Part:BBa_K5226064

porin42

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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

E. coli was cultured at 37 °C in an LB medium containing (g L−1) 10 NaCl, 10 tryptone, and 5 yeast extract. H. bluephagenesis was cultured at 37 °C in a 60LB medium, namely, the LB medium supplemented with 60 g L−1 NaCl. A 20-LB medium indicates the LB medium containing 20 g L−1 NaCl. Moreover, 15 g L−1 agar was added before autoclaving for preparing solid media in Petric plates. Ampicillin (100 μg mL−1), chloramphenicol (25 μg mL−1), kanamycin (50 μg mL−1), or spectinomycin (100 μg mL−1) were added to the above media whenever necessary.

shake flask studies

H. bluephagenesis TD80 and its derivatives were cultivated in 50MMF medium in shake flask studies. The 50MM medium was composed of (g/L): NaCl 50, sodium formate 15, yeast extract 1, CO(NH2)2 0.25, MgSO4 0.2, Na2HPO4·12H2O 9.65, KH2PO4 1.5, trace element solution I 10 mL/L and trace element solution II 1 mL/L. The composition of trace element solution I was (g/L): Fe(III)-NH4-citrate 5, CaCl2 2, HCl 1 M. The trace element solution II was composed of (mg/L): ZnSO4·7H2O 100, MnCl2·4H2O 30, H3BO3 300, CoCl2·6H2O 200, CuSO4·5H2O 10, NiCl2·6H2O 20 and NaMoO4·2H2O 30. The pH-value of the medium was adjusted to approximately 9.0 using 5 M NaOH.

the microbial glycerol stocks were resuscitated by streaking on fresh plates. Then constructed plasmid was transferred into Halomonas TD80 through modified conjugation method using E.coli S17-1 as donor cells. Single colonies from newly-conjugated plates were picked and inoculated in the 60LB liquid medium for 12 h at 200 rpm to acquire the first seed culture, which was further grown on a fresh 60-LB liquid medium at a volume ratio of 1%. The second seed culture was inoculated for 12 h at 200 rpm. Afterward, it was inoculated into 150mL conical flasks containing 20 mL of the defined minimal medium at a volume ratio of 5% and cultivated for 48 h at 200 rpm. Antibiotics were added if needed. The temperature for all cultivations was 37 °C.

experimental design

Experimental group:
Using 15g/L sodium formate as the sole carbon source
Set IPTG induction concentration gradients of 0 and 2mg/L
1. Importing pSEVA321 Vib/AM1-C1M into TD80
(add 25 μ g mL-1 chlorocatechol):
Observing and comparising of the effect of Vib/AM1 C1M on enhancing TD80 formic acid assimilation ability
2. Importing pSEVA321 Vib/AM1-C13M and pSEVA341 Vib/AM1-C2M into TD80:
(Add 25 μ g mL-1 chloramphenicol and 100 μ g mL-1 spectinomycin)
Observing the effect of introducing the entire formate assimilation pathway on enhancing the formate assimilation ability of TD80, and comparing the effect of increasing C23M on enhancing the formate assimilation ability of TD80.

Control group:
1. Wild TD80 without carbon source, using only 50MM as culture medium:
eliminating the influence of other factors on TD80 growth
2. Wild TD80 containing 15g/L sodium formate:
observe the effect of introducing the entire formate assimilation pathway on TD80 assimilation of formate;
3. TD80 containing 15g/L sodium formate as the sole carbon source, introduced into pSEVA341:
eliminating the error of pSEVA341 affecting TD80 growth;

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 BBa_K5226073

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.