229
Biodesulfurization of fossil fuels
Kevin A Gray, Gregory T Mrachkoyz and Charles H Squiresy§
Biotechnological techniques enabling the specific removal of
sulfur from fossil fuels have been developed. In the past three
years there have been important advances in the elucidation of
the mechanisms of biodesulfurization; some of the most
significant relate to the role of a flavin reductase, DszD, in the
enzymology of desulfurization, and to the use of new tools that
enable enzyme enhancement via DNA manipulation to influence
both the rate and the substrate range of Dsz. Also, a clearer
understanding of the unique desulfinase step in the pathway has
begun to emerge.
Addresses
Diversa Corporation, 4955 Director’s Place, San Diego, CA 92121, USA
e-mail: [email protected]
y
The Dow Chemical Company, 5501 Oberlin Dr, San Diego,
CA 92121, USA
z
e-mail: [email protected]
§
Corresponding author: Chuck Squires
e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:229–235
This review comes from a themed issue on
Ecology and industrial microbiology
Edited by Bernard Witholt and Eugene Rosenberg
1369-5274/03/$ – see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1369-5274(03)00065-1
Abbreviations
DBT
dibenzothiophene
DBT-MO
DBT monooxygenase
DBTO2-MO DBT-sulfone monooxygenase
FMNH2
reduced flavin mononucleotide
HBP
2-hydroxybiphenyl
HPBS
2-(20 -hydroxyphenyl)benzenesulfinic acid
PBS
2-phenylbenzene sulfinate
RACHITT
random chimeragenesis on transient templates
TPL
tyrosine-phenol lyase
Introduction
The development of biotechnologies aimed at upgrading
the quality of fossil fuels has been an area of interest for at
least two decades (for recent reviews see [1,2]). Much of
this attention has focused on microbiological/biochemical
means of desulfurizing fossil-fuel streams. The impetus
for this work was the discovery of a highly specific
metabolic pathway in the organism Rhodococcus erythropolis strain IGTS8. This organism is capable of obtaining
nutritional sulfur from common heterocyclic molecules in
a fuel without causing oxidative loss of carbon from the
fuel itself. A biological approach to desulfurization has
also been motivated by difficulties in reducing sulfur to
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very low levels using current petroleum refinery practices
(hydrodesulfurization). These low levels are required by
governmental regulations, and in the next few years the
regulations are expected to become even more stringent.
Crude oil contains a large number of molecules that
contain sulfur; however, the major sulfur-containing
molecules in the middle distillate fraction (diesel fuel)
are alkylated dibenzothiophenes (DBT). The complete
removal of sulfur from DBT-like molecules requires four
enzymes. Two of these enzymes, DBT monooxygenase
(DszC or DBT-MO) and DBT-sulfone monooxygenase
(DszA or DBTO2-MO), are flavin-dependent. These
both require a third enzyme (the flavin reductase DszD)
for activity. The fourth enzyme, HPBS desulfinase
(DszB), completes the reaction sequence, which results
in a phenolic product and SO32 (Figure 1). The genes for
these enzymes have been cloned, sequenced and engineered from a variety of microorganisms.
Within the past few years several new flavin reductases,
including thermo-tolerant enzymes, have been discovered and a more comprehensive picture of how these
enzymes participate in the oxygenation reaction is emerging. In addition, the least well understood enzyme in the
pathway, HPBS desulfinase, is becoming better characterized. On the basis of both published and unpublished
data, we present a speculative model of the mechanism of
action of this enzyme as a starting point for further
research.
A significant stumbling block to the commercialization of
biodesulfurization is the rate at which whole bacterial
cells can remove sulfur. The throughput of substrates in
this pathway may be hindered at several steps, including
substrate acquisition [3], the supply of reducing equivalents [4,5] and enzyme turnover rates for specific substrates [6]. Recently, directed evolution techniques have
been applied to improve both the rate and extent of
biodesulfurization by derivatives of R. erythropolis strain
IGTS8 [7,8]. Application of these techniques, together
with the progress cited above, promises to improve both
the range and robustness of this microbiologically catalyzed chemical transformation.
In this review, we summarize published and unpublished
results of the past three years on the flavin reductase
required for monooxygenase activity, the recent discovery
of additional Dsz pathways and enzymes, the use of
selective and in vitro genetic tools to recover improved
or novel Dsz phenotypes, and on the final enzyme in the
pathway, which has a novel catalytic activity and for
which we propose some enzyme reaction mechanisms.
