for other purposes in their projects.
In 2015 for example, the team Nankai used taurine to modified Poly-γ-glutamic acid (γ-PGA), grafting it to the side chain of γ-PGA to obtain sulfonated γ-PGA. This was done with the objective of loading more Superoxide dismutase on treatment gels, which is used as an antioxidant for decreasing the content of reactive oxygen species in injured tissues, and to finally develop γ-PGA hydrogels to promote wound healing.
Dominy, J., Simmons, C., Karplus, P., Gehring, A., & Stipanuk, M. (2006). Identification and Characterization of Bacterial Cysteine Dioxygenases: a New Route of Cysteine Degradation for Eubacteria. Journal Of Bacteriology, 188(15), 5561-5569. doi:10.1128/jb.00291-06
Nakamura, H., Yatsuki, J., & Ubuka, T. (2006). Production of hypotaurine, taurine and sulfate in rats and mice injected with L-cysteinesulfinate. Amino Acids, 31(1), 27-33. doi:10.1007/s00726-005-0277-7
Nankai iGEM team. (2015) Recovered from https://2015.igem.org/Team:Nankai/pudding_health_kit
Ubuka, T., Okada, A., & Nakamura, H. (2008). Production of hypotaurine from l-cysteinesulfinate by rat liver mitochondria. Amino Acids, 35(1), 53-58. doi:10.1007/s00726-007-0633-x
This project aims to bioengineer E. coli in order to produce hypotaurine, the predecessor of taurine. We chose this project because the idea has not been developed yet using synthetic biology and because of the market potential that has emerged around taurine.
There are several ways in which hypotaurine is biosynthesized from cysteine, but the simplest and shortest one found was the one that acts via oxidation of cysteine with the enzyme cysteine dioxygenase (CDO), followed by the decarboxylation of cysteinesulfinate using the enzyme cysteine sulfinic acid decarboxylase (CSAD) to produce hypotaurine.
When the genetically modified E. coli is cultured in a cysteine-rich medium these enzymes will be in charge of catalyzing the reaction to produce hypotaurine, which leads to the opportunity to the production, purification and commercialization of this precursor of taurine.
Proteins
CDO
Cysteine dioxygenase (CDO, 2 EC 1.13.11.20) is a non-heme mononuclear iron metalloenzyme that catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid (CSA) with addition of molecular oxygen. This enzyme has a length of 200 aminoacids and a mass of 22,972 Daltons (UniProt, 2005).
The product of the reaction catalyzed by CDO, cysteine sulfinic acid, is either decarboxylated to hypotaurine, which is further oxidized to taurine by a poorly understood mechanism, or transaminated to the putative intermediate 3-sulfinylpyruvate that spontaneously decomposes to pyruvate and sulfite, with sulfite being further oxidized to sulfate by sulfite oxidase. Thus, CDO not only removes excess cysteine but is necessary for hypotaurine/taurine and sulfite/sulfate production from cysteine. It is likely that both roles of CDO have physiological significance and that the major role of CDO may vary with cell type. CDO is one of the most highly regulated metabolic enzymes responding to diet that is known (Stipanuk et al., 2008).
CDO is known to be expressed in the brain, kidney, and lung, with significantly high levels in liver tissue, where this enzyme has an important role in maintaining the hepatic concentration of intracellular free cysteine (Ye et al., 2007).
It is believed that CDO is involved in a number of neurodegenerative diseases, like Parkinson and Alzheimer, and also in autoimmune diseases, like systemic lupus erythematosus and rheumatoid arthritis, due to its function in regulating free cysteine levels (Ye et al., 2007).
CSAD
The cysteine sulfinic acid decarboxylase enzyme (CSAD), also known as sulfoalanine decarboxylase, is an enzyme involved in the hypotaurine biosynthesis. It is in charge of catalyzing the reaction that decarboxylates cysteine sulfinate generating hypotaurine and CO2. CSAD is a liase, and more specifically a carboxylase (Sumizu, K., 1962). This enzyme has a length of 493 aminoacids, and has a weight of 55,023 Daltons (UniProt, 2006).
GADL1 (Glutamate Decarboxylase Like 1) is a Protein Coding gene. Diseases associated with GADL1 include Gadl1-Related Altered Drug Metabolism. Among its related pathways are Pantothenate and CoA biosynthesis and Taurine and hypotaurine metabolism. GO annotations related to this gene include pyridoxal phosphate binding and sulfinoalanine decarboxylase activity. An important paralog of this gene is DDC.
GADL1 encodes a member of the group 2 decarboxylase family. A similar protein in rodents plays a role in multiple biological processes as the rate-limiting enzyme in taurine biosynthesis, catalyzing the decarboxylation of cysteine sulfinate to hypotaurine. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene.
Stipanuk, M. H., Ueki, I., Dominy, J. E., Simmons, C. R., & Hirschberger, L. L. (2008). Cysteine dioxygenase: A robust system for regulation of cellular cysteine levels. Amino Acids, 37(1), 55-63. doi:10.1007/s00726-008-0202-y
Ye, S., Wu, X., Wei, L., Tang, D., Sun, P., Bartlam, M., & Rao, Z. (2006). An Insight into the Mechanism of Human Cysteine Dioxygenase: KEY ROLES OF THE THIOETHER-BONDED TYROSINE-CYSTEINE COFACTOR. Journal of Biological Chemistry, 282(5), 3391-3402. doi:10.1074/jbc.m609337200
The assembly for this project is mainly composed for two enzymes into several constructs, all of them in pSB1C3. The plasmids shown below have different purposes, like only for the expression of the required enzymes, and plasmids that codify the sequence with poly his-tag to ease the purification of CDO and CSD.
