Part:BBa_K4274000

ClSS_S533A

ClSS_S533A is an optimized biobrick part encoding the gene for alpha-santalene synthase from Clausena lansium. The enzyme catalyzes the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the alpha-santelene in a single step. It is reported that ClSS's basic amino acid residue S533’s mutation to typical nonpolar amino acid Ala could result in a 1.7-fold increase in production of alpha-santalene compared to the nonmutated strain (Jia Z.et al., 2022) .

This year, we are using ClSS_S533A to construct composite parts ptac-RiboJ-B0034-Cl</i>SS_S533A-B0034-ERG20-B0015 (part: BBa_K4274020), ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20_F96W-B0015 (part: BBa_K4274021) and ptac-RiboJ-B0034-ClSS_S533A-FL-ERG20_F96W-B0015 (part: BBa_K4274023). This will allow engineering E.coli DH5a (tnaA-) to heterologously express ClSS_S533A. Other teams can utilize this part for E. coli alpha-santalene production.

Usage and Biology

ClSS_S533A is an optimized biobrick part encoding the gene for alpha-santalene synthase from Clausena lansium. It was firstly characterized by Jia Z.in 2022, who mutated ClSS's basic amino acid residue S533 to increase the production of alpha-santalene in E. coli (Jia Z.et al., 2022).

Natural ClSS is an alpha-santalene synthase which could catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the alpha-santelene in a single step. It has been successfully heterologously expressed to produce functional terpene product in both yeast (Wenlong Z.et al., 2020) and E. coli (Jia Z.et al., 2022). But the mutation of residue S533 to Ala led to the addition of two hydrogen bonds near this site (<4 Å), which resulted in a high α-santalene production E.coli strain.

In our study, ClSS_S533A could be heterologously expressed in E. coli, and allows for fused proteins to be bound to ERG20 (Part: BBa_K849001) and ERG20_F96W (Part: BBa_K4274002)

Source

Clausena lansium

Characterization

With the help of the co-transformation of pMVA plasmid with various pW1 plasmids, including pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL, different strains were successfully constructed and utilized for santalene production. The yield of santalene from strains CE, CEM, TCEM, CEM_FL could be used to characterize composite parts BBa_K4274030, BBa_K4274031, BBa_K4274032, BBa_K4274033. Eventually, strain CEM produces the maximal level of α-santalene, which elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%. It substantiated the prominent performance of ERG20_F96W in enhancing the supply of FPP and α-santalene production in E. coli. However, hydrophilic tag of CmR29M2 resulted in a significantly decrease in santalene production.

Figure 1: Characterization of BBa_K4274030, BBa_K4274031, BBa_K4274032, BBa_K4274033. (a) Measurement santalene production of different strains by GC/MS results. (b) Schematic representing the structure of pMVA, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL transformed into E.coli DH5α ∆TnaA.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 183
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Wenlong Z., Tianyue A., Ting L., et al. Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast. ACS Synth. Biol. 9 (2), 449-456 (2020). https://doi.org/10.1021/acssynbio.9b00479
[2] Jia Z., Xun W., Xinyi Z., et al. Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia coli. J. Agric. Food Chem. 70 (17), 5377-5385 (2022). https://doi.org/10.1021/acs.jafc.2c00754