Current Opinion in Microbiology 2003, 6:229–235
230 Ecology and industrial microbiology
Figure 1
DBT
S
FMNH2
DBT-MO (DszC)
O2
NAD+
DszD
NADH
FMN
DBT-sulfoxide
S
O
FMNH2
DBT-MO (DszC)
O2
DszD
NAD+
NADH
FMN
DBT-sulfone
S
O
DBTO2-MO (DszA)
O2
O
FMNH2
DszD
NAD+
NADH
FMN
HO
HPBS
SO2−
HPBS desulfinase
(DszB)
HO
HBP
+
HSO3−
Current Opinion in Microbiology
The desulfurization pathway of R. erythropolis strain IGTS8.
DszD: the flavin reductase
Oxidative desulfurization of dibenzothiophene requires
reducing equivalents in the form of NADH and reduced
flavin mononucleotide (FMNH2). As with other biological
reactions that involve oxygen insertion, reducing equivalents are necessary to activate molecular oxygen. Presumably, the activated oxygen species is a flavin hydroperoxide
that catalyzes the transformation of DBT to DBT sulfoxide and DBT sulfone via DBT-MO and the subsequent
conversion of DBT sulfone to HPBS via DBTO2-MO.
Neither DBT-MO nor DBTO2-MO are ‘flavoproteins’, in
that the purified proteins do not bind FMN very tightly,
nor do these enzymes oxidize NADH, distinguishing
them from the well-described flavin monooxygenases
[9]. An auxiliary protein, termed DszD, was identified
from R. erythropolis; this protein reduces FMN to FMNH2
using NADH as the reducing agent [10,11]. In vitro and in
vivo experiments showed that DszD supports the activity
Current Opinion in Microbiology 2003, 6:229–235
of DBT-MO and DBTO2-MO. Other non-native flavin
reductases are also able to supply reduced flavin to the
monooxygenases [4,5,12]. Using the N-terminal sequence
from purified DszD [10], the dszD gene was cloned and
sequenced from R. erythropolis IGTS8 [11]. The gene
encodes a protein of 20 kDa and is present on the
chromosome of IGTS8 rather than on the 120 kbp plasmid
that contains dszABC. Insertional inactivation of the dszD
gene leads to the loss of both flavin reductase activity and
the Dsz phenotype (J Childs, personal communication).
However, there is no apparent effect on strain viability
when it is grown on another sulfur source, implying that
there is no other required physiological function for DszD.
The Dsz-negative phenotype is complemented by the
dszD gene provided in trans. Overexpression of dszD or
another flavin reductase enhances the overall rate of
desulfurization in R. erythropolis (J Childs, personal communication), Escherichia coli [12] and Pseudomonas [4],
suggesting that the supply of reduced flavin to the monooxygenase is a rate-limiting step in the complete in vivo
pathway. McFarland [2] indicates that the ThcE protein of
strain IGTS8 is also involved in desulfurization; however, a
thcE null mutant is still Dszþ (C Squires and J Childs,
unpublished data) suggesting that this protein does not
have a direct role.
As DszD uses FMN as a dissociable substrate it has been
classified as a member of the class I flavin reductases
[13]. This class is further subdivided on the basis of
sequence similarities. One subgroup, typified by the wellcharacterized E. coli protein Fre, is involved in the
activation of ribonucleotide reductase [13,14,15]. Other
members of this subgroup include the flavin reductases
from bioluminescent bacteria [16,17,18]. A second subgroup, to which DszD belongs, contains enzymes
involved in the synthesis of certain antibiotics; these
enzymes include ActVB [13,19,20], SnaC [21], VlmR
[22], HpaC for the oxidation of 4-hydroxyphenylacetate
in E. coli [23] and cB for the degradation of nitriloacetic
acid in Chelatobacter heintzii [24,25]. Detailed structural
and mechanistic information is becoming available for
these classes of flavin reductases [13,14,15]. The data
suggest that the enzymatic mechanism of the ActVB and
DszD proteins is ordered-sequential, where NADH binds
first [13] (J Emmanuele, personal communication). Free
reduced flavin is very unstable and autooxidizes extremely quickly; it is therefore being proposed [18,26] that
some sort of protein–protein interaction occurs between
the reductase and its cognate oxygenase to stabilize the
reduced flavin and allow oxygen insertion.
