Part:BBa_K3846006

Bifunctional cytochrome P450/NADPH-P450 reductase

This part contains the codon-optimised codon sequence of Bifunctional cytochrome P450/NADPH--P450 reductase CYP102A1 from Bacillus megaterium.

It functions as as a fatty acid monooxygenase and catalyzes hydroxylation of fatty acids at omega-1, omega-2 and omega-3 positions. CYP102A1 shows activity toward medium and long-chain fatty acids, with optimum chain lengths of 12, 14 and 16 carbons (lauric, myristic, and palmitic acids). Able to metabolize some of these primary metabolites to secondary and tertiary products. Marginal activity towards short chain lengths of 8-10 carbons was also observed. The enzyme hydroxylates highly branched fatty acids, which play an essential role in membrane fluidity regulation. It also displays a NADPH-dependent reductase activity in the C-terminal domain, which allows electron transfer from NADPH to the heme iron of the cytochrome P450 N-terminal domain. CYP102A1 is involved in inactivation of quorum sensing signals of other competing bacteria by oxidazing efficiently acyl homoserine lactones (AHLs), molecules involved in quorum sensing signaling pathways, and their lactonolysis products acyl homoserines (AHs) (PubMed:18020460).

For our project we were mainly interested in the NADPH-P450 reductase (BM3R) part of this protein. The related phytobrick containing this sequence is this one. BBa_K3846105

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]

iGEM Hamburg 2021 part collection

Terpenoids are an important group of natural products used as biofuels, drugs or fragrances. Naturally occuring in plants it has been shown that microbial terpene production in microorganisms like yeast, E. coli or cyanobacteria is possible. Nevertheless iGEM projects seem to rarely focus on this interesting class of natural products which is correlated with a lack of useful parts inside the iGEM registry.

Fortunately we were able to change that and designed a novel golden gate based toolbox which allows.

1. production of terpenoid precursors and simple terpenoids
2. creation of CYP P450-reductase fusion enzymes to optimise processing of terpenoid precursors and production of bioactive target products
3. modularity of the system to enable exchange of linker sequences/promoters/etc. (MoClo-compatible toolbox)

MoClo-based Part Design 2.0

To improve the usefulness of our parts, we then aimed to make our parts compatible with the MoClo standard of goten gate based IIS restriction enzyme assembly. Thereby we expanded the Common Genetic Syntax for fusion sites to allow the creation of a) fusion proteins connected by linker sequences and b) multiple CDS expressed in an operon. More useful information and an overview of all our parts can be found on our wiki.

Figure 1: MoClo syntax of the part collection.