Part:BBa_K4274032

ptac-RiboJ-B0034-CmR29M2_ClSS_S533A-B0034-ERG20_F96W-B0015

This composite part was used for the pathway IPP ⇄DMAPP → GPP → FPP → alpha-santalene, which ultimately creates alpha santalene, the precursor of santalol. In this part, we encoded the CmR29M2_ClSS_S533A (C. lansium santalene synthase with a hydrophilic DNA fragment) (Part: BBa_K4274001) and ERG20_F96W (Part: BBa_K4274002), an enzyme that catalyzes the formation of FPP (farnesyl diphosphate). Besides, the IPTG inducible promoter ptac-RiboJ (Part: BBa_K3552015) and the ribosome binding site B0034 (Part: BBa_B0034) were utilized for the expression of CmR29M2_ClSS_S533A and ERG20_F96W. Then, the composite part ends with the terminator B0015 (Part: BBa_B0015).

Our parts collection aims to optimize the production of alpha-santalene. There are two parts in this collection aiming to optimize the activity of FPP (farnesyl diphosphate), respectively ERG20_F96W (Part: BBa_K4274002) and ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20-B0015 (Part: BBa_K4274030). Then, we also composed parts that secretes ClSS and ERG20, comprising this composite part (BBa_K4274032) and CmR29M2_ClSS_S533A (PART: BBa_K4274001). Finally, we have two parts exploring the effects of adding a flexible linker and the production of alpha-santalene, consisting of ClSS_S533A-FL-ERG20_F96W (Part: BBa_K4274033) and (Part: BBa_K4274003).

By organizing this parts collection, we hope to hope the future teams by establishing our results and explorations on alpha-santalene production, providing some inspirations for in-depth investigation and utilization.

Usage and Biology

CmR29M2_ClSS_S533A is a gene sequence that expresses C. lansium santalene synthase, an enzyme that catalyzes the transformation from FPP (farnesyl diphosphate) to alpha-santalene. Two modifications have been made that made this part. To begin with, there was an artificial mutation that mutated the 533_rd amino acid of ClSS from serine to alanine. Then, we have also attached a DNA fragment derived from Synechocystis, performing the role of a solubility enhancer (Nico Betterle et al., 2018), hoping to increase the expression of ClSS by improving its solubility. It was concluded by research that adding CmR29M2 can lead to 131% production of alpha-santalene comparing to the ones with no tag (Jia Z et al, 2022). However, after conducting a series of experiments and tests, our results demonstrate that adding a tag to enhance solubility does not increase the production of alpha-santalene. On the contrary, it decreases the production to only 1/3 of the production without a tag strain. Therefore, the addition of CmR29M2 is not effective at optimizing the production of alpha-santalene.

Figure 1: 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.

In addition, we have also expressed ERG20_F96W in this composite part. ERG20_F96W is an enzyme that catalyzes the formation of FPP (farnesyl diphosphate). Its 96th amino acid was artificially mutated from phenylalanine to tryptophan.

Source

ClSS_S533A (Part: BBa_K4274000) is from Clausena lansium, ERG20_F96W (Part: BBa_K849001) is from S. cerevisiae, and CmR29 is from Synechocystis.

Sequence and features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4012
  Illegal BglII site found at 4073
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1628
  Illegal AgeI site found at 3728
  
• 1000
  COMPATIBLE WITH RFC[1000]