biooxidation of n-butane to 1-butanol by engineered p450 monooxygenase under increased pressure

Journal of Biotechnology 191 (2014) 8692

Contents lists available at ScienceDirect

Journal of Biotechnology

j ourna l ho me pa ge: www.elsev ier .com/ locate / jb io tec

Biooxid neemonoo

Bernd A. ttinMichael elb,Detlef Kra Institute of Te y1b BASF SE, 6705

a r t i c l

Article history:Received 8 ApReceived in reAccepted 19 AAvailable online 26 August 2014

Dedicated to the 60th birthday of Prof.Jaeger.

Keywords:CYP153AFusion proteinButane hydrox1-ButanolHigh pressure

ol proe P4s an

ane oquantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvementof the native redox system, a highly -regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12 g/L butanol,of which 2.99 g/L comprise for 1-butanol, has been obtained after 20 h reaction time. Due to the poor sol-ubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase theconversion. After optimization a maximum product content of 4.35 g/L 1-butanol from a total amount

1. Introdu

1-Butanproductionvarious appfor paints, cical procesof propenerecent yearalternative.sugar, starcroute. Espeby Clostridibutanol proobic behavithe limited

CorresponE-mail add

1 Tel.: +49 6

http://dx.doi.o0168-1656/ ylation

bioconversion

of 4.53 g/L butanol catalyzed by the self-sufcient fusion monooxygenase has been obtained at 15 barpressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced vefoldusing the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.

2014 Elsevier B.V. All rights reserved.

ction

ol is a versatile basic chemical with a worldwide of more than 2.8 million tons per year and used forlications from components in plasticizers to solventsoating and varnishes (Green, 2011). The major chem-

s for the production of butanol is the oxo-synthesis (hydroformylation) (Weissermel and Arpe, 2003). Ins 1-butanol has been considered as an ideal biofuel

In this light, new fermentation processes based onh and cellulose are investigated as a second alternativecially the acetone-butanol-ethanol (ABE) fermentationum acetobutylicum represents an additional option forduction (Green, 2011). Nevertheless the strict anaer-or of Clostridia and their slow growth rates, as well as

sustainability and restraints inherent to the microbe,

ding author.ress: [email protected] (B. Hauer).85 63193.

such as sporulation, are problematic for industrial fermentations(Durre, 2008, 2011; Lutke-Eversloh and Bahl, 2011; Papoutsakis,2008). Recently a new innovative attempt has been reportedfor the production of 1-butanol in metabolically engineeredEscherichia coli (E. coli) strains comprising a set of genes involvedin the biosynthesis of metabolic pathways to produce up to 1.2 g/L1-butanol (Atsumi et al., 2008a, 2008b; Dellomonaco et al., 2011).

Hence the selective -hydroxylation of gaseous n-butane con-stitutes a potential biotechnological alternative for the synthesisof 1-butanol. The use of gaseous substrates in biocatalysis, like n-butane, which remain untapped as energy and chemical feedstockis a relatively young research area. In literature various in vivo bio-transformations to convert n-butane into 1-butanol are reported.

Several soil and marine microorganisms such as Pseudomonasand Mycobacteria have naturally evolved metal-enzymes thatdegrade alkanes into alcohols using gaseous aliphatic alkanes ascarbon and energy source (van Beilen and Funhoff, 2007). Thesemicrobes contain oxygenase enzymes that can introduce oxygeninto the terminal, non-activated position of different aliphatic sub-strates. Alkane -hydroxylases are oxygenases, whose activitiesdepend on the chain-length of the hydrocarbon. In general, short

rg/10.1016/j.jbiotec.2014.08.0222014 Elsevier B.V. All rights reserved.ation of n-butane to 1-butanol by engixygenase under increased pressure

Nebela, Daniel Schepsa, Sumire Honda Malcaa, BeBreuerb, Hans-Gnter Wagnerb, Boris Breitscheidatzb, Bernhard Hauera,

chnical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, German6 Ludwigshafen, Germany

e i n f o

ril 2014vised form 6 August 2014ugust 2014

a b s t r a c t

In addition to the traditional 1-butanmentation of biomass, the cytochromprimary alcohol 1-butanol constitutechemical. Moreover the use of n-butred P450

a M. Nestl a,

duction by hydroformylation of gaseous propene and by fer-50-catalyzed direct terminal oxidation of n-butane into the

alternative route to provide the high demand of this basicffers an unexploited ubiquitous feed stock available in large

B.A. Nebel et al. / Journal of Biotechnology 191 (2014) 8692 87

chain alkanes (C1C4) are hydroxylated by methane, propane andbutane monooxygenases (Hamamura et al., 1999). Medium-chainalkanes (C5diiron moncontaining (van Beilenpropane mthan C4, walkane hydthe 153A soxidize n-bet al., 2011class I P450of a catalytteins, nameFAD-contaiessary for active site (Hand Eiben, ized butanein vitro 1-(Cooley et aUnlike otherecombinanredox partnmonooxygea yield of 63lution of th1-butanol (ther exampCYP153A6 and its mutbutanol, resvan Beilen a

We recalkanes, prfrom Mycobolei VT8 anet al., 2011)screened tousing CamAfer partneralkane subsand C12, wistrates. Thewith low acCYP153A fr307, which reported asincrease theoctane alka(Mestres, 20

Aiming tfused CYP1reductase dOxygenatiofusion protecells. We alof gas ow rsoluble in wcult to supmedium attemperaturlimitation wtem to incremedium, w

