Part:BBa_K1497014

Pelargonidin producing operon (low yield)- T7-B0034-F3H-B0034-DFR-B0034-ANS

Pelargonidin is an anthocyanidin. Anthocyanidins are vacuolar pigments that appear yellow to dark-red (pH-dependent), which are responsible for color of flowers and fruits and are health-promoting for humans. The iGEM Team TU Darmstadt 2014 constructed a pelargonidin producing operon under the control of a T7 promoter. (K1497014 and K1497015, respectively). The operon consists of 3 genes (flavonon-3beta-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase) each with strong RBS (Fig.1) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 2). The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided from Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E. coli coding optimized and synthesized by MWG Eurofins.

Figure 1 A: Genetic map of pelargonidin producing operon R: RBS; F3H: flavonon-3beta-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanidin synthase.B: Reaction scheme of a pelargonidin producing operon.

Functional Parameters

To analyze the pelargonidin production operon (K1497014), we transformed it into E. coli Bl21(DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan et al., (2007). After the induction with 1 mM Isopropyl-β-D-thiogalactopyranosid (IPTG) E. coli BL21 (DE3) cells were transferred into M9-media and fermented for 48h at 37°C in present of 0.1 mM naringenin.

Figure 2 E. coli BL21 (DE3) pellet containing the pelargonidin producing operon after the fermentation. According to Yan et al. (2007) a pelargonidin producing E. coli should be red after a pelargonidin production. The operon with the engineered anthocyanidin synthase produces more pelargonidin.

After the expression of pelargonidin producing operon with engineered ANS (K1497015) in present of 0.5 mM narigenin we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet and supernatant and verified the pH-dependency of pelargonidin (Fig. 3).

Figure 3 Extracted pelargonidin from E. coli BL21 (DE3) under day light. The color of pelargonidin depends on pH value and solvent. This indicates the present of pelargonidin. Left: Methanol extraction; right: Dichlormethane extraction.

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 239
  Illegal BamHI site found at 729
  Illegal BamHI site found at 1535
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1287
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1272
  

References

1. Yan Y, Chemler J, Huang L, et al. (2005) Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli. 71:3617–3623. doi: 10.1128/AEM.71.7.3617

2. Yan Y, Li Z, Koffas M a G (2008) High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnology and bioengineering 100:126–40. doi: 10.1002/bit.21721