Team:GZ HFI/Design

Design | iGEM GZ_HFI


Probiotics Selection

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

Conversion Pathway

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.

The metabolism pathway of ammonia conversion. The figure is modified from figure 1.A in Ref. Kurtz (2019).

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.

The metabolism pathway of hydrogen sulfide conversion. The figure is modified from figure 1 in Ref. Nakatani (2012).

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).

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).

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).

Intestine Homeostasis

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.


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