Figure N° 1. pSB1C3 with the construct for constitutive expression of cysteine dioxygenase (CDO) and cysteine sulfinic acid decarboxylase (CSAD).
PC+RBS+CDO+HIS+TT
Figure N° 2: pSB1C3 with the construct for constitutive expression of CDO enzyme with a poly-his tag attached downstream to ease the protein purification.
PC+RBS+CSAD+HIS+TT
Figure N° 3: pSB1C3 with the construct of constitutive promoter, CDA enzyme sequence with a poly-his tag attached downstream to ease its purification.
PC+RBS+CDO+RBS+GFP+TT
PC+RBS+CSAD+RBS+SPISPINK+TT
Figure N° 4: These constructs ease the identification of the respective enzymes because contains the sequence attached a reporter gene, GFP with CDO and SPISPINK with CSAD.
PC+RBS+SPIS+TT
PC+RBS+SPIS+HIS+TT
Figure N° 5: Assembly shown above contain the sequences for a modified and improved version of the chromoprotein Spis Pink designed by BIOSINT_Mexico 2015 iGem team. The device in the left correspond to the current chromoprotein sequenced with an improved stop site to end the transcription process. Meanwhile device in the right side is the chromoprotein with a poly-his tag attached downstream for a better protein purification.
Several devices were designed with different purposes, some of them just correspond to the protein expression, CDO and CSAD, meanwhile other devices were built for help the protein purification and the protein expression identification in the wet lab by using chromoproteins like reporters.
Figure shown above explains how the entire system works. From L-cysteine metabolism to be converted in 3-Sulfino-L-alanine to finally convert this substrate in Hypotaurine.
Below are shown different constructs the team designed to ease the identification of a functional device, or right expression of the enzymes. A culture with a proper expression of the CDO construct (BBa_K2004011), will be green because the reporter gene located downstream of this sequence, which codifies to Green fluorescent protein (GFP).
Meanwhile, the CSAD construct (BBa_K2004012) will produce this enzyme and a pink chromoprotein because both gene sequences are together next to its constitutive promoter. It should be noted that the sequence used to the pink chromoprotein is a new and improved version of SpisPink part (BBa_1684000) designed by iGEM BIOSINT_Mexico 2015 team.
In this way, a brief step to confirm the chromoprotein expression is purifying it, so to facilitate this process we fuse a improved version of the sequence (BBa_K2004004) that codifies to pink chromoprotein with another registered part, the poly his tag (BBa_K844000). The last version of this chromoprotein(BBa_K1684000), designed by the previous Biosint team, was analyzed according to its sequence and all the parameter that must meet to be part of a standardized biobrick. This device (BBa_K2004013) allows to test the fusion, because contains a constitutive promoter and a RBS upstream and the double terminator downstream.
In order to get as a final result, the hypotaurine production, both enzymes CDO and CSAD must be together. For carry out this project, we took the CDO1 sequence from Enterobacter aerogenes and we optimized the sequence to be able to express a functional enzyme in Escherichia coli (BBa_K2004007), besides the sequence was cleaned up of whatever restriction site, that is not part of the BBF RFC 10, that could generate troubles with the standard 3A Assembly. The same happened with the GADL sequence to codify the CSAD enzyme, which is part of the Homo sapiens sequence; this sequence has been widely used in a experimental way to study the expression of this enzyme by using microorganism, so we cleaned up the sequence of restriction enzymes, optimize the sequence to be used into E. coli ((BBa_K2004008). Finally we added the corresponding prefix and suffix sequence to both enzyme sequence to be assembled together with an RBS (BBa_J61101) sequence between them.
For this device (BBa_K2004009), both enzyme sequences were joined to a RBS sequence (BBa_J61101) at the 5’ side, a constitutive promoter sequence (BBa_J23106) upstream at the 5’ end and a double terminator sequence (BBa_B0012) downstream at the 3’ end.
Furthermore, we have noticed that protein purification is an essential step to further experiments and analysis of the system behavior, because of this we decided to fuse the optimized enzyme sequences (CDO and GADL) with another registered part which is a poly his tag (BBa_K844000) designed by iGEM12_Utah_State team. In this way, there are two new modified parts, the first is BBa_K2004002, the fusion between CDO (BBa_K2004007) with Polyhis tag, and the respective constitutive promoter, RBS and double terminator. The second device BBa_K2004010 is the corresponding to the GADL1 sequence, in the same conditions, assembled with its constitutive promoter, RBS and double terminator.
Bioproduction of hypotaurine: Metabolic pathway insertion in Escherichia coli
BIOSINT MEXICO 2016
Instituto Tecnologico y de Estudios Superiores de Monterrey Campus Queretaro Epigmenio Gonzales 500, Fracc. San Pablo Email: biosintqro@gmail.com Queretaro, QRO