2. Materials and methods

chemicals, solvents and buffer components were obtainedigma-Aldrich (Schnelldorf, Germany). All gases for the exper-

werA poiogaln-Ro

rains

mid

netic

wildsysteferreAA-

f theoligoCG ATG Gt (CYs pET3AP.sligonAC ATG AC-5

3A-Ces 5GA ATA Cp theed oateda heeque-direP153ol. Cor traes. VucleoT GA

GT CAG-5aninenI-trons (

tran

ltiva

cel3A ccribets wem fomizeion (oli BL

L ErediaycinC16) are oxidized by integral-membrane non-hemeooxygenases (alkB) or alternatively by heme iron-cytochrome P450 monooxygenases (P450s or CYPs)

and Funhoff, 2005). Non-engineered methane andonooxygenases tend to be specic for alkanes shorterhile the soluble butane monooxygenases especiallyroxylases and cytochrome P450 monooxygenases ofubfamily (CYP153A) were recently found to naturallyutane (Funhoff et al., 2007; Koch et al., 2009; Scheps; van Beilen and Funhoff, 2007). CYP153s are bacterials that operate as three-component systems, comprisedic unit (P450 domain) and two additional redox pro-ly an iron-sulfur electron carrier (ferredoxin) and a

ning reductase (ferredoxin reductase), which are nec-the transfer of electrons from NAD(P)H to the P450lavica and Lehnerer, 2010; Munro et al., 2007; Urlacher

2006; Urlacher et al., 2004). One of the rst character- monooxygenases from Thauera butanivorans producedbutanol with a conversion rate of 40.5 nM/min/mgl., 2009; Dubbels et al., 2009; Hamamura et al., 1999).r alkane monooxygenases, CYP153A and alkB have beently expressed with physiological or non-physiologicalers in E. coli and Pseudomonas sp. Using the AlkB-nase wild type enzyme from Pseudomonas putida Gpo13 M 1-butanol could be obtained. After directed evo-is protein the yield could be increased up to 1586 MJohnson and Hyman, 2006; Koch et al., 2009). A fur-le for the conversion of n-butane is reported by themonooxygenase from Mycobacterium sp. HXN-1500ant CYP153A6BMO1 producing 277 M and 393 M 1-pectively (Funhoff et al., 2006, 2007; Koch et al., 2009;nd Funhoff, 2007).

ently reported -hydroxylations of medium chainimary alcohols and fatty acids by CYP153 enzymesacterium marinum (CYP153A16), Marinobacter aquae-d Polaromonas sp. (Honda Malca et al., 2012; Scheps. CYP153A16 and CYP153A from Polaromonas sp. wereward C5C12 alkanes and C6C12 primary alcohols

and CamB from Pseudomonas putida as electron trans-s within a cofactor regenerating enzyme system. Thetrate range of the biocatalyst oscillated between C5

th pentane and hexane being the preferred alkane sub-y were converted to the corresponding primary alcoholstivity but absolute -regioselectivity. Furthermore, inom Marinobacter aquaeolei VT8, a glycine at positioncorresponds to position 254 in Polaromonas sp., could be

mutation hot spot to change substrate specicity and to activity for terminal hydroxylation reactions against n-ne, 1-heptanol as well as short chain fatty acids (C8C9)05; Seifert et al., 2009).o increase the efciency of electron transfer, we herein53A from Polaromonas sp. JS666 (CYP153AP.sp.) to theomain of P450-BM3 from Bacillus megaterium (CPRBM3).n reactions of gaseous n-butane to 1-butanol with thein and variants thereof were carried out in E. coli wholeso optimized our biotransformation assembly in termsate and pressure. At normal pressure n-butane is poorlyater (61 mg/L at 20 C) and as a consequence, it is dif-ply large amounts of this gaseous substrate in aqueous

ambient conditions (atmospheric pressure and roome) to saturate the enzyme. In order to overcome thise performed transformations using a pressurized sys-ase the butane substrate concentration in the reactionhich is a common strategy in chemical synthesis.

All from SimentsPfu DN-d-th(St. Leo

2.1. St

Plas

2.2. Ge

Theredox and a ATCC Bsites ousing GAA GGTG Cmutanated aCYP15with oGTA ATAG GCAT AGCYP15cleotidGGC AGAG Ting stedescribThe ligcells viDNA-s

Siteing CYprotocused fmixturoligonGGC AAGCG GGCC Cacid althe Dpmutatiused to

2.3. Cu

TheCYP15as desvarian250 rpto optiformatof E. cout in 1(TB) mkaname obtained from Air Liquide and were at least 99.5% pure.lymerase, endonucleases, T4 DNA ligase and isopropylactopyranoside (IPTG) were purchased from Fermentast, Germany).

and plasmids

s and strains used in this study are listed in Table 1.

manipulation

type CYP153AP.sp. (Bpro 5301) and the correspondingm with a FAD-dependent oxidoreductase (Bpro 530)doxin (Bpro 299) from Polaromonas sp. strain JS666500 was introduced into the NdeI and HindIII cloning

pET-28a(+) vector. The operon was amplied by PCRnucleotides 5-GGT CAT ATG AGA TCA TTA ATG AGTTT GTG GTA AAC AAC C-3 and 5-AGCT AAG CTT TCACC GAG CGG-3. The fusion (CYP153AP.sp.-CPRBM3) andP153AP.sp.(G254A)-CPRBM3) fusion chimeras were cre-28a(+) vector constructs. The enzymatic part for the

p.-CPRBM3 constructs was created by PCR amplicationucleotide 5-GGT GCT AGC ATG AGT GAA GCG ATT GTGAC CAA AAC G-3 and 3-TAG CAG ACT GTT CAG TGCAG GAA TAG CGT TGA TGC GGA CGG GCA GCG ACTfor the construct with the original linker sequence. ThePRBM3 redox system was amplied by PCR with oligonu--CAC TGA ACA GTC TGC TAA AAA AGT ACG CAA AAAAA CGC TCA TAA TAC GCC GCT GC-3 and 3-CAT CTCCC AGC CCA CAC GTC TTT TGC GTA TC-5. In a follow-

matching amplied products were assembled at theirverlapping sections by PCR and ligated into pET-28a(+).

