H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase

Article abstract of DOI:10.1039/c5cc06078h

An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD⁺-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H₂-driven NADH cofactor regeneration even in the presence of O₂, the co-substrate of monooxygenase.

Full text of DOI:10.1039/c5cc06078h

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H2-driven biotransformation of n-octane to
1-octanol by a recombinant Pseudomonas putida
strain co-synthesizing an O2-tolerant hydrogenase
and a P450 monooxygenase†
Cite this:DOI: 10.1039/c5cc06078h
Received 21st July 2015,
Accepted 11th September 2015
Thomas H. Lonsdale,‡ab Lars Lauterbach,‡*a Sumire Honda Malca,c
DOI: 10.1039/c5cc06078h
Bettina M. Nestl,c Bernhard Hauerc and Oliver Lenz*a
www.rsc.org/chemcomm
An in vivo biotransformation system is presented that affords the a sufficient supply of the reduced cofactor NADH is required. In
hydroxylation of n-octane to 1-octanol on the basis of NADH- whole-cell systems, NAD(P)H is generated either from the basic
dependent CYP153A monooxygenase and NAD+-reducing hydro- metabolic routes or via a recombinant regeneration system,
genase heterologously synthesized in
a bacterial host. The generally based on glucose dehydrogenase relying on the sup-
hydrogenase sustains H2-driven NADH cofactor regeneration even plementation of the growth medium with glucose.6 Whole-cell
in the presence of O2, the co-substrate of monooxygenase.
catalysis for chemical synthesis is attractive because enzymes are
generally more stable in their natural environment. Recently, a
Cytochrome P450 monooxygenases (CYPs) have the extraordinary hydrogenase that oxidizes dihydrogen (H2) and transfers the
capability to introduce oxygen into non-activated C–H bonds in released electrons to NAD+ has complemented the so-far esta-
a regio- and stereoselective manner, which is still a challenging blished systems for NADH regeneration.7–10 In contrast to glucose,
task for synthetic catalysts. The continuous discovery of novel H2 readily diffuses through cell membranes, and hydrogenase-
CYPs and their modification by protein engineering paves new based NADH recycling has the advantage of proceeding without a
avenues for their utilization as biocatalysts in the production of carbon-based reducing agent. Moreover, when coupled with CYP-
pharmaceuticals and fine chemicals.1–3
mediated catalysis water is the only by-product (Fig. 1).11
Exposure of most hydrogenases to O2 leads to their irreversible
In particular the regioselective terminal hydroxylation of
alkanes by CYPs is of significant interest because the corres- inactivation.12 However, a few O2-tolerant [NiFe]-hydrogenases
ponding alkanols can be used as surfactants, plasticizers, sustain catalytic activity in the presence of O2. One of the most
solvents or polymer precursors.4 In this study we used the tri- prominent examples is the NAD+-reducing hydrogenase (SH) from
partite alkane-oxidizing CYP153A system from Polaromonas the ‘‘Knallgas’’ bacterium Ralstonia eutropha H16 consisting
sp. JS666 for selective oxidation of n-octane to 1-octanol.5 The of two functionally distinct heterodimeric moieties.13 The
system is composed of the catalytically active cytochrome H2-dependent NAD+ reduction activity of the SH is unaffected
(CYP153A), the electron carrier ferredoxin (Fd) and the FAD- even at ambient O2 concentrations.14 These features make the
containing ferredoxin reductase (FRN). The latter two proteins SH particularly promising for NADH regeneration in cascade
are necessary for electron transfer from NADH to the P450 active reactions requiring O2, e.g. those involving CYPs.
site (Fig. 1, see ESI,† for details). CYP153 enzymes are promising
biocatalysts as they can be heterologously produced in active and
soluble form.4 For efficient use of CYPs both in vitro and in vivo,
a
¨
Department of Chemistry, Technische Universitat Berlin, Straße des 17. Juni 135,
10623 Berlin, Germany. E-mail: lars.lauterbach@tu-berlin.de,
oliver.lenz@tu-berlin.de
b Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory,
Fig. 1 H2-driven conversion of n-octane to 1-octanol in recombinant
Pseudomonas putida KT2440 cells producing the soluble NAD+-reducing
hydrogenase (SH) from Ralstonia eutropha in addition to CYP153A (CYP),
NADH-ferredoxin reductase (FRN), and ferredoxin (Fd) from Polaromonas
sp. JS666. The SH contains two flavin mononucleotide (FMN) molecules, and
FRN contains one flavin adenine dinucleotide (FAD). Electron-conducting
iron–sulfur clusters are shown as cluster-forming spheres coloured in
brown and yellow.
