Part:BBa_K4144011

Optimized S-adenosyl methionine synthetase type-2 (opSam2)

Background and purpose

S-adenosyl-L-methionine (also called SAMe, S-adenosyl methionine) is a natural metabolite in all eukaryotes, which is sold in the United States as a dietary supplement. Abnormal levels of SAMe in the body have been reported in depression. Recent days, some studies have proved its beneficial efficiency as a natural anti-depressant. Unlike other usual antidepressants, side effects of SAMe are uncommon, and when they do occur, they are usually minor problems. So we choose it as our antidepressant molecule to relieve depression symptoms. And we plan to choose a SAMe synthetase to produce SAMe in our engineered bacteria.

Design of opSam2

Sam2, a SAMe synthetase from Saccharomyces cerevisiae (S. cerevisiae) which won’t be regulated by feedback inhibition, is our best choice. This property has been utilized to produce SAMe industrially. It has capacity of catalyzing production of SAMe from ATP and methionine (Fig. 1). There has been a part named SAM2 registered as BBa_K3817013 by 2021 team UCAS-China, but they didn’t characterize it at all. When we utilize this part, we consider that our engineered bacteria are purposed to be gram-positive. So to improve Sam2 expression efficiency, we used a codon optimization tool ExpOptimizer to optimize and we gained a new coding sequence opSam2. And we constructed it into the MCS of the shuttle vector pHT01K to express opSam2 in Bacillus subtilis. The coding sequence of opSam2 is downstream of Pgrac, a lactose inducible promoter.

Figure. 1 Enzymatic Reaction catalyzed by opSam2

Result

We have successfully expressed opSam2 in E. coli and B. subtilis using Pgrac to activate and His-tag to verify through Western Blot (Fig. 2).

Figure 2 Expression verification of opSam2 in E. coli and B. subtilis through WB.

Meanwhile, we used SAMe quantification kit to measure the concentration of SAMe synthesized in pHT-Sam2 and Empty Vector group, and verified the normal enzymatic activity of opSam2 in B. subtilis (Fig. 3).

Figure. 3 Enzymatic Activity Verificatio of opSam2 in Bacillus subtilis.

Improvement and Analysis

To have a better understanding of opSam2 and to improve its activity, we used Alphafold2 and SWISS-Model to analyze this homodimer’s structure (Fig. 4). We can figure out that opSam2 has several ligand binding sites, such as magnesium ion, potassium ion, PPK, adenosine and SAM. The binding of such ion’s residues can be found according to the structural prediction model. For example, the SAMe is adjacent to 20 residues within 4å. We decided to collect the relative residue sites and perform point mutation to construct a mutation library. Through SAMe production ability selection, we can gain a m-opSam2 with a better enzymatic ability.

Figure. 4 Structural analysis of opSam2 through AlphaFold2 and SWISS-Model.

Reference
[1]Galizia I, Oldani L, Macritchie K, Amari E, Dougall D, Jones TN, Lam RW, Massei GJ, Yatham LN, Young AH. S-adenosyl methionine (SAMe) for depression in adults. Cochrane Database of Systematic Reviews 2016, Issue 10. Art. No.: CD011286.
[2]Chu J, Qian J, Zhuang Y, Zhang S, Li Y. Progress in the research of S-adenosyl-L-methionine production. Appl Microbiol Biotechnol. 2013 Jan;97(1):41-9. doi: 10.1007/s00253-012-4536-8. Epub 2012 Nov 8. PMID: 23135229..
[3]iGEM21_UCAS-China https://parts.igem.org/Part:BBa_K3817013.

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]