plasmid was used to transform competent E. coli DH5at shock. Successful cloning was veried by automatedncing (GATC-Biotech, Konstanz, Germany).cted mutagenesis using the plasmid pET28a(+) harbor-AP.sp.-CPR was mutated using the QuikChange standardhemical competent E. coli HMS174 (DE3) cells werensformation reactions with the DpnI-treated QC-PCRariant G254A was created by PCR amplication withtide 5-CTG GGC AAC CTC ATT TTG CTG ATC GTC GCGC ACG ACC CGC-3 and its complementary sequence 3-

GT GTC ATT GCC CGC GAC GAT CAG CAA AAT GAG GTT, which contains the codon GCG covering the amino. Competent E. coli DH5 cells were transformed with

eated PCR mixtures. Isolated plasmids with the desiredsequencing by GATC-Biotech, Konstanz, Germany) weresform in competent E. coli HMS174 (DE3) cells.

tion of biomass

l cultivation and protein expression for the threeonstructs ITB379, ITB413 and ITB408 was conductedd below. E. coli BL21(DE3) cells expressing CYP153Are pre-cultured in LB medium at 37 C with shaking atr 24 h. 1 mM of the non-gaseous n-hexane was added

the adaption toward alkanes and to increase productKoch et al., 2009; van Beilen et al., 2006). Cultivations21(DE3) whole cells for bioconversions were carriedlenmeyer shake asks containing 200 mL terrifc broth

supplemented with the appropriate antibiotic (1 mL/L solution, 50 g/mL). Cultivation of E. coli was generally

88 B.A. Nebel et al. / Journal of Biotechnology 191 (2014) 8692

Table 1Host strains and plasmid construct used in this work.

Construct abbreviation Strain or plasmid Characteristics Reference

E. coli strainsITB379 BL21 (DE3) pET28a(+):CYP153A-operon from Polaromonas sp. This studyITB413 HMS174 (DE3) pET28a(+):CYP153A-CPRBM3 from Polaromonas sp. This studyITB408 HMS174 (DE3) pET28a(+):CYP153A(G254A)-CPRBM3 from Polaromonas sp. This study

PlasmidpET28a(+) Expression vector Novagen, Wisconsin, USA

performed aerobically in orbital shakers (Multitron, Infors HT,Bottmingen, Switzerland) at 180 rpm and 37 C.

The growth was carried out on a shaker to an OD600 of1.11.3. Expression was induced by the addition of 0.25 mM IPTG.The culture was supplemented with 4 g/L glycerol, 0.5 mM 5-aminolevulinic acid (-ALA) and 100 mg FeSO4 in E. coli. The cellswere incubated for 24 h at 28 C and 180 rpm and harvested by acentrifugation step at 4.000 g and 4 C for 30 min. Due to vari-ations in the expression level of the different CYP153A variants,23 independent cultures were prepared to assure a high enzymeconcentration. The pellets were washed with 100 mM potassiumphosphate buffer (pH 7.4). After this procedure the cells were con-centrated into 100 mM potassium phosphate buffer (pH 7.5) to anal concentration of 50 gcww/L buffer (18.7 gcdw/L).

2.4. Biotransformation

After the cells were provided with 1% glycerol (v/v) and 20 mMglucose as carbon source, the gaseous substrate was added to thereaction mixture as illustrated in Fig. 1.

2.4.1. In vivAll biotr

with CYP15pH 7.5. For pension anin a 250 mLto the reac

n-butane and 95.8 vol.% synthetic air. The n-butane gas concentra-tion was settled at 4.2 vol.% because of safety reasons (explosionrange 1.49.4 vol.% n-butane). The total gas ow rate was adjustedto 12.5 L/h by using mass ow units. Butane/air gas supply intothe cell slurry was guaranteed through a continuous ow rate andthe use of a sparger after mixing in a dispenser nozzle (Fig. 1). Tominimize product loss, a back ow gas collection system was used.The collection asks were lled with 150 mL ddH2O. After denedtime points, samples from the bioreactor ask and the collectionasks, which were installed downstream of the fermentation askto avoid product discharge, were taken and after a fast and tightsealing procedure analyzed by GC/MS-headspace chromatography.For the quantication of the formed C4-product the amounts of 1-butanol and 2-butanol in the bioconversion asks and downstreamasks were combined.

2.4.2. In vivo biotransformation of n-butane to butanol underincreased pressure

The terminal hydroxylation of the gaseous C4-substrate wasinvestigated in a high pressure reactor. Into the 250 mL glass insert

cellded.d inAfterI) watic aited ps als

Fig. 1. Schemsparger unit aproduct lost (othe ow and greasons.o biotransformation of n-butane to butanolansformations were carried out with resting cells (E. coli3A, 50 gcww/L) in 100 mM potassium phosphate bufferbioconversions of gaseous n-butane, 100 mL of cell sus-d 15 L of antifoam 204 (SigmaAldrich) were stirred

Schott-ask at room temperature. n-Butane was addedtion mix with following an inlet gas ratio of 4.2 vol.%

100 mLwas adresulte5 C. clave Isynthea selecensureatic illustration of the atmospheric pressure continuous gas ow assembly. A 250 mL rend a gas outlet connected in serial with two 250 mL gas collection vessels. The two collenly one gas collection ask is illustrated). The inlet gas ow was controlled by a masteras composition. The gas mix was settled at 4.2 vol% n-butane to avoid an explosive atmo suspension was provided and a magnetic stirring bar 10 g of cold liquid n-butane was lled in excess which

the formation of a second phase at a temperature ofwards the pressure tank (Carl Roth, high-pressure auto-s sealed and connected via a high pressure line to ar gas cylinder. This setup offers the opportunity to applyressure of 1100 bar (nal pressure 120 bar). This stepo the supply of sufcient oxygen for the reaction. Theaction vessel was equipped with a magnetic stirrer, a gas inlet withction aks were lled with water and installed downstream to avoid

ow and two mass ow units (n-butane and synthetic air) to adjustsphere. The assembly was installed in a fume hood because of safety


compression step at the beginning of each reaction was made asslowly as possible to avoid physical stress. For each time point anew reaction setup was necessary because the used high pressureassembly did not allow continuous sampling.