South Parks Rd, Oxford, OX1 3QR, UK
c
¨
Institute of Technical Biochemistry, Universitat Stuttgart, Allmandring 31, 70569,
Stuttgart, Germany
† Electronic supplementary information (ESI) available: Construction of SH and
CYP153A production systems, GC analysis, CYP153A and SH production in
recombinant strains and catabolism assays. See DOI: 10.1039/c5cc06078h
‡ These authors contributed equally to this work.
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In this study, we have designed an electron transfer pathway
for the H2-driven o-hydroxylation of n-octane to 1-octanol in
recombinant Pseudomonas putida.§ Pseudomonads provide an
attractive platform for new metabolic pathways due to their
solvent resistance, metabolic versatility and simple genetic
accessibility.15 Biosynthesis of the [NiFe] active site of the SH is
highly complex and requires a set of auxiliary proteins involved in
nickel uptake, metal centre assembly and insertion, site-specific
endoproteolysis and transcriptional regulation.16 The corres-
ponding set of 13 genes is arranged as an operon and was set
under control of the alkB promoter that controls alkane degrada-
tion in Pseudomonas putida GPo1.17 The three genes required for
biosynthesis of the CYP153A system from Polaromonas sp. JS666
were likewise equipped with the alkB promoter. Both recombi-
nant operons were inserted into a broad-host-plasmid replicating
in P. putida KT2440 (see footnote § and ESI,† for details). In order
to obtain the optimal growth conditions for the biotransforma-
tions, both the SH activity and the CYP concentration were
determined dependent on the length of the induction period.
Recombinant P. putida cells were first grown for 12 hours in a
glucose–glycerol (GGN) minimal medium.
Fig. 2 H2-supported production of 1-octanol by recombinant P. putida
KT2440 cells synthesizing CYP153A and SH. Bioconversions were moni-
tored either in the absence (open bars) or presence (dashed bars) of H2. The
bar height represents the mean value of three replicates, and the corres-
ponding standard deviation is indicated.
Gene expression was induced through addition of dicyclo- Table 1 H2-supported conversion of n-octane to 1-octanol, octanoic
acid and 2-octanol/octanal. The measurement replicates were done in
triplicate. Mean values and the corresponding standard deviations
propyl ketone (DCPK), which cannot be metabolized by P. putida.
In the course of the induction period (24 h), the CYP concen-
tration showed an almost continuous increase reaching a final
are shown
value of 0.58 nmol mgÀ1 (of total protein) after 24 h (Table S1,
ESI†). The SH activity, by contrast, showed a considerable
increase during the first 4 h of induction and then stayed
relatively stable at 0.15 U mgÀ1 for a further 4 h. However, less
than 50% of the maximal activity was left after 24 h (Table S1,
ESI†). Henceforth, all subsequent biotransformations were carried
out with resting cells derived from a recombinant P. putida culture
1-Octanol
2-Octanol/octanal
(mg LÀ1
Octanoic acid
Time (h)
(mg LÀ1
)
)
(mg LÀ1
)
2
4
6
24
28 Æ 3
4 Æ 0.1
8 Æ 0.6
15 Æ 1
32 Æ 9
8 Æ 3
9 Æ 3
29 Æ 10
37 Æ 8
16 Æ 5
32 Æ 14
101 Æ 1
grown for 12 hours in GGN minimal medium followed by an From these observations, it seems likely that the accumulation
induction period of 6 h. of octanoic acid and 2-octanol/octanal resulted from a combi-
After induction the cells were centrifuged, and the cell pellet nation of direct overoxidation by CYP153A and native catabolic
was re-suspended in carbon-free H16 minimal medium con- pathways for 1-octanol degradation present in P. putida. Indeed,
taining n-octane. Biotransformations with 15% (v/v) n-octane in contrast to wild-type P. putida KT2440, the recombinant
were performed in gas-tight reaction vessels containing a gas strain synthesizing the CYP153A system was able to grow with
mixture of H2 and air in the headspace. The same setup without n-octane as the sole carbon and energy source (Fig. S2, ESI†).
H2 was used to evaluate the biotransformation capacity of the This clearly indicates catabolic degradation of 1-octanol in vivo.
system without support of the SH.
Future mutagenesis studies18 are envisaged to identify potential
The addition of H2 led to a significantly higher yield of alcohol dehydrogenases involved in 1-octanol conversion.
