Introduction
Many anaerobes carry out carbon fixation by Wood-Ljungdahl pathway. verified by Harland G. Wood and Lars G. Ljungdahl. This pathway, also known as the reductive acetyl-CoA pathway, is an anaerobic acetogenic pathway. One of the core enzymes in this pathway is carbon monoxide dehydrogenase/acetyl-CoA synthetase (CODH/ACS), a bifunctional enzyme which is able to employ H2 as reducer to reduce CO2 into CO, and then converted to acetyl-CoA, the central molecule in metabolism (Fig. 1)[1]. In this part, we will transform the gene of CODH/ACS into E.coli to make it autotrophic, that is, they are able to grow without carbon source.
Fig. 1. Wood-Ljundahl Pathway
In our project, related genes in the genome of the model acetogenic bacteria Moorella thermoacetica were chosen as basic components in gene circuit construction, because the structures and functions of these genes have been elucidated very well.[2]
Carbon monoxide dehydrogenase/ acetyl-CoA synthase (CODH/ACS) is The core enzyme in this pathway. It is a protein consists of 2 types of subunits. It is remarkable that correct folding of both these 2 subunits require involvements of certain cofactors. The overall reaction also requires the involvement of Fe(4)-Ni(1)-S(4) Cluster located in active site, methylene-H4folate reductase and the corrinoid iron-sulfur protein (CFeSP).[3]
Results
Ni concentration influence testing
The active site of CODH/ACS contain a Ni metallo-cluster, which means that the environmental concentration of Ni in medium is critical. Thus, we tested the influence of different Ni concentration on E.coli growth. According to the result, (Fig. 2) the required concentration of Ni will not obviously affect E.coli growth.
Fig. 2. The results of Ni concentration influence testing.The absorbrance (OD467) is proportional to bacteria concentration. H2O and C represent pure water and empty LB broth respectively. The numbers represent different Ni concentration (x 0.1mM).
Pre-experiment with Acetyl-CoA synthase alpha subunit C-terminal domain
Before we got started, we did a pre-experiment with Acetyl-CoA synthase α subunit C-terminal domain, which is the subunit the Ni ion binds to. (Fig. 3) We hoped to construct the expression vector of this gene to prove the reliability of this gene. According to our design, we will integrate this gene into expression plasmid pET28b, and purify protein product through His-Tag method. Figure 4 shows that we cloned the gene successfully. But unfortunately, we didn’t realize that there is an unexpected restriction site located in the gene. Thus, the PCR product was digested incorrectly. (Fig. 5)We have to gave up our pre-experiment and start our experiment directly.
Fig. 3. The gene circuit used in pre-experiment with CODH/ACS α subunit C-terminal.
Fig. 4. PCR results shows that we have cloned target gene successfully. The data of each lane is the temperature gradient for annealing.
Fig. 5. Enzymatic digestion result shows that the PCR product was digested incorrectly. Lane 2 is the digestion product digested for 5min; Lane 3 is the digestion product digested for 15min; Lane 4 is the digestion product digested for 30min.
Part standardization and expression
In this part, 6 parts were standardized, they are: CODH/ACS β subunit A, CODH/ACS α subunit M, CODH chaperone, ACS chaperone, methylene-H4 folate reductase, CFeSP. (Fig. 6) Then we inserted each gene into expression vector pET-28b, and expressed each part in E.coli respectively. The results of SDS-PAGE are shown in Figure 7. (Others are not shown) We also planed to verify their effective expression with His-tag method, which will be finished in the future.
Fig. 6. Gene circuit used in part expression.
Fig. 7. The results of SDS-PAGE for CODH/ACS β subunit A (Above) and ACS chaperone (Below). Lanes 1 and 2 are E.coli with empty plasmid. Lane 3-6 are E.coli with target genes and induced by IPTG with a concentration gradient (0mM,4mM，8mM，16mM).
For both CODH/ACS β subunit A and ACS chaperone, the 4 lanes with target genes showed exactly same pattern, which means the protein expression was not successful. Theoretically, The lane 3 should have 1 band (target proteins) less than lane 4-6 because its IPTG concentration is 0.
Theoretical Modeling in aquatic environment
The theoretical model of the whole protein complex in aquatic environment was made with software NAMD2 (NAnoscale Molecular Dynamics 2)(Fig. 8). It will be the reference model when we are capable to analyze the whole structure, and it will be used to prove the function of CODH chaperone and ACS chaperone, which play critical roles in protein folding.
Fig.8. The theoretical model of the whole protein complex in aquatic environment.
Reference
[1] Lindahl PA, Chang B. The evolution of acetyl-CoA synthase. Orig Life Evol Biosph, 2001, 31:403-434.
[2] Doukov TI, Iverson TM, Seravalli J, et al. A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Science, 2002, 298: 567-572.
[3] Pierce E, Xie G, Barabote RD, et al. The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum). Environ Microbiol, 2008, 10: 2550–2573.