2.5. Analysis

2.5.1. P450 activity assayConcentrations of the P450 enzymes were determined by the

carbon monoxide (CO) differential spectral assay, based on theformation of the characteristic FeII-CO complex at 448 nm. Thecells were disrupted by sonication on ice (4 2 min, 2 min inter-vals). Enzymes in cell-free extracts were reduced by the additionof 10 mM sodium dithionite from a freshly prepared 1 M stocksolution, and the carbon monoxide complex was formed by slowbubbling with CO gas for approximately 30 s. The concentrationswere calculated using the absorbance difference at A450 and A490(Ultrospec 3100pro spectrophotometer, Amersham Biosciences)and an extinction coefcient of 91 mM1 cm1 (Omura and Sato,1964).

2.5.2. HeadTo overc

headspace mSamples wment (Shim(30 m 0.25pler operatsyringe. Hewith a splitand the masinterface anof the biotrvial. To oveture was dilinternal staheadspace 40 C, hold 5For quantiinternal stacentrationspotassium p

3. Results

3.1. Engine

With th1-butanol,

the knowledge from directed evolution strategies by the groupof F. H. Arnold on CYP153A6BMO1 (Koch et al., 2009). Followingthese results of the CYP153A6BMO1 and its ability to -oxidizealkanes, cycloalkenes and alicyclic compounds (Funhoff et al.,2006), by using a homology-based approach we were able toidentify CYP153AP.sp. possessing a valine at position 95, which isequivalent to alanine residue 97 in CYP153A6BMO1. The increasedactivity of CYP153A6BMO1 toward n-butane gas has been attributedto a single amino acid substitution Ala97Val (Koch et al., 2009). Withregard to the high amino acid sequence similarity (85%) betweenCYP153AP.sp. and CYP153A6 and the preference of CYP153AP.sp. foralkanes over primary alcohol and fatty acids (Honda Malca et al.,2012), the biocatalyst CYP153AP.sp. was therefore considered to bepromising starting candidate for the hydroxylation of n-butane to1-butanol.

In biocatalytic reactions with oxygenases whole cells are pre-ferred over isolated enzymes primarily because cells are capableof regenerating NAD(P)H. Cells may also confer higher oxygen-ase stability by providing a protected compartment or a betterorganization of its components. Native CYP153A enzymes dependon a ferredoxin reductase and a ferredoxin to obtain reducing

lentsprotP mies. Aepenfciease in tratly ss (Guhemte to

Robinono

M3 004;

the Pely h

2002ent ef thrcomSche

desin fro3, ITBn of

tha

Fig. 2. Establi ncreaself-sufcient roteincontaining red oredusp.space GC/MS analysisome time consuming extraction procedures a GC/MS-ethod for 1-butanol products analysis was established.

ere analyzed by using a GC/MS QP-2010 instru-adzu, Japan) equipped with a FS-Supreme-5-column

mm 0.25 m) in combination with a CombiPal Sam-ed in headspace mode and a 2.5 mL heated headspacelium as carrier gas (ow rate 0.69 mL/min) was applied-ratio of 15:1. Electron impact (EI) ionization was useds range from 20 to 200 m/z was detected. Both detectord injector temperature were set at 250 C. One milliliteransformation mix was lled into a 20 mL headspacercome detector saturation, the biotransformation cul-uted with the appropriate buffer medium. 100 L of thendard solution (10 mM 1-hexanol) was added and thevials were sealed tightly. Temperature program: start

min, 5 C/min to 85 C, hold 1 min, 60 C/min to 300 C.cation of 1-butanol, the system was calibrated with thendard 1-hexanol. Standard samples with different con-

(0.012 mM of 1-butanol and 2-butanol) in 100 mMhosphate buffer were measured via GC/MS.

and discussion

ering of biocatalysts

e goal of evaluating an alternative route to producewe started the engineering of the catalyst based on

equivaredox the CYenzymare indself-sureductelectrois mossystemto the substra2011; P450 mP450-Bet al., 2sists ofextrem1997, inefciment ofusion 2009;

WedomaiCPRBMdomaishown

shing an optimized self-sufcient fusion complex comprising mutation G254A for ifusion protein with a CPRBM3 redox protein from the CYP153AP.sp. natural multiple puctase domain) from Bacillus megaterium, FdR and Fdx: natural FAD-containing oxid from NAD(P)H. Low expression levels of the separateeins or inability to interact in equimolar ratios withght impair whole cell biotransformations using theselthough most P450 and electron-transferring proteinsdent proteins encoded by different genes, there arent monooxygenases that contain both P450 and P450n a single polypeptide chain (Munro et al., 2007). Thensfer from the redox complex to the catalytic domainlow and one of the rate-limiting aspects in many CYPengerich, 2002). The improvement of electron deliverye-domain is important for the efcient conversion of

product by cytochrome P450 enzymes (OReilly et al., et al., 2009). A typical example of such a self-sufcientoxygenase is the long-chain fatty acid hydroxylase

from Bacillus megaterium (Davis et al., 1996; Gustafsson Narhi and Fulco, 1986, 1987). This monooxygenase con-450 (CYP102A1) and P450 reductase domain and showsigh molecular turnover of >1500 min1 (Munro et al.,