1-octanol (Fig. 2), demonstrating the supportive role of SH- A subsequent knock-out of the corresponding gene(s) is expected
generated NADH during the biotransformation. After 24 h, to reduce the amount of overoxidation products.
101 mg LÀ1 and 36 mg LÀ1 of 1-octanol were formed in the
Recombinant P. putida KT2440 cells synthesizing both the
presence and absence of H2, respectively. The maximum concen- CYP153A system and SH grew even slightly faster when H2 was
tration of 1-octanol in the biotransformation mixture was approxi- added (Fig. S3, ESI†). This result in combination with the increased
mately five times higher than that achieved previously in in vitro 1-octanol production in the presence of H2 point out that NADH-
experiments with purified Polaromonas sp. JS666 CYP153A.5
regeneration in carbon-free minimal medium is significantly facili-
Further oxidation products such as octanoic acid and tated by the SH. This is in marked contrast to the results obtained
2-octanol/octanal were found to constitute 39% of the total for cells grown in glucose-supplemented mineral medium,
octane oxidation products (Table 1). Overoxidation is frequently where P. putida is able to adjust its energy and reductant demand
observed in biotransformations involving P450 enzymes, and in according to the metabolic burden.15,19
a previous in vitro study, up to 10% of the octane was shown to
In our proof-of-concept study, we investigated the applicability
be overoxidized by CYP153A.5 In contrast to the latter study, of the R. eutropha SH to increase the NADH supply in whole cell
however, no a,o-diols were detected in our in vivo approach. systems. H2-supported NADH recycling led to an approximately
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re-suspended to a final OD436 of 20 in carbon-free, five times concentrated,
mineral salts medium.14 After addition of 15% (v/v) n-octane and 2% (v/v)
DMSO the flasks were sealed with a rubber bung. The headspace of the
flasks was filled with a gas mixture of 20% H2 in air. Control experiments
were carried out under air. Aliquots of the biotransformation suspensions
were analyzed using GC-FID, see ESI,† for details.
3-fold increase in the 1-octanol yield in a whole-cell biotrans-
formation system, which uses a dedicated P450 monooxygenase
as octane-converting biocatalyst. In order to prevent formation
of hazardous gas mixtures during in vivo biotransformation, the
application of sub-critical gas concentrations, such as o8 vol%
O2 and o5 vol% H2, is recommended.20,21 Our study clearly
shows that the SH is capable of overcoming potential bottlenecks
of cofactor supply in whole-cell systems. Moreover, H2 represents
a viable alternative to carbon-based reductants currently used for
in vivo cofactor recycling strategies. Thus, our H2-driven in vivo
cofactor regeneration system holds considerable potential for
application in other cascades reactions that rely on sustainable
supply of NAD(P)H as the reducing agent.
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This work was supported by an European Research Council
(ERC) proof of concept grant 297503 (to L.L.) and the Deutsche
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would like to thank the Deutscher Akademischer Austauschdienst
(DAAD) for a one year scholarship. We are indebted to Andreas
Schmid and Bruno Bu¨hler for providing us plasmid pSPZ10. We
thank Kylie A. Vincent, Holly A. Reeve and Leland B. Gee for
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Notes and references
§ The heterologous overproduction system for the SH from R. eutropha
and the CYP153A system from Polaromonas sp. JS666 was assembled as 16 J. Schiffels and T. Selmer, Biotechnol. Bioeng., 2015, DOI: 10.1002/
described in the ESI.† Main cultures were grown at 30 1C in baffled bit.25658.
2 L-Erlenmeyer flasks including 300 mL of mineral salts medium14 17 J. B. van Beilen, S. Panke, S. Lucchini, A. G. Franchini, M. Rothlisberger
containing 0.2% (w/v) glucose and 0.2% (v/v) glycerol as carbon and and B. Witholt, Microbiology, 2001, 147, 1621–1630.
energy sources (GGN medium). Inoculation was done with 3 mL of a 18 H. A. Vrionis, A. J. Daugulis and A. M. Kropinski, Appl. Microbiol.
starter culture grown previously for 48 h in mineral salts medium with Biotechnol., 2002, 58, 469–475.
0.4% (w/v) glucose. Induction of the alkB promoter was carried out by 19 T. Vallon, M. Glemser, S. H. Malca, D. Scheps, J. Schmid, M. Siemann-
addition of 0.05% (v/v) dicyclopropyl ketone, and CYP production was
enhanced through addition of 0.5 mM 5-aminolevulinic acid and 1 mM
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NADH at 365 nm.14 Protein concentration was determined using the 21 Z. M. Shapiro and T. R. Moffette, US. Department of Energy, 1957,
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