Recently, Ishii et al. [27] have isolated and cloned a
flavin reductase (tdsD) involved in desulfurization by the
thermophilic desulfurizing bacterium, Paenibacillus sp.
A11-2. TdsD has high sequence similarity to other flavin
reductases like Fre, but there is no detectable sequence
similarity between TdsD and the Rhodococcus DszD.
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Biodesulfurization of fossil fuels Gray, Mrachko and Squires 231
In addition, the tdsD gene is not located in the flanking
region of the tds operon encoding TdsA and TdsC
monooxygenases.
In certain cases it has been shown that the rate-limiting
factor in complete desulfurization is flavin reductase
activity; hence, overexpression of an appropriate reductase enhances desulfurization. Interestingly, Ohshiro et al.
[28] report that different flavin reductases have different
abilities to couple to the monooxygenases. In fact, they
show that the flavin reductases from a non-desulfurizing
bacterium are superior to those from a DBT-desulfurizing
bacterium, suggesting more efficient transfer of FMNH2
from the reductase to the monooxygenases. This information may be beneficial in developing the most efficient
enzymatic desulfurization system and in elucidating the
mechanisms of interaction between the monooxygenases,
reductases and reduced flavin species.
Recent discovery of additional dsz
pathways and enzymes
Although R. erythropolis is the most extensively studied
example, several other mesophillic microorganisms have
also been shown to catalyze oxidative desulfurization;
these include Sphingomonas [29], Corynebacterium [30],
Gordona [31], Klebsiella [32] and Nocardia [33]. Duarte
et al. [34] have performed genetic analyses (using PCR
and denaturing gel electrophoresis) of the bacterial community found in oil-contaminated soils. They observed
that highly polluted samples contain organisms related
to actinomycetes, Arthrobacter and an unidentifiable
microorganism. The dsz genes (A, B and C) were present
in all soil samples and the dszA sequences obtained
following PCR revealed a great similarity (>95%) to dszA
from strain IGTS8. However, the PCR primers used in
these experiments were designed using Rhodococcus dsz
sequences, potentially biasing the results. These genes
did not appear to be present in experiments on noncontaminated soils.
In the past few years it has been recognized that the use
of a thermophilic organism might be extremely advantageous for a large-scale industrial process. Elevated temperatures may provide the following advantages: first,
the potential for improved enzymatic rates; second, a
decrease in bacterial contamination; and third, overall
improvements in biocatalyst stability. Thermophilic
desulfurization has been shown to occur in Paenibacillus
sp. [27,35–38] and Mycobacterium phlei [39–41]. Both of
these strains function at temperatures >508C. It is interesting to note, however, that the dszABC genes of
M. phlei GTIS10 are identical in DNA sequence to those
from IGTS8 even though the temperature optima and
stability of the strains differ by almost 208C [39]. This
implies that there are other factors in the whole-cell
reaction that contribute to the thermostability of the
reaction pathway.
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The use of selective and in vitro genetic tools
to recover improved or novel Dsz
phenotypes
The emergence of new in vitro tools for mutation and
genetic rearrangement [42,43] and the clever application of some older ones [7] have enabled the limits of the
biodesulfurization system to begin to be explored. Fossil
fuels contain, in addition to alkylated DBTs, a variety of
alkyl and aryl sulfides, thiophenes and benzothiophenes
[44,45]. Arensdorf et al. [7] used model compound assays
to explore the limits of biodesulfurization for these classes
of non-DBT sulfur compounds. They chose several models (5-methyl benzothiophene [5-MBT], 2,3,5-trimethylthiophene, and octyl sulfide) that were not substrates for
the R. erythropolis strain IGTS8 Dsz system. Gain-offunction mutations were isolated for octyl sulfide utilization (dszA Q345A) and 5-MBT utilization (dszC V261F).
Further, saturation mutagenesis of dszC at amino acid
residue 261 showed that none of the other 19 possible
substitutions resulted in the ability to use 5-MBT.
Coco et al. [8] introduced a method of in vitro recombination to generate libraries of evolved enzymes — random chimeragenesis on transient templates (RACHITT).
In this method, randomly cleaved parental DNA fragments are annealed to a transient polynucleotide scaffold.