). To address the problems of low catalytic efciency,lectron transfer and complicated inner cellular arrange-ee single proteins, a functional self-sufcient CYP153Aplex was created and further engineered (Robin et al.,ps et al., 2013).gned and characterized a fusion construct of the heme-m CYP153AP.sp. from Polaromonas sp. (CYP153AP.sp.-413) containing a C-terminally-linked CPR reductase

P450-BM3 (Fig. 2). Furthermore, a previous study hast a single point mutation of glycine at position 307

sed activity (CYP153AP.sp.(G254A)-CPRBM3). Construction of a functional complex. Abbreviations: CPR: cytochrome P450 reductase (FAD/FMN-ctase and [2Fe-2S] ferredoxin of CYP153A enzyme from Polaromonas


Fig. 3. Produc tural and accumula e) durdetermined by sion (pH 7.5) over 2 assem12.5 L/h, 4.2 vo

in CYP153Aposition 25-hydroxylthe termin2012). To rCYP153AP.sgained by tCO differenCYP153AP.syielded in sindicates thfunctionally

Furthermalkane hydthrough prgrowth on tCYP153 genBased on thused duringactivity on aand produc

3.2. In vivo

In orderduce 1-butenzymes inatmospheriformed. Totthe biotrangas ow ra12.5 L/h. Th12.5 L/h and

With imassembly a mined. Fig. at a total airtime scale odecreased alection askline). Basediments werthe quantiasks and d

rthereacttionr prot al.,

ed to cofa

Dueactolank

glyce of

wertermr buter tht moM b

atmooselenol a

the tante sys

diffese iml prtion and product release of 1-butanol in the reaction vessel by the CYP153AP.sp. nation of 1-butanol in the gas collection ask #1 and #2 (dashed line and hashed lin

GC-HS/MS. The reactions were performed in the presence of 100 mL cell suspen4 h reaction time. The biotransformation was done in a continuous reactor systeml% n-butane and 95.8 vol% synthetic air).

from Marinobacter aquaeolei, which corresponds to4 in Polaromonas sp. caused not only higher in vitroation activity but also improved regioselectivity foral position (Chen et al., 2012; Honda Malca et al.,einforce the lessons learned, we created the mutantp.(G254A)-CPRBM3 (ITB408) by leveraging the knowledgehe fusion enzyme CYP153AP.sp.-CPRBM3 (ITB413). P450ce spectral analyses showed that cell extracts of all

p. constructs (ITB379, ITB413 and ITB408; 0.2 gcww/mL)oluble and active enzymes between 2.8 and 3.1 M. Thisat all cytochrome P450 monooxygenase systems were

expressed in similar yields.ore, previous efforts to engineer CYP153 genes for

roxylation resulted in a host that has been adaptedolonged cultivation on alkanes to obtain signicanthese substrates without any mutations occurring in thees themselves (Koch et al., 2009; van Beilen et al., 2006).e results of Koch et al. and van Beilen et al. n-hexane was

the growth phase potentially enabling enrichment forlkanes and adaption of the production host to substratet as small as butane and butanol.

biotransformation

to exploit the ability of the E. coli host system to pro-anol with the heterologously expressed CYP153AP.sp.

A fuof the prevenof polaSteen ereportamide2012).for coflysts (B0.52%absenc0.5 mMwas detion fo

Undmutanof 42 munder -regi1-butason tothe muenzymat vestepwibutanocluding ITB379, ITB413 and ITB408, bioconversions atc pressure and continuous gas ow for 20 h were per-al butanol concentration was enhanced by improvingsformation assembly through the increase of the inlette and aeration. The total gas ow was adjusted ate maximum product concentration was observed at

a butaneair ratio of 4.2 vol.% to 95.8 vol.%.plementation of gas collection asks into the reactionproduct release out of the reaction vessel could be deter-3 shows the 1-butanol release out of the reaction vessel

ow of 12 L/h at room temperature (dotted line). Over af 24 h the 1-butanol concentration in the reaction vesselnd a signicant accumulation of the compound in col-

one and two could be observed (dashed line and hashed on these results and to overcome product lost all exper-e performed with two gas collection asks. Furthermorecation the product concentration in the bioconversionownstream asks were combined.

further optdomain enz

Moreoveselective aoxidation tbutanediol

3.3. Pressur

The solurather low (eter for a bavailabilityusing the wThe best pr15 bar, expdecrease inmultiple protein complex (ITB379) into reaction vessel (dotted line)ing process. The 1-butanol production (mM butanol formation) was50 gcww/L, 15 L antifoam 204, 100 mM potassium phosphate bufferbled with two washing asks to avoid product discharge (total ow

r positive aspect of the continuous product removal oution vessel is the enhanced 1-butanol production by the

of cell damage and cell death due to an accumulationducts in the cell membrane (Isken and de Bont, 1998;

2008). Also the addition of a glycerol/glucose mixture, have a benecial effect on cell function and nicotin-ctor regeneration, was investigated (Gudiminchi et al.,

to the fact that glycerol is known to be a driving forcer regeneration in whole cell-mediated redox biocata-

et al., 2008), media containing either 0.050.3% glucose,erol or a mixture of glucose/glycerol were tested. In theglycerol or glucose, butanol concentrations less thane detected. A mixture of 20 mM glucose and 1% glycerolined to be the most efcient carbon source concentra-anol production (data not shown).e improved conditions we observed that the optimizednooxygenase enzyme system produced a maximumutanol (3.12 g butanol/L using 50 gcww resting cells)spheric pressure after 20 h (Table 2). Due to the highctivity of 96% of the mutant, a product titer of 40 mMnd 2 mM 2-butanol has been obtained. In compari-wild type system, the concentration of butanol using

enzyme system could by increased vefold. All three-tems were tested under the same reaction conditionsrent time points (Fig. 4). On the basis of the describedprovement of the wild type a signicant increase of

oduction by the self-sufcient fusion complex and a

imized product concentration by the mutated multipleyme system could be achieved.r, the engineered monooxygenase systems are verynd products do not suffer from overoxidation. Noo butanal or butanoic acid and further reaction to 1,4-was detected.

ized in vivo biotransformation

bility of n-butane gas in water or aqueous media is61 mg/L at 20 C) and thus, constitutes a critical param-iocatalytic process. In an attempt to enhance substrate, we performed additional in vivo biotransformationsild type under different pressure conditions (120 bar).oduct concentrations were obtained at a pressure oferiments carried out at more than 15 bar caused a

total butanol production (Fig. 5).


