As with ethylene, isoprene is a medium-value biochemical that is produced through steam cracking of oil. It is actually an important by-product of ethylene production and is almost entirely used for production of a synthetic substitute for natural rubber. It is also natu­rally produced by many plants as a heat stress response, where it was shown to increase the stability of photosyn­thetic membranes at high temperatures (Sharkey et al.,

2001) . It can represent as much as 2% of all carbon fixed by oak leaves at a temperature of 30 °C (Sharkey, 1996), showing the physiological importance of this com­pound. The enzyme isoprene synthase (ispS) was shown to produce isoprene in plants, converting one of the products of the methylerythritol phosphate (MEP) pathway, dimethylallyl-diphosphate (DMADP), into isoprene (Silver and Fall, 1991; Silver and Fall, 1995). Prokaryotes were suggested to be able to produce isoprene after reports of the detection of this compound in the headspace of culture broth on many species (Kuzma et al., 1995), with emphasis on Bacillus subtilis. Not surprisingly, sequence analysis of bacterial genome could not identify any gene homologous to the ispS. found in plants (Withers et al., 2007). So far, functional genomics has also failed to identify the pathway for isoprene pro­duction in prokaryotes. Sequence-independent methods showed that 19,000 E. coli clones transformed with DNA fragments from B. subtilis in an environment where DMADP and IPP (isopentenyl pyrophosphate) levels were selectively toxic, showed that no single enzyme was sufficient to convert DMADP to isoprene, where the few clones that managed to survive, preferably converted it to a prenyl alcohol (Withers et al., 2007). As all isopre — noids are thought to be solely produced from DMADP and IPP (Xue and Ahring, 2011), the conversion of the metabolites involved in MEP or mevalonate pathway

Coa-dependent pathway

FIGURE 22.4 Pathway alternatives for n-butanol bioproduction. The alcohol n-butanol is naturally produced in different microorganisms in small quantities, where it can be synthesized either through the CoA-dependent pathway or the keto acids pathway. (For color version of this figure, the reader is referred to the online version of this book.)

to isoprene in bacteria could be a phenotype derived from convergent evolution using a multistep reaction diverged from those pathways (Izumikawa et al., 2010; Withers et al., 2007; Xue and Ahring, 2011).

Bioproduction of isoprene is feasible and has already been demonstrated in E. coli expressing heterologous ispS (Miller et al., 2001; Zhao et al., 2011). Of course pro­ductivity is an issue and different strategies were tried to increase isoprene production. Simultaneous expres­sion of heterologous enzymes involved in MEP or meval — onate pathways was shown to be effective in both cases (Yang et al., 2012; Zhao et al., 2011). Julsing et al. also showed that the individual expression of the genes encoding enzymes involved in the MEP pathway did not affect isoprene production with the exception of the dxs gene, encoding the enzyme that catalyzes the first re­action of the MEP pathway, which significantly improved isoprene production (Julsing et al., 2007). Cya­nobacteria produce DMAPP through the MEP pathway for secondary metabolites and, albeit with no natural pro­duction of isoprene being reported yet, transformation and expression of heterologous ispS were shown to be sufficient for production of isoprene. Lindberg et al. re­ported isoprene production using Synechocystis sp. PCC 6803 as a model organism harboring ispS from Pueraria montana (Kudzu) (Lindberg et al., 2010). The transgene was inserted at the psbA2 locus and mutants did not
show any disturbance in growth when compared to the wild type. This was a well-achieved proof of concept, and the low productivity reported, 50 mg per gram of dry cell weight per day, can be much improved through metabolic engineering. However, the use of cyanobacte­ria to produce isoprene has issues different from meta­bolic yield: to develop a production system of a molecule with a half-life of only a couple hours in the presence of light is particularly challenging in a photo­synthetic organism. To overcome this issue, the develop­ment of special photobioreactors is made in parallel to the molecular research, where the properties of isoprene as a volatile hydrophobic compound, easily separated from a culture broth and concentrating in the headspace, are exploited (Lindblad et al., 2012). The production of a gas in microorganisms is an interesting strategy because one does not need to harvest the cells, the product is concentrated in the gaseous phase of the culture. Howev­er, the cultivation techniques and the purification of this gas from a complex mixture represents an important step in the production chain and, as shown in this case, should develop together.