Nicotinamide Cytosine Dinucleotide Cofactor Synthesis

We initially employed commercially available competent Escherichia coli and introduced the pET-28(a) plasmid, which contains the gene encoding the Ncds-2 protein, into the E. coli BL21 strain. Subsequently, all E. coli cultures grown in LB medium containing kanamycin were confirmed to contain both the pET-28(a) plasmid and the target gene.

After induction with 0.2 mM IPTG, we collected two sets of Escherichia coli cultures: one with the Ncds-2 insert and another without it. Following cell lysis, we measured the intracellular cofactor concentrations. Interestingly, we observed that E. coli containing the Ncds-2 gene insert had an NCD concentration of 0.31 mM, while the group lacking Ncds-2 did not exhibit any detectable NCD (Figure 1).

Figure 1.Intracellular cofactor concentrations

(Notably, in the strains capable of synthesizing NCD, the synthesis of NAD was reduced. The NAD concentration was reduced from 0.93 mM to 0.45 mM. This could potentially be attributed to the fact that both NAD and NCD synthesis are dependent on NMN.)

To enhance the efficiency of NCD synthesis while minimizing its impact on intracellular NAD production, we introduced the FtNadE and CtCTPs genes(Figure 2). The enzymes encoded by these genes play a crucial role in catalyzing the precursor molecules NMN and CTP(Figure 3), which are vital for NCD synthesis. Consequently, the inclusion of the FtNadE and CtCTPs genes is considered essential.

Figure 2. The genetic circuit of NCD synthesis

(The synthesis of NCD within the cell has been verified, and we now need to purify the Ncds-2 protein to lay the groundwork for subsequent extracellular experiments.)

We induced our engineered strain to express the target protein and subsequently purified it using a nickel column. Following purification, we performed ultrafiltration and passed the protein solution through a molecular sieve. As a result, we obtained the Ncds-2 protein at a concentration of 5.8 mg/ml. The protein concentration was determined using the Bicinchoninic Acid (BCA) method(Figure 4). Notably, during the measurement process, the purified Ncds-2 protein was diluted tenfold.

Figure 4. Standard curve for determination of protein content by BCA method

Formaldehyde Metabolic Pathway Construction and Verification

To confirm the activity of formaldehyde dehydrogenase, formate dehydrogenase, and malic enzyme, we introduced the respective plasmids into the E. coli BL21 strain for induced expression, using the method previously described.

Following the purification of formaldehyde dehydrogenase and formate dehydrogenase, we conducted the procedures(Figure 5) to assess their activity.

Figure 5. The protocols of measuring FalDH and FDH activity

The variations in OD values serve as an indicator of dehydrogenase activity. Notably, we observed a more significant change in OD values in the Formate Dehydrogenase (FDH) system compared to the Formaldehyde Dehydrogenase (FalDH) system. As an example, when utilizing NMN and CTP at 5 mM, the change in mOD value per minute in the FDH system was 0.78 higher than that in the FalDH system (Figure 6). This observation suggests that the formate generated during formaldehyde metabolism is rapidly converted into carbonate, effectively reducing its toxicity to the engineered bacteria.

Figure 6. The degree of change in OD values increases with the rise in the concentration of NCD synthesis substrates.

In summary, we successfully confirmed the activity of formaldehyde dehydrogenase and formate dehydrogenase in relation to the non-natural cofactor NCD. This achievement has enabled us to construct an orthogonal pathway for the bioconversion of C1 molecules, holding promise for applications in both synthetic and chemical biology research.

To facilitate NCD regeneration, we introduced malic enzyme as a crucial catalyst for the conversion of NCDH to NCD. Furthermore, this enzyme exhibits the capability to synthesize malic acid from carbon dioxide and pyruvate. We have determined the activity of malic enzyme, proceed as Figure 7.

Figure 7. The protocol of measuring malic enzyme activity

Over time, we observed a gradual increase in the malic acid content(Figure 8). In our system, where NADH is absent, malic enzyme exclusively utilizes NCDH as a cofactor. Hence, the rising malic acid content serves as evidence that malic enzyme is indeed capable of catalyzing the synthesis of malic acid from pyruvate, carbon dioxide, and NCDH.

Figure 8. Reductive carboxylation of pyruvate in vitro

Build of Kill Switch

To ensure that our engineered bacteria does not pose any risk to the environment or users, we implemented a kill switch. We adopted the well-established MazF/MazE toxin/antitoxin system, a robust and widely-used mechanism within iGEM competition.

In our system, the expression of the toxin MazF is governed by the powerful T7 promoter, while the expression of the antitoxin MazE is regulated by the arabinose operon.

Figure 9. The genetic circuit of kill switch

When L-arabinose is introduced into the culture medium, the engineered bacteria commence the expression of the antitoxin MazE. This antitoxin can bind to the constitutively expressed toxin MazF, forming a complex that is non-toxic to cells^[1]. Consequently, in a culture medium supplemented with L-arabinose, the engineered bacteria can thrive as usual.

However, if these bacteria inadvertently enter an environment lacking L-arabinose, they will rapidly perish due to the presence of the toxin protein MazF, which prevents the expression of the protective antitoxin MazE. This meticulous design ensures that the engineered bacteria can only survive within a specified environment, thus preventing any potential adverse effects on external ecosystems.

References

[1] Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nature Reviews Microbiology, 9(11), 779–790.