Table 2Product formation after bioconversion of n-butane in CYP153A resting cells.

ITB379 ITB413 ITB408 ITB379 at 15 bar ITB408 at 15 bar

Total butano 42.2 6.3 19.8 2.2 61.2 5.4Final 1-buta 2Initial produ 6Specic activ 5Space time y 0

* No oxidati by GC** The total b 20 h r

calculated wit sion c

70

30

40

50

60

utanol[m

M]

0

10

20

30

0

total b

Fig. 4. Compstructs (ITB37CYP153AP.sp.(G2The total butaHS/MS. The wcell suspensionpH 7.5). The bow 12.5 L/h, reactor assemexperiments.

15

20

tano

l[m

M]

5

10

0

totalbut

Fig. 5. Pressu(ITB379). The tby GC-HS/MS.ence of 100 mL7.5) and 10 g oThe pressure wunit to a synthnecessary becacontinuous samexperiments.

Moreovewas increasreaction timconcentratiselectivity oenzymes (Trate by a fal titer [mM/50 gcww/20 h]* 10.2 1.8 27.9 5.2 nol titer [g/L]** 0.72 0.13 1.98 0.38 ct rate [mM/h]** 1.4 4.3 ity [mg/h/M P450] 1.37 4.22 ield [g/L/h] 0.04 0.007 0.10 0.019 on to butanal or butanoic acid and further reaction to 1,4-butanediol was detectedutanol (1-butanol and 2-butanol) and nal 1-butanol titer was determined after hin the rst four hours of the hydroxylation of n-butane using wild type ITB379, fu4 8 12 16

me [h]

arison of total butanol titer with different CYP153AP.sp. con-9, ITB413 and ITB408) at atmospheric pressure and the mutant54A)-CPRBM3 (ITB408) under 15 bar pressure over 20 h reaction time.nol production (mM butanol formation) was determined by GC-hole-cell bioconversions were carried out in the presence of 100 mL

(50 gcww/L, 15 L antifoam 204, 100 mM potassium phosphate bufferiotransformation was done in a continuous reactor system (total4.2 vol% n-butane and 95.8 vol% synthetic air) and a high pressurebly. The values represent means standard deviation of triplicate

5 10 15 20pressure [bar]

re-dependency of butane oxidation with CYP153AP.sp. wild typeotal butanol concentration (mM butanol formation) was determined

The reactions were performed at the indicated pressure in the pres- cell suspension (50 gcww/L, 100 mM potassium phosphate buffer pHf cold liquid n-butane performed in a high pressure reactor system.as adjusted between 1 and 20 bar, by connecting the high pressure

etic air gas cylinder. For each pressure point a new reaction setup wasuse due to the fact that the used high pressure assembly did not allowpling. The values represent means standard deviation of triplicate

r, the bioconversion of n-butane catalyzed by mutanted from 42.2 mM (1 bar) to 61.2 mM (15 bar) after 20 he using 50 gcww resting cells. This results in a maximumon of 4.35 g/L 1-butanol at 15 bar which reects a 96%f the overall process using the monooxygenase mutantable 2). These results represent a raise in initial productctor of 2.8 in a time course of 14 h (linear increase of

product waof the butan

This tenstrate n-butthe produchigher-presvation of ththe systemniques for the deactiv

4. Conclus

To conclneering strareaction sycell activityof a self-suevolution sgenase wilsystem by K1-butanol, fobtained bygations in tbeen carrien-butane ingies led to production4.35 g/L 1-bthe optimizcould be mrun an econmore than are requirethat there iing the percould be inimprovemeet al., 2009mation, thea continuoustudies.

In sumincreased the implantto the wild

Acknowled

BASF SEMagin, BASsure reacto.99 0.47 1.41 0.16 4.35 0.40

.0 2.9 8.2

.88 2.84 8.04

.15 0.023 0.06 0.008 0.23 0.020HS/MS.eaction time directly by GCHS/MS and the initial product rate wasonstruct ITB413 and mutant variant ITB408.

s measurable) without further oxidation and reactionol product.

dency reects that the increased solubility of the sub-ane at higher pressure is an important factor to increaset concentration. The lower butanol concentration atsure conditions (>15 bar) might be explained by deacti-e enzyme system due to physical stress of pressurizing. Further experiments using even more rened tech-examination will be necessary to take a closer look atation of the biocatalytic system.

ion

ude, we have shown that the utilization of enzyme engi-tegies as well as the implementation of a high pressurestem resulted in improvements of the overall whole

and nal 1-butanol concentration by the combinationfcient fusion complex and a domain-based directedtrategy. In comparison to the CYP153AP.sp. monooxy-d type and the previous published CYP153A6-BMO1och et al., an increase of activity of the production ofrom 0.72 and 0.86 g/L, respectively, to 2.99 g/L has been

the fusion protein and its mutant. Moreover, investi-he use of a pressure-reactor system up to 20 bar haved out in order to overcome the substrate limitation of

an aqueous system. These process engineering strate-an additional increase of the enzymatic regioselective

of butanol up to 4.53 g/L which results in a yield ofutanol at 15 bar. The high regioselectivity of >96% ofed CYP153AP.sp.(G254A)-CPRBM3 fusion protein (ITB408)aintained during the entire experimental process. Toomical feasible process a nal 1-butanol concentration50 g/L as well as a space time yield higher than 3 g/L/hd. To reach the goals our preliminary studies indicates the opportunity to dene various options for improv-formance of the system. The activity of the enzymecreased by applying different reductase units and thent of the linker system (OReilly et al., 2011; Robin). The optimization of the C-source during biotransfor-

increase of the cell stability and the implementation ofs pressure system are topics waiting for more detailed

mary the nal 1-butanol concentration could bevefold by using the optimized self-fusion construct and

ation of the high pressure reactor system in comparisontype system at atmospheric pressure.

gements

, Germany is acknowledged for nancial support. EdgarF SE for technical assistance in setting up the high pres-r.


