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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

After engineering, E. coli could utilize both MEP pathway and MVA pathway for the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), then synthesize santalene with the help of FPP Synthase (FPPS) and santalene synthase (SS). Except heterologously expressed MVA pathway and ERG20 of Saccharomyces cerevisiae and santalene synthase of Clausena lansium (ClSS), several modifications upon ERG20 or ClSS by amino acid mutation, binding to a hydrophillic tag and the construction of fusion protein were tested for the higher yield of santalene. Therefore, 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 like CE, CEM, TCEM, CEM_FL were successfully constructed (Figure 1). The complete pathway we designed for producing santalene in E. coli is illustrated in Figure 1.

Figure 1: Construction and expression of santalene. (a) Enzymes and some of the reaction intermediates necessary for the production of santalene through the MEP pathway and MVA pathway. (b) Schematic representing the structure of pMVA, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL transformed into E.coli DH5α ∆TnaA.

Afterwards, the various engineering of E.coli DH5α ∆TnaA mentioned above were used for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for verification of α-santalene production. It turned out that all samples from four strains appeared a significant peak at the retention time of 26-27 min, and various peak area of different samples exhibited santalene production with differing levels, indicating the general success of E. coli engineering. It can be concluded that the E. coli strain CEM (with pW1_CEM plasmid) produces the maximal level of α-santalene compared to other strains (73.93 mg/L). Furthermore, our study elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%, substantiating the prominent performance of ERG20_F96W in enhancing the supply of FPP and α-santalene production in E. coli (Figure 2).

Figure 2: Quantification analysis of α-santalene production. (a) Measurement santalene production of different strains by GC/MS results. (b) Quantification of α-santalene is analyzed with 0.475 g/L α-humulene as an internal standard. And the GC/MS results demonstrate that the peaks at the retention time of 26-27 min and 28-29 min respectively were α-santalene and α-humulene. (c) GC/MS results of samples originated from CE, CEM, TCEM and CEM-FL strains.

Sequence and Features

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