This technique appears to have advantages in the diversity it can generate, the fact that closely linked alleles are
easily recombined, and the fact that parental sequences
are virtually assured to be absent in the recombinant pool
[43,46]. Coco et al. demonstrated the method by carrying out in vitro recombination of dszC genes derived from
two distinct genera, Rhodococcus and Nocardia. The DBTMO derived from Nocardia had been shown to have a
higher substrate affinity and/or substrate range for complex alkylated derivatives of dibenzothiophene that were
otherwise poorly converted. The DBT-MO from Rhodococcus, by comparison, had been shown to have a higher
specific reaction rate for sparsely alkylated DBTs [7].
Coco et al. were able to isolate recombinant clones from a
dszC-family recombinant library encoding altered DBTMO proteins with significantly improved rates of substrate turnover. Of 175 unselected recombinants tested,
the six best were confirmed by further testing to have
significantly higher activity than the parents. All were
found to be chimeras by DNA sequencing. One clone was
found to be better at desulfurizing diesel, but not the
model compound, DBT. Variants from the same library
were isolated that displayed both higher rates and more
extensive substrate oxidation, showing the ability of
RACHITT to generate clones with multiple simultaneous improvements.
HPBS desulfinase
The last step in the pathway, which is catalyzed by HPBS
desulfinase, is the actual desulfurization of condensed
thiophenic substrates. A unique aspect of this reaction is
Current Opinion in Microbiology 2003, 6:229–235
232 Ecology and industrial microbiology
that it results in carbon–sulfur bond breakage between a
phenyl and a sulfinic acid group. There are reports in the
literature of enzymatic carbon–sulfur bond breakage
between a phenyl and a sulfonic acid group; however,
this is proposed to be catalyzed by a monooxygenase [32].
There are also reports of enzymatic carbon–sulfur bond
breakage between aliphatic carbon and the sulfinic acid
group; however, this tends to be the result of alternative
activities of enzymes that have an absolute catalytic
requirement for pyridoxal 50 -phosphate (PLP) [47,48].
HPBS desulfinase is not a monooxygenase, nor does it
require PLP.
except in the presence of HPBS, 2-hydroxybiphenyl(HBP)
and 2-(20 -hydroxyphenyl)benzenesulfonate (HPBSo)
(GT Mrachko, unpublished data) [50]. Therefore, a catalytic or binding role is implicated for cysteine.
The use of alternative substrates and/or inhibitors of the
desulfinase can provide a substantial amount of information. 2-Phenylbenzene sulfinate (PBS) is a substrate, as
evidenced by the measurement of the product biphenyl
by GC-MS [49]. The kcat was similar to that measured
for HPBS, but the Km was nearly eightfold greater. An
interesting inhibition result in light of the catalytic efficiency of the desulfinase on PBS is the observation that
naphthosultam inhibits the desulfinase, whereas naphthosultone is not an inhibitor [50]. The specific difference
between the sultam and sultone lies in the fact that the
sultam has a proton available for hydrogen bonding with
an active site residue. The fact that the catalytic efficiency of the desulfulfinase is eightfold diminished with
Nakayama et al. [49] cloned, expressed, purified and
characterized an HPBS desulfinase from R. erythropolis
strain KA2-5-1. They also report on the inactivation of
the enzyme by mutation of the sole cysteine to serine.
Chemical modification of cysteine has also been performed, which resulted in inactivation of the enzyme
Figure 2
H
(a)
E-B:
H
E-B
O
O
OH
O
TPL
Tyrosine
E-A-H
CO2−
PLP
E-A:
CO2−
H
NH
H
NH
PLP
(b)
CO2−
PLP
E-B:
E-B:
H
H
O
O
3HC
CH3
S
O
H
O
3HC
CH3
−
3 HC
CH3
−
E-A:
O
E-A-H
E-B:
H
S
O
H
SO2
H2O
O
E-A-H
HSO3−
(c)
E-B:
H
O
CH3
S
3HC
3HC
CH3
CH3
O−
O
H
O
O
3HC
E-A-H
E-B:
E-B-H
H
E-A:
S
O
O−
H
E-A:
Current Opinion in Microbiology
The proposed mechanism for TPL [52], which is used as a model for two working hypotheses for the desulfinase. (a) The proposed mechanism for
TPL. (b) One working hypothesis for the desulfinase, general acid-catalyzed desulfination, in which the substrate hydroxyl group is involved in
substrate recognition and binding. (c) Another hypothesis, proton-transfer-assisted desulfination, in which the hydroxyl group is involved in catalysis.
The reaction is shown using the substrate 5,50 -dimethyl-2-hydroxybiphenyl-20 -sulfinate to illustrate the inductive effect of a para methyl group on the
stabilization of the carbocation formed during the proposed protodesulfination.