References

Atsumi, S., Cann, A.F., Connor, M.R., Shen, C.R., Smith, K.M., Brynildsen, M.P., Chou,K.J., Hanai, T., Liao, J.C., 2008a. Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. 10, 305311.

Atsumi, S., Hanai, T., Liao, J.C., 2008b. Non-fermentative pathways for synthesis ofbranched-chain higher alcohols as biofuels. Nature 451, 8689.

Blank, L.M., Ebert, B.E., Buhler, B., Schmid, A., 2008. Metabolic capacity estimation ofEscherichia coli as a platform for redox biocatalysis: constraint-based modelingand experimental verication. Biotechnol. Bioeng. 100, 10501065.

Chen, M.M.Y., Snow, C.D., Vizcarra, C.L., Mayo, S.L., Arnold, F.H., 2012. Compari-son of random mutagenesis and semi-rational designed libraries for improvedcytochrome P450 BM3-catalyzed hydroxylation of small alkanes. Protein Eng.Des. Sel. 25, 171178.

Cooley, R.B., Dubbels, B.L., Sayavedra-Soto, L.A., Bottomley, P.J., Arp, D.J.,2009. Kinetic characterization of the soluble butane monooxygenase fromThauera butanivorans, formerly Pseudomonas butanovora. Microbiology 155,20862096.

Davis, S.C., Sui, Z., Peterson, J.A., Ortiz de Montellano, P.R., 1996. Oxidation of omega-oxo fatty acids by cytochrome P450BM-3 (CYP102). Arch. Biochem. Biophys. 328,3542.

Dellomonaco, C., Clomburg, J.M., Miller, E.N., Gonzalez, R., 2011. Engineered reversalof the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 476,355359.

Dubbels, B.L., Sayavedra-Soto, L.A., Bottomley, P.J., Arp, D.J., 2009. Thauera butanivo-rans sp. nov., a C2C9 alkane-oxidizing bacterium previously referred to asPseudomonas butanovora. Int. J. Syst. Evol. Microbiol. 59, 15761578.

Durre, P., 2008. Fermentative butanol production: bulk chemical and biofuel. Ann.N.Y. Acad. Sci. 1125, 353362.

Durre, P., 201Curr. Opin

Funhoff, E.G., Ba soluble P188, 5220

Funhoff, E.G., ation and eTechnol. 4

Green, E.M., 20Curr. Opin

Gudiminchi, RJ., Smit, Mstrains ex1507151

Guengerich, F.383, 1553

Gustafsson, MA., Fulco, Asion, puriCYP102A2Bacillus me

Hamamura, Nmonooxyg4586459

Hlavica, P., Lecytochromspecicity85104.

Honda Malca, Hauer, B., 2hydroxyla

Isken, S., de Bo229238.

Johnson, E.L., Hyman, M.R., 2006. Propane and n-butane oxidation by Pseudomonasputida GPo1. Appl. Environ. Microbiol. 72, 950952.

Koch, D.J., Chen, M.M., van Beilen, J.B., Arnold, F.H., 2009. In vivo evolution of butaneoxidation by terminal alkane hydroxylases AlkB and CYP153A6. Appl. Environ.Microbiol. 75, 337344.

Lutke-Eversloh, T., Bahl, H., 2011. Metabolic engineering of Clostridium ace-tobutylicum: recent advances to improve butanol production. Curr. Opin.Biotechnol. 22 (5), 634667.

Mestres, J., 2005. Structure conservation in cytochromes P450. Proteins 58, 596609.Munro, A.W., Daff, S.N., Turner, K.L., Chapman, S.K., 1997. Redox characterisation of

avocytochrome P-450 BM3 from Bacillus megaterium. Biochem. Soc. Trans. 25,S628.

Munro, A.W., Leys, D.G., McLean, K.J., Marshall, K.R., Ost, T.W., Daff, S., Miles, C.S.,Chapman, S.K., Lysek, D.A., Moser, C.C., Page, C.C., Dutton, P.L., 2002. P450 BM3:the very model of a modern avocytochrome. Trends Biochem. Sci. 27, 250257.

Munro, A.W., Girvan, H.M., McLean, K.J., 2007. Cytochrome P450 redox partnerfusion enzymes. Biochim. Biophys. Acta 1770, 345359.

Narhi, L.O., Fulco, A.J., 1986. Characterization of a catalytically self-sufcient119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates inBacillus megaterium. J. Biol. Chem. 261, 71607169.

Narhi, L.O., Fulco, A.J., 1987. Identication and characterization of two functionaldomains in cytochrome P-450BM-3, a catalytically self-sufcient monooxy-genase induced by barbiturates in Bacillus megaterium. J. Biol. Chem. 262,66836690.

Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver micro-somes. II. Solubilization, purication, and properties. J. Biol. Chem. 239,23792385.

