Latest revision as of 14:11, 18 September 2015

Harmonised Ethene Monooxygenase

EtnABCD construct consisting of 4 ORFs corresponding to the 4 monooxygenase subunits, with a RBS upstream of each ORF.

Background

The ethene monooxygenase (MO) enzyme converts alkenes to epoxides, and is found in Mycobacterium strains that grow on ethene (ethylene) as their carbon and energy source ¹. This enzyme is of intense interest for bioremediation and biocatalysis ², but to date it has never been expressed in a heterologous hots outside the genus Mycobacterium ³. A major focus of our project was to enable expression of ethene MO enzyme in Pseudomonas putida as an alternative expression host.

The etnABCD genes were modified using a novel approach called [http://2015.igem.org/Team:Sydney_Australia/TransOpt TransOpt], to give sequences that we expected would be effectively translated in Pseudomonas putida.

Experimental Validation

Sequences modified using TransOpt were ordered as three Gblocks, and cloned by [http://2015.igem.org/Team:Sydney_Australia/goldengate Golden Gate] cloning into pSB1C3; we sequenced several clones. While two clones gave bad sequence reads, and one had several large mutations, one clone was very close to the the expected sequence, with just one base change; this resulted in a single amino acid change converting glutamate residue 91 of EtnD to aspartate (E91D). Note that this change is conservative (both negatively charged aa’s), and also an aspartate is found at this position in the EtnD enzyme of other mycobacteria, so we expect the cloned EtnABCD to be functional, if given the correct host environment.

The etnABCD genes were cloned from pSB1C3 into the broad host range vector pBBR1MCS-2, where they are expressed from the lac promoter, and electrotransformed into P. putida KT2440. Note that in this host that lacks LacI, the lac promoter is constitutive. The culture was exposed to ethene, but none of the expected product (ethene oxide) was formed, based on testing with the colorimetric NBP assay. In a similar experiment, the wild-type etnABCD genes cloned in the same plasmid yielded detectable ethene oxide, so all the experimental setup and reagents were working.

We believed that the constitutive expression of ethene MO straight after transformation would put too much stress on the cells, and may have resulted in the growth of deletion mutants or other unexpected incorrect plasmid derivatives. Hence, we introduced LacI to control the expression of the system on a second plasmid, an RSF1010 derivative called pUS44, and reintroduced the pBR1MCS-2/etnABCD ligation mixture. However, after IPTG induction, the NBP assay still failed to detect ethene oxide, which suggests that the inactivity lies within the enzyme itself, not the cells. Testing the assay reagents with a pure epoxide solution (styrene oxide) yielded a strong purple colour, which confirmed our suspicion that the EtnABCD enzyme itself was to blame.

One reason for the ethene MO inactivity could be that our in-house harmonisation tool TransOpt did not yield a sequence which could be properly folded and active in Pseudomonas putida, since this was the result unexpectedly obtained in parallel experiments with different codon-optimised forms of a fluorescent protein BsFP (see [http://2015.igem.org/Team:Sydney_Australia/Results results and discussion]).

Part Description

While we could not demonstrate successful enzyme activity with this Pseudomonas putida-harmonised sequence variant of the ethene MO, we still deposit it as a Part. Our rationale for Part submission was twofold:

1. The EtnABCD sequence was 99.99% correct, and that the single mutation present was a conservative change which was consistent with sequences of other variants of this enzyme.
2. This enzyme is notoriously difficult to express in heterologous hosts, and thus further optimisation of elements like host strain, RBS’s, promoter, and accessory proteins may enable functional expression from the Part submitted here

We hope that future iGEM teams can further develop this system to yield a functional monooxygenase enzyme.

Important notes concerning this part:

• Due to an oversight in Gblock design, the pSB1C3-EtnABCD part submitted is missing the XbaI and NotI cut sites in the BioBrick prefix, but an EcoRI is present here.
• A mutation (E91D) exists in the sequence due to an accidental change introduced while removing BsaI cut sites, however, the change is conservative and is present in ethene MO of other Mycobacterium strains, which means that it is unlikely to have had an effect on the function of the enzyme.
• There are two slightly different alternative C-terminal ends quoted in GenBank for the EtnD subunit of this enzyme, it is unclear which is the correct annotation.

Structure of pSB1C3-EtnABCD plasmid construct submitted to the registry. Note that RBS are placed upstream of each ORF as shown in the above figure.

Sequence and Features

References

1 Coleman, N. V. and J. C. Spain (2003). Epoxyalkane:coenzyme M transferase in the ethene and vinyl chloride biodegradation pathways of Mycobacterium strain JS60.Journal of Bacteriology 185: 5536-5545.

2 Mattes, T. E., A. K. Alexander and N. V. Coleman (2010). Aerobic biodegradation of the chloroethenes: pathways, enzymes, ecology, and evolution. FEMS Microbiol Rev 34: 445-475.

3 Ly, M.A. et al. (2011). Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control. J Microbiol Methods 86: 320-326.