When producing any molecule of commercial interest with a microorganism, prospecting for the key gene is as important as the choice of the host to be used. The evolu­tionary convergence of ethylene production is high­lighted by the three pathways delineated above: Yang cycle, KMBA and 2-oxoglutarate, with the last having been found to be the most efficient when overproduc­tion is desired. The evolutionary radiation of the mobile plasmid encoding the efe gene among the different species and strains might have produced a naturally optimized gene that could be used in commercial pro­duction. A comparison between 20 P. syringae strains revealed a high amino acid sequence similarity between five pathovars, with P. syringae pv. phaseolicola PK2 being the most efficient, giving a twofold higher production of ethylene (Weingart et al., 1999). However, these varia­tions are likely to be due to differences in regulation as the sequence of amino acids of efe of these five strains differs by only one codon.

Using the efe gene encoded by an indigenous plasmid from P. syringae pv. phaseolicola PK2, ethylene production was reported in E. coli with a tenfold increase when compared to the original strain, P. syringae (Fukuda et al., 1992a; Ishihara et al., 1995), showing that the efe gene alone was sufficient for ethylene production. When a high-copy-number plasmid containing efe was transconjugated into Pseudomonas putida and P. syringae, ethylene production was increased, but surprisingly, production was 27- and 8-fold higher, respectively, than the wild type, whereas the amount of protein pro­duced in the cloned P. syringae was 20-fold higher (Ishihara et al., 1996), suggesting the presence of a post­transcription regulatory system. Using cellulose as sub­strate, Tao et al. showed the production of ethylene in Trichoderma viride through the heterologous expression of efe from P. syringae pv. glycinea. Thus, the use of agri­culture wastes as substrate for ethylene production was proved to be feasible, but the recombinant filamentous fungus produced only very small amounts of ethylene (Tao et al., 2008).

So far, cyanobacteria have been shown to be the best model for the bioproduction of ethylene. Of course, many barriers still have to be crossed and commercial production is far from reality at present, but the last few years have seen encouraging reports where the pro­ductivity was increased several fold without compro­mising cell fitness, suggesting that the true production limit might be much higher. The efe (EFE) from P. syrin­gae was originally cloned into Synechococcus elongatus PCC 7942 (Fukuda et al., 1994; Sakai et al., 1997; Taka — hama et al., 2003). The first problem area, the production of only trace amounts of ethylene by the transformants (Fukuda et al., 1994), was later shown to be due to the nature of the promoter used. A systematic evaluation of different promoters showed the psbA1 promoter is more efficient for efe expression than those (lac and efe) previously used in other reports, achieving production rates up to 240nl/mlh or 451 nl/ml h OD730 (Taka — hama et al., 2003). However, these recombinants showed high genetic instability. Sequencing of the heterologous gene from mutants that had ceased to produce ethylene showed punctual mutations at a defined sequence of five nucleotides, suggested to be a possible hot-spot site for spontaneous mutagenesis (Takahama et al.,

2003) . Nevertheless, active ethylene-producing strains showed signs of metabolic stress, evidenced by their yellow-green color. When these strains had ceased ethylene production due to spontaneous mutation of efe (genetic instability), they recovered the normal blue-green phenotype.

In another strategy (Ungerer et al., 2012), Synechocys — tis sp. PCC 6803 was used as model organism. Toxicity to ethylene was tested, efe was codon optimized and artifi­cially synthesized, eliminating the bases at the putative mutational hot spot by conservative substitution. As well, efe was placed under the control of the psbA1 pro­moter. A semicontinuous culture using a clone contain­ing two copies of efe was sustained over a three-week period, reaching a constant production of 3100 nl/ml h, compared to the previous result of 240 nl/ml h (Taka- hama et al., 2003). The peak of the specific productivities was 380 nl/ml h OD730 for one efe copy and 580 nl/ mL h OD730 for two copies, respectively, and when in semicontinuous culture, the average rate was 200 nl/ ml h OD730. The additional copy of the efe gene pre­sented some production improvement when compared with the previous work from Takahama et al., (451 nl/ ml h OD730 compared to 580 nl/ml h OD730) but the real advance for the field can be seen from the healthy state of the culture. The growth rate, the color of the cul­ture and the growth curve were the same for wild type and the mutants containing one or two copies of efe. This shows that there is no toxicity either by the product or by the metabolic route used to produce ethylene. In addition to the zero toxicity, the release of five carbons per ethylene formed does not seem to present a burden to the cell, as shown by the growth pattern of the single and double mutant when compared with the wild type. Nevertheless, the metabolic consequences to the cell of a higher rate of ethylene production are unknown and a physiological approach would help to understand how far ethylene production can be pushed and what to target to improve the final yield.