Current Opinion in Microbiology 2003, 6:229–235
www.current-opinion.com
Biodesulfurization of fossil fuels Gray, Mrachko and Squires 233
PBS as compared with HPBS as a substrate, and that the
naphthosultam inhibits the enzyme, is a possible indication that the hydroxyl group of HPBS has a role in
binding, at least, as depicted in Figure 2b. Another result
in support of this working hypothesis is the finding that
HPBSo is a competitive inhibitor, with a Ki comparable to
the Km for HPBS (GT Mrachko, unpublished data), but
that biphenyl-2-carboxylate was found not to be an inhibitor [50]. A catalytic role for the substrate hydroxyl
involving electron localization by a proton-transfer step
to facilitate electrophilic substitution of the sulfinate by a
proton, as described in Figure 2c, may not be the case,
given the comparable kcat for HPBS and PBS and the
apparent requirement of a proton-donating substituent
for enzyme inhibition. The alkyl-substituted HPBS compounds 3,30 -dimethyl-2-hydroxybiphenyl-20 -sulfinate and
5,50 -dimethyl-2-hydroxybiphenyl-20 -sulfinate, which are
derived from the action of the two Dsz monooxygenases on
4,6-dimethyldibenzothiophene and 2,8-dimethyl-dibenzothiophene, respectively, are eight times more reactive
with the desulfinase than is HPBS (GT Mrachko, unpublished results). These results lend themselves in support of
the reaction mechanisms depicted in Figure 2, as a methyl
group ortho or para to the substitution site is an activating
substituent for this proposed protodesulfination electrophilic substitution. The regioisomeric distribution of
methylated HBP produced from asymmetrically methylated DBT could provide further enlightenment with
respect to the working hypotheses in Figure 2 [38].
[51,52]. Tyrosine has been implicated by chemical modification studies in the reaction catalyzed by the desulfinase [49,50]. Further experiments aimed at identifying
a tyrosine as the catalytic acid in the desulfinase, which
generate mutations at that position and couple these
results to pH dependence studies, should provide the
next level of understanding of this unique enzyme.
Naturally, a crystal structure, preferably with a bound
inhibitor such as HPBSo, would provide a detailed picture
of structure–function relationships.
A couple of working hypotheses are presented to describe
an electrophilic substitution of the aryl sulfinate by a
proton, with or without the assistance of a proton transfer
involving the substrate hydroxyl group. The enzyme
mechanism proposed for tyrosine-phenol lyase (TPL),
as illustrated in Figure 2a, has been used as a model for
such an enzyme reaction mechanism [51,52]. The results
in the literature summarized here favor the reaction
mechanism without a proton transfer step involving the
hydroxyl group of the substrate; however, several
mechanistic details remain to be elucidated to complete
the proposal for the desulfinase modeled after TPL. A
crucial experiment missing from the data is the determination of a pH-rate profile describing the dependence of
V/K and Vmax on pH; such a profile would indicate the pKa
values for binding and catalysis, which might aid in the
elucidation of structure–function relationships. A comparison of the pH dependence of the chemical modification
of cysteine with the dependence of V/K and Vmax on pH
would aid in the illumination of the role for cysteine in the
enzyme reaction mechanism. Furthermore, a pH-rate
profile for the desulfination of PBS compared with HPBS,
in addition to a determination of the pH dependency of
the inhibition by HPBSo, could aid in the description of
the involvement of the substrate hydroxyl in binding.
The catalytic base and the catalytic acid proposed in the
TPL reaction are arginine and tyrosine, respectively
References and recommended reading
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Conclusions
Our understanding of how microorganisms metabolize
sulfur heterocyclic compounds through the Dsz pathway
is improving rapidly. However, we still need a far better
understanding of all aspects of this pathway and of the
attendant substrate-acquisition issues if this pathway is
ever to be turned into a commercial process. It is evident
that a more in-depth view of the enzymology of the
process will be required, particularly as this relates to
the supply of reducing equivalents to the monooxygenase
steps. To some extent this knowledge will dictate the
genetic approaches required to improve both the rate of
desulfurization of the multiple thiophenic species present
in fossil fuels and the extent to which these can be
catalytically removed. Enzymology and microbiology
together may offer a unique path to the total removal
of sulfur from these petroleum streams if these problems
can be overcome.
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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234 Ecology and industrial microbiology
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