Efficient Production of Active Polyhydroxyalkanoate Synthase inEscherichia coli by Coexpression of Molecular Chaperones

Nicholas M. Thomson,a Azusa Saika,b Kazunori Ushimaru,b Smith Sangiambut,a Takeharu Tsuge,b David K. Summers,c Easan Sivaniaha

Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdoma; Department of Innovative and Engineered Materials, Tokyo Institute of Technology,Yokohama, Japanb; Department of Genetics, University of Cambridge, Cambridge, United Kingdomc

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with si-multaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter�1), coexpressionwith chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble frac-tion. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greaterenzyme yields (82.37 mg liter�1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme wasunaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction ofsynthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production ofpolymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical proper-ties of the polymer.

Polyhydroxyalkanoates (PHAs) are one of the leading candi-dates to replace petrochemical plastics in many everyday

applications (1). The key enzyme in production of PHAs is poly-hydroxyalkanoate synthase (PhaC), as its substrate specificity de-termines which monomers can be polymerized.

PHA synthases are divided into four groups depending on thenumber of subunits constituting an active enzyme and on theirspecificity for monomers of different chain lengths (2). The PHAsynthase from Cupriavidus necator has been adopted as the arche-type for group I synthases and consequently is very well studied. C.necator was previously called Ralstonia eutropha, and the synthaseprotein is still widely known as PhaCRe, a convention that is fol-lowed here.

As a type I synthase, PhaCRe preferentially catalyzes the polym-erization of short-chain (R)-hydroxyalkanoic acids (4 to 6 carbonatoms), particularly (R)-3-hydroxybutyrate-coenzyme A (3HB-CoA) to poly(hydroxybutyrate) (PHB) (3). 3HB-CoA is producedfrom acetyl-CoA by the sequential action of two enzymes: �-ke-tothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB). Thegenes encoding these three enzymes constitute an operon in thegenome of C. necator and are sufficient to allow production ofPHB up to more than 90% of dry cell weight (DCW) when heter-ologously expressed in Escherichia coli (2).

Unfortunately, no crystal structure exists for any PHA syn-thase, because the enzyme tends to form inclusion bodies whenoverexpressed in a bacterial host or to aggregate during purifica-tion. Therefore, the catalytic mechanism is not fully understood,and studies of the enzyme must be carried out with the smallquantities that can be produced.

There is interest in developing in vitro PHA production pro-cesses (4). This would facilitate the development of continuousproduction systems, eliminate the costly and environmentallydamaging process of PHA recovery from within bacterial cells,allow the polymerization of monomeric units that are not easilyproduced by bacteria, and extend the range of physical propertiesof the final product. For in vitro production to be feasible on anindustrial scale would require production of far larger quantitiesof PhaC than is currently possible. Therefore, it is important to

improve upon the existing methods for PhaC expression both toassist scientific understanding and for more efficient industrialproduction.

Inclusion body formation is a well-known phenomenon inheterologous protein production, particularly in E. coli (5). Manymethods to combat the problem have been suggested, includingreducing the concentration of protein by modulating inducerquantities, expression at reduced temperatures (�30°C), use ofspecialized “folding” strains such as E. coli Origami, and in vitrorefolding of proteins from isolated inclusion bodies (6, 7). How-ever, these techniques increase the time required to obtain activeprotein and suffer from drawbacks, including reduced proteinyield and high cost.

Despite the difficulties involved with PhaC production, littlework on improving the efficiency of the process has been reported.Reduced temperatures (typically 30°C) are often used to preventinclusion body formation, and addition of a mild nonionic deter-gent such as 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopy-ranoside (Hecameg) is essential to prevent agglomeration of theenzyme during and after purification (3). Some attempts have alsobeen made to resolubilize inclusion bodies formed by the type IIsynthases from Pseudomonas oleovorans using S-Sepharose (8)and from Pseudomonas putida by denaturing with 6 M guanidinehydrochloride, followed by refolding (9).

A widely used technique to aid production of biologically ac-tive heterologous proteins is simultaneously to overexpress one ormore chaperone proteins, which constitute a diverse family, manyof which belong to the group of heat shock proteins and are up-regulated when the cell is under stress (7, 10, 11). This has beenused with success for many different proteins, particularly eukary-

Received 19 September 2012 Accepted 28 December 2012

Published ahead of print 18 January 2013

Address correspondence to Easan Sivaniah, es10009@cam.ac.uk.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02881-12

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otic or membrane-bound proteins (reviewed in reference 12), al-though the appropriate expression conditions must usually be de-termined experimentally on a case-by-case basis.

We report here the results of a study on chaperone-assistedPhaCRe expression and PHA production. Three chaperone sys-tems were chosen for coexpression with PhaCRe: GroEL/GroES(plasmid pGro7), trigger factor (Tf; plasmid pTf16) and DnaK/DnaJ/GrpE (plasmid pKJE7). Additionally, Tf and GroEL/GroESwere expressed together (plasmid pG-Tf2), as were GroEL/GroESand DnaK/DnaJ/GrpE (plasmid pG-KJE8). The Tf and DnaK/DnaJ/GrpE systems both bind to hydrophobic residues on nascentproteins, preventing aggregation until other folding steps havecompleted (13, 14). Poorly folded proteins can be targeted to theGroE complex, composed of a barrel with a hydrophobic core ofGroEL and a lid of GroES. Conformational changes in GroEL,driven by ATP, force proteins within the barrel into more com-pact, properly folded conformations (7).

MATERIALS AND METHODSStrains, plasmids, and genetic manipulation. The strains and plasmidsused in this study are described in Table 1.

To construct plasmid pASG1phaCRe, the StarGate cloning system(IBA, Germany) was utilized according to the manufacturer’s instruc-tions. The phaCRe gene was amplified by PCR with Phusion DNA poly-merase (Fisher Scientific) using 5=-phosphorylated primers to allowblunt-ended ligation into the linearized and dephosphorylated vectorpENTRY10. pET15phaCRe was used as the template. The forward primersequence was AATGGCGACCGGCAAAGGC. The reverse primer se-quence was TCCCGTGGTGGTGGTGGTGGTGTGCCTTGGCTTTGACGTATCG. Underlined bases represent partial recombination sites thatare not expressed in the protein. Bases in bold type encode the His6 tag.Following verification of gene entry by digestion with XbaI and HindIII,the His6-tagged phaCRe gene was transferred to plasmid pASG-IBAwt1 byrecombination to create pASG1phaCRe. The integrity of the new plasmidwas verified by Sanger sequencing.

Growth conditions. For PhaCRe expression, strains were grown in1-liter Erlenmeyer flasks with 200 ml of Luria-Bertani (LB) medium sup-plemented with ampicillin (100 �g ml�1) and chloramphenicol (30 �gml�1) as necessary for plasmid selection. Overnight cultures in LB me-dium (2 ml) were used for inoculation, and the cultures were then incu-bated at 37°C while shaking at 250 rpm until the optical density at 600 nm(OD600) was approximately 0.6. At this point, PhaCRe production was

induced as described below, and the incubation temperature was reducedto 30°C for a further 6 h. The cells were harvested by centrifugation at4,000 � g for 15 min, washed once with deionized water, and stored at�80°C with 100,000 U lysozyme and 10 