OReilly, E., Kohler, V., Flitsch, S.L., Turner, N.J., 2011. Cytochromes P 450 as usefulbiocatalysts: addressing the limitations. Chem. Commun. 47, 24902501.

akis, E204., Robch, S.L0camD., MomegYP1536. OrgD., Hohesis truct.., Vom09. Rry wi861..J., Chling, Juctio, V.B.,hetic , V.B.,gy. Apen, J.Bmes an, J.B.adatioen, J.Bothliss of thgral mmel, K.1. Fermentative production of butanol the academic perspective.. Biotechnol. 22, 331336.auer, U., Garcia-Rubio, I., Witholt, B., van Beilen, J.B., 2006. CYP153A6,450 oxygenase catalyzing terminal-alkane hydroxylation. J. Bacteriol.5227.

Salzmann, J., Bauer, U., Witholt, B., van Beilen, J.B., 2007. Hydroxyl-poxidation reactions catalyzed by CYP153 enzymes. Enzyme Microb.0, 806812.11. Fermentative production of butanol the industrial perspective.. Biotechnol. 22, 337343..K., Randall, C., Opperman, D.J., Olaofe, O.A., Harrison, S.T., Albertyn,.S., 2012. Whole-cell hydroxylation of n-octane by Escherichia colipressing the CYP153A6 operon. Appl. Microbiol. Biotechnol. 96 (6),6.P., 2002. Rate-limiting steps in cytochrome P 450 catalysis. Biol. Chem.1564.

.C.U., Roitel, O., Marshall, K.R., Noble, M.A., Chapman, S.K., Pessegueiro,.J., Cheesman, M.R., von Wachenfeldt, C., Munro, A.W., 2004. Expres-cation, and characterization of Bacillus subtilis cytochromes P450

and CYP102A3: avocytochrome homologues of P450 BM3 fromgaterium. Biochemistry 43, 54745487.., Storfa, R.T., Semprini, L., Arp, D.J., 1999. Diversity in butaneenases among butane-grown bacteria. Appl. Environ. Microbiol. 65,3.hnerer, M., 2010. Oxidative biotransformation of fatty acids byes P450: predicted key structural elements orchestrating substrate, regioselectivity and catalytic efciency. Curr. Drug Metab. 11,

S., Scheps, D., Kuhnel, L., Venegas-Venegas, E., Seifert, A., Nestl, B.M.,012. Bacterial CYP153A monooxygenases for the synthesis of omega-

ted fatty acids. Chem. Commun. 42, 51155117.nt, J.A., 1998. Bacteria tolerant to organic solvents. Extremophiles 2,

Papouts19, 4

Robin, AFlits[P45

Scheps, tive by CJS66

Scheps, Syntcons

Seifert, AJ., 20libra853

Steen, EKeasprod

Urlachersynt

Urlachernolo

van Beilenzy

van Beiledegr

van BeilR., Rlaseinte

Weisserheim.T., 2008. Engineering solventogenic clostridia. Curr. Opin. Biotechnol.29.erts, G.A., Kisch, J., Sabbadin, F., Grogan, G., Bruce, N., Turner, N.J.,., 2009. Engineering and improvement of the efciency of a chimeric-RhFRed reductase domain] enzyme. Chem. Commun., 24782480.alca, S.H., Hoffmann, H., Nestl, B.M., Hauer, B., 2011. Regioselec-a-hydroxylation of medium-chain n-alkanes and primary alcohols

enzymes from Mycobacterium marinum and Polaromonas sp. strain. Biomol. Chem. 9, 67276733.nda Malca, S., Richter, S.M., Marisch, K., Nestl, B.M., Hauer, B., 2013.of -hydroxy dodecanoic acid based on an engineered CYP153A fusion

Microbial. Biotechnol. 6, 694707.und, S., Grohmann, K., Kriening, S., Urlacher, V.B., Laschat, S., Pleiss,

ational design of a minimal and highly enriched CYP102A1 mutantth improved regio-, stereo- and chemoselectivity. ChemBioChem 10,

an, R., Prasad, N., Myers, S., Petzold, C.J., Redding, A., Ouellet, M.,.D., 2008. Metabolic engineering of Saccharomyces cerevisiae for then of n-butanol. Microb. Cell Fact. 7, 36.

Eiben, S., 2006. Cytochrome P450 monooxygenases: perspectives forapplication. Trends Biotechnol. 24, 324330.

Lutz-Wahl, S., Schmid, R.D., 2004. Microbial P450 enzymes in biotech-pl. Microbiol. Biotechnol. 64, 317325.., Funhoff, E.G., 2005. Expanding the alkane oxygenase toolbox: newnd applications. Curr. Opin. Biotechnol. 16, 308314., Funhoff, E.G., 2007. Alkane hydroxylases involved in microbial alkanen. Appl. Microbiol. Biotechnol. 74, 1321.., Funhoff, E.G., van, L.A., Just, A., Kaysser, L., Bouza, M., Holtackers,berger, M., Li, Z., Witholt, B., 2006. Cytochrome P450 alkane hydroxy-e CYP153 family are common in alkane-degrading eubacteria lackingembrane alkane hydroxylases. Appl. Environ. Microbiol. 72, 5965.., Arpe, H.P., 2003. Industrial Organic Chemistry. Wiley-VCH, Wein-

Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure1 Introduction2 Materials and methods2.1 Strains and plasmids2.2 Genetic manipulation2.3 Cultivation of biomass2.4 Biotransformation2.4.1 In vivo biotransformation of n-butane to butanol2.4.2 In vivo biotransformation of n-butane to butanol under increased pressure

2.5 Analysis2.5.1 P450 activity assay2.5.2 Headspace GC/MS analysis

3 Results and discussion3.1 Engineering of biocatalysts3.2 In vivo biotransformation3.3 Pressurized in vivo biotransformation

4 ConclusionAcknowledgementsReferences

biooxidation of n-butane to 1-butanol by engineered p450 monooxygenase under increased pressure

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