Environmental Engineering Reference
In-Depth Information
2. State of the Science
2.1. Microbial Desulfurization
A promising alternative biotechnological approach employs the unusual abilities of a
microbial enzymatic pathway to oxidize thiophenic sulfur atoms and subsequently cleave
them from carbonaceous rings, releasing the sulfur and leaving the carbon essentially
untouched [7]. This biodesulfurization (Dsz) pathway was first discovered in Rhodococcus
erythropus strain IGTS8 in the late 1980s and has been studied in detail both in this organism
and in analogous form in several others, including several other members of the Rhodococci
as well as Agrobacterium MC501, Mycobacterium G 3 , Gordona CYKS1, Sphingomonas
AD109, and strains of Klebsiella, Xanthomonas, Nocardia globelula, and thermophilic
Paenibacillus and Bacillus [2, 7]. Physiological studies of these organisms have been
conducted primarily with the model compound dibenzothiophene (DBT) and have made great
progress in establishing the platform of understanding necessary to allow further
improvements through enzyme and pathway engineering [8].
2.2. Genes and Pathways
2.2.1. DBT uptake . In the native rhodococcal transformation (Figure 21), the
dibenzothiophene first gains access to the cell through apparently passive means that are
nevertheless assisted greatly by the tendency of rhodococci, in contrast with many other
bacteria, to collect at oil-water interfaces and even to partition into the oil phase in fine
emulsions [1, 9].
Despite their tendency to gather at oil drop surfaces, Rhodococci nevertheless rank low in
solvent tolerance [7], in contrast to Pseudomonas strains that are typically quite solvent-
tolerant [2]. To address this problem, as well as that of enzyme expression (below),
researchers cloned the dszABC genes of R. erythropolis IGTS 8 behind the constitutive tac
promoter into P. putida and P. aeruginosa species. The resulting strains grew more rapidly
than the rhodococci with DBT as the sole sulfur source and converted DBT to HBP
quantitatively, showing that this approach could provide strains that are more useful
commercially [2].
2.2.2. Sulfur oxidation. Once within the cell, the DBT sulfur atom is first oxidized by a
mono-oxygenase known as DszC. This enzyme makes use of a noncovalently-bound
FMNH2, provided in reduced form by the flavin reductase DszD, to activate the molecular
oxygen [8]. Notably, the availability of FMNH 2 is a crucial control on the rate of the
desulfurization, as discussed below. A second oxidation of the sulfur atom is also catalyzed
by DszC, again using FMNH2 provided by DszD, forming dibenzothiophene sulfone. A
second mono-oxygenase, DszA, catalyzes the transformation of the sulfone to the sulfinate,
again using FMNH2 and cleaving one carbon-sulfur bond. The remaining carbon-sulfur bond
is then cleaved by a most unusual enzyme, the desulfinase DszB, releasing sulfite and
hydroxybiphenyl, HBP [1, 2, 7, 8].
The availability of FMNH2 for the two mono-oxygenases appears to be a crucial rate-
limiting factor in microbial desulfurization. FMNH 2 activity depends on the activity of DszD,
the NADH-dependent FMN reductase, although DszD can be replaced in vitro by other FMN
reductases [2], and FMN reduction in turn depends on the availability of NADH from cellular
metabolism. This is an energy-intensive process, with ~4 NADH required per DBT
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