Part:BBa_K1497002

B0034-eANS (engineered anthocyanidin synthase with strong RBS)

The anthocyanidin synthase from Fragaria x ananassa (ANS, EC 1.14.11.19) catalyzes many reactions in the anthocyanidin pathway. The TU Darmstadt iGEM team 2014 used its functionality by catalyzing the conversion of leucoanthocyanidin (2R,3S,4S)-cis-lucopelargonidin to pelargonidin. It also catalyzes the conversion of the leucoanthocyanidin to flavonol (kampferol). In order to avoid this side reaction and enhance metabolic flux, they designed a protein scaffold (BBa_K1497033). Earlier studies hypothesized that the used enzymes may be involved in metabolic channeling in their original organisms. By modelling the mechanical movements of ANS, they discovered a strong flexibility at the C-terminus. Subsequently they modelled ANS' structure and its movements. Thereby they detected a tail of the enzyme fluctuating and covering the active site.

Figure 1 Elastic network model of the anthocyanidin synthase. Only the C-terminus is very flexible and the rest of the ANS is highly rigid.

Figure 2 A: Visualization of the linear response theory results of the ANS. B: Linear response theory model (LRT): The RMSF (ref. RMSF Plots) computations reproduced the results derived from the coarse grained simulations. This underlines the complexity and importance of coarse grained simulations for rational protein design. With the RMSF we can clearly bring to proof that the C Terminal region is highly flexible and thus a obstacle to the active site of the ANS.

Visit the model page of the (iGEM Team TU Darmstadt 2014) and see the whole model results for the ANS.

Conclusion: It was necessary to open the active site by cutting of the C- Terminal region. Only with this modification it was able to increase the turnover of the ANS.

Please use this engineerd eANS. You will get a higher yield of your product!

Functional Parameters

In oder to improve the enzyme the iGEM Team TU Darmstadt 2014 decided to remove the tail and to construct a pelargonidin operon with this engineered ANS (eANS). The laboratory results confirmed the previous modeling results. The engineered ANS exhibited better yields than the original one when used in an operon producing pelargonidin (BBa_K1497015).(Fig. 3)

Figure 3 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 pelargenidin production. The operon with the engineered anthocyanindin synthase produces more pelargonidin

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]

References

1. Turnbull JJ, Nakajima J-I, Welford RWD, et al. (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase. The Journal of biological chemistry 279:1206–16. doi: 10.1074/jbc.M309228200

2. Welford RWD, Clifton IJ, Turnbull JJ, et al. (2005) Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism. 3117–3126.