Part:BBa_K5226063
Mmp1 Vib C1M-porin58 Vib C3M
Contents
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1199
Illegal EcoRI site found at 3904
Illegal EcoRI site found at 5277
Illegal PstI site found at 4846 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1199
Illegal EcoRI site found at 3904
Illegal EcoRI site found at 5277
Illegal NheI site found at 2161
Illegal NheI site found at 4200
Illegal PstI site found at 4846 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1199
Illegal EcoRI site found at 3904
Illegal EcoRI site found at 5277 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1199
Illegal EcoRI site found at 3904
Illegal EcoRI site found at 5277
Illegal PstI site found at 4846 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1199
Illegal EcoRI site found at 3904
Illegal EcoRI site found at 5277
Illegal PstI site found at 4846
Illegal NgoMIV site found at 465 - 1000COMPATIBLE WITH RFC[1000]
Introduction
One of the goals of iGEM24-SCUT-China-A is to use synthetic biology tools to obtain Halomonas TD strains that can metabolize formate. We chose to introduce the formate assimilation pathway to enable it to utilize formate, a difficult-to-recover product in CDE.
For the first method, based on previous studies obtained from literature research,[1][2][3][4]we selected the tetrahydrofolate (THF) cycle imported from Methylobacterium extorquens AM1 and strengthened the endogenous C2 and C3 modules of Halomonas TD.
As a second approach, based on the homology between Vibrio natriegens and Halomonas TD [5], we chose to import the C1, C2, and C3 modules from Vibrio natriegens.
Usage and Biology
After obtaining the fermentation data of C1M imported into TD80(BBa_K5226061), we were eager to explore whether the overexpression of C2M and C3M would further enhance the ability of TD80 to assimilate formate. However, it should be noted that bacteria do not allow us to do whatever we want. In the world of Halomonas TD, it cannot accept plasmids that are too large, nor can it accommodate two plasmids containing the same replication origin. In addition, we must recognize that we commonly use two types of plasmids in our laboratory: pSEVA321 and pSEVA341. Due to its high copy characteristics and antibiotic resistance genes, pSEVA341 poses a challenge for the growth of Halomonas TD. Therefore, considering the size and importance of the C1, C2, and C3 modules, we have decided to combine C1M with C3M on pSEVA321 and to place C2M on pSEVA341.
Among them,
C1M is composed of three key enzymes: formate tetrahydrofuran ligase, methyltetrahydrolase, and methylnetetrahydrofolate dehydrogenase, which work together to convert formic acid to 5,10-Methylene-THF;
C2M consists of four essential enzymes: dihydrolipoyl dehydrogenase, aminomethyltransferase, glycine recycling system protein H, and glycine dehydrogenase, which convert 5,10-Methylene-THF into glycine;
C3M is made up of two critical enzymes, Serine hydroxymethyltransferase and L-serine dehydration, which transform glycine into pyruvate.
At this stage, thanks to the collective efforts of the entire formate assimilation module, formate has been successfully converted into pyruvate, enabling entry into both material metabolism and energy metabolism.
Experimental characterisation
growth conditions
shake flask studies
experimental design
Post fermentation treatment
To ensure the measurement accuracy of the spectrophotometer, we diluted the bacterial solution 5 times and measured OD600.
Data Processing and Analysis
pSEVA341 significantly affects the growth of the strain, and its assimilation effect on formate is even weaker than that observed without introducing the entire pathway. Therefore, we aim to avoid using plasmid 341. Considering the importance of C1M, we have decided to integrate it into the genome while placing C23M on pSEVA321.
Prior to this integration, data analysis revealed that IPTG induction did not show a consistent trend. As a first step, we will focus on transforming the inducible promoter of C1M into the most effective constitutive promoter based on existing experimental results, and subsequently integrate it into the genome. For further experiments, please refer to <a href="https://parts.igem.org/Part:BBa_K5226074">BBa_K5226074</a>.
References
[1] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.
[2] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.
[3] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.
[4] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.
[5] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.
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