It is important to choose a safe probiotic microorganism because the final product is designed to be orally taken. We selected E.coli Nissle 1917 (EcN) as the engineered chassis. EcN has been used as probiotics in Europe for a long time in history (Wessenaar, 2016). Moreover, by examining the excretion of healthy volunteers who orally taken EcN, researchers found out that the median clearance time of EcN is one week, and for 80% of healthy volunteers, the clearance time is less than three weeks (Kurtz, 2018). Thus, EcN does not cause long-term harm to the human digestive system.
EcN also serves as the agent for probiotic therapy. Various studies have used E.coli to treat relapse of ulcerative colitis and bowel disease, which showed positive consequences. Those studies further ensure the biosafety of EcN in humans (Kruis, 1917; Rembacken, 1999).
Construction of the Metabolic Pathways
The genetic design for ammonia metabolic pathway is inspired by a 2019 research in which the researchers genetically engineered EcN strain to convert ammonia to arginine in responding to an intestinal anaerobic environment. It shows a positive result in mice treated with hyperammonemia (Kurtz, 2019). Arginine is a crucial intermediate in the urea cycle, and more importantly, its chemical formula contains four nitrogen, which makes it an ideal option for converting ammonia efficiently (Urea Cycle, n.d.; Arginine, n.d.).
The ammonia that enters into E.coli is converted to glutamate along with α-Ketoglutarate (Fig.1). The series of reactions that convert glutamate into arginine are catalyzed by N-acetyl glutamate synthetase (NAGS), encoded by gene argA, to acetylize glutamate (Cunin, 1986).
However, the arginine synthesis pathway is repressed through two regulatory pathways. First, arginine itself has feedback inhibition on NAGS (Vyas, 1963). In order to maximize the conversion of ammonia to arginine, feedback-resistant (fbr) NAGS enzymes is required, whose argA gene is mutated into argA^fbr by substituting the sequences TAT (Tyr) at 19th position into TGT (Cys), and the GCG (Ala) at 389th position into GCT (Ala) (Rajagopal, 1998). Second, arginine synthesis is restricted through the arginine regulation, because the formation of crucial enzymes is not only controlled by the conjunction of arginine but also arginine repressor encoded by the regulatory gene argR (Lim, 1987). Thus, argR is designed to be knocked out in the genome.
Figure 1: The metabolism pathway of ammonia conversion. The figure is modified from figure 1.A in Ref. Kurtz (2019).
In the pathway of converting hydrogen sulfide to L-cysteine, L-serine and Acetyl-CoA form O-acetylserine catalyzed by L-serine O-acetyltransferase (SAT) (Fig. 2). SAT is encoded by gene cysE, and its feedback is inhibited by the final product L-cysteine (Kondoh, 2019). In order to induce overproduction of L-cysteine, we need to develop a mutant gene of cysE which will code for feedback inhibition-insensitive SAT. One study suggests turning the ATG (Met) at the 256th position into TGG (Trp) by mutation to obtain cysE-256 (Nakamori, 1998). Another study created several different cysE mutants that overexpress non-feedback-resistant cysE, and the best two mutants are selected(Kai Y, 2006), named as cysE-5, to substitutes GTC (Val), GAT (Asp) at the 95th and 96th positions for AGA (Arg), CCC (Pro), and cysE 11-2, which substitutes CGT (Arg), ACC (Thr) for CAT (His), GTA (Val) at 89th, 90th position, and CCG (Pro), GCA (Ala) for GCT (Ala), ACA (Thr) at 93th and 94th position (Kai, 2006).
To figure out whether there exists a better mutant, new types of cysE mutants are created by combining different mutants together (cysE 5-11-2; cysE 256-5; cysE 256-11-2; cysE 256-5-11-2).
The efficiency of different mutants to overproduce cysteine is tested and the most optimal mutant is selected into product design.
Figure 2: The metabolism pathway of hydrogen sulfide conversion. The figure is modified from figure 1 in Ref. Nakatani (2012).
Myrcene Synthesis Pathway
We also aim to construct the E.coli to produce a fragrant substance to mask the acrid smell. For the purpose of both producing aroma and being safe for humans, we selected terpenes, which can be made naturally in plants, to be the agent masking the acrid smell (Berger, 2007). Myrcene, with the lowest toxicity among the monoterpene family, is a type of terpenes-kind substance that can be naturally produced. Researches also prove that the E.coli pierced with myrcene almost exhibits no growth inhibition (Kim, 2015). Thus, we design to engineer pathways to produce myrcene. Three plasmids are constructed containing essential genes: pMevT, pMBIS, and pTYT-GPPS-MS (Fig. 3). The myrcene pathway MevT contains three genes that encode for necessary enzymes to catalyze the reaction that transforms acetyl-CoA into mevalonate (Fig.3 A). These three genes are AtoB from E.coli and HMGS and HMGR from Saccharomyces cerevisiae (Woo, 2013). MBIS pathway contains five different genes that encode for necessary enzymes to catalyze the reaction that transforms mevalonate into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are ERG12, ERG8, MVD1, idi, and ispA from Saccharomyces cerevisiae (Fig.3 B) (Martin, 2003). Next, IPP and DMAPP are catalyzed by geranyl pyrophosphate synthase (fig.3 B), which is encoded by gene GPPS, to form geranyl diphosphate, the precursor of monoterpene. Then geranyl diphosphate is converted into β-myrcene via myrcene synthase encoded by MS gene (Fig. 3 B) (Kim, 2012). In one study that engineered E.coli to produce myrcene, the researchers test the performance and efficiency of GPPS and MS gene from different kinds of organisms, and from their results, we select the MS(Qi) gene from Quercus ilex L. and GPPS2 from Abies grandies (Kim, 2015).
Figure 3: The metabolism pathway of myrcene. (A) The MevT pathway. (B) MBIS Pathway and genes GPPS and MS. The figure is modified from figure 1.A in Ref. Kim (2012) and figure 1 in Ref. Matin (2003).
In the human digestive system, a delicate bioregulatory system maintains intestinal homeostasis. In the urea cycle, arginine is broken down into urea and ornithine by arginase (Walker, 2014). The gut bacterium, such as E.coli, Salmonella enterica, Clostridia, and Enterobacter aerogenes, can convert cysteine back to hydrogen sulfide (Kumagai, 1975). Human intestinal tissue also conducts the conversion by the cystathionine beta‑synthase and cystathionine gamma‑lyase (Martin, 2010).
Therefore, the natural mechanism of homeostasis will prevent arginine and cysteine from affecting the intestinal balance in the long term. In other words, the engineered E.coli would only temporarily lower the concentration of hydrogen sulfide in the intestine.
Berger RG: Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Springer; 2007.
Cunin R, et al. Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 1986;50(3):314-52.
Jiang Y, et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015;81(7):2506-14.
Kai Y, et al. Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine. Protein Eng Des Sel 2006;19(4):163-7.
Kim EM, et al. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli. J Agric Food Chem 2015,13;63(18):4606-12.
Kondoh M, et al. L-Cysteine production by metabolically engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 2019;103(6):2609-19.
Kruis W, et al. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997;11(5):853-8.
Kumagai H, et al. Crystallization and properties of cysteine desulfhydrase from Aerobacter aerogenes. FEBS Lett 1975;52(2):304-7.
Kurtz C, et al. Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates. Clin Transl Sci 2018;11(2):200-7.
Kurtz CB, et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med 2019;11(475):eaau7975.
Lim DB, et al. Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the argininerepressor. Proc Natl Acad Sci USA 1987;84(19):6697-701.
Martin VJJ, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nat Biotechnol 2003;21(7):796-802.
Matin GR, et al. Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract. Dig Liver Dis 2010;42(2):103-9.
Nakamori S, et al. Overproduction of L-Cysteine and L-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase. Appl Environ Microbiol 1998; 64(5):1607-11.
Nakatani T, et al. Enhancement of thioredoxin: glutaredoxin- mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. Microb Cell Fact 2012;11:62.
Rajagopal BS, et al. Use of Inducible Feedback-Resistant N-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered Escherichia coli K-12 Strains. Appl Environ Microbiol 1998;64(5):1805-11.
Rembacken BJ, et al. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999;354(9179):635-9.
Urea Cycle. NYU School of Medicine. http://education.med.nyu.edu/mbm/aminoAcids/
ureaCycle.shtml. Accessed September 13, 2020.
Vyas S, et al. Feedback inhibition of acetylglutamate synthetase by arginine in Escherichia coli. Arch Biochem Biophys 1963; 100(3), 542-6.
Sun V, et al. Surviving Colorectal Cancer: Long-Term, Persistent Ostomy-Specific Concerns and Adaptations. J Wound Ostomy Continence Nurs 2013; 40(1): 61-72.
Trans5α Chemically Competent Cell. TransGene. https://www.transgen.com.cn/chemically/
278.html. Accessed 14th September, 2020.
Trans10 Chemically Competent Cell. TransGene. https://www.transgen.com.cn/chemically/
278.html. Accessed 14th September, 2020.
Walker V. Ammonia Metabolism and Hyperammonemic Disorders. Adv Clin Chem 2014;67:73-150
Wassenaar TM. Insights from 100 Years of Research with Probiotic E. Coli. Eur J Microbiol Immunol (Bp) 2016;6(3):147-61*.*
Woo HM, et al. Application of targeted proteomics and biological parts assembly in E. coli to optimize the biosynthesis of an anti-malarial drug precursor, amorpha-4,11-diene. Chemical Engineering Science 2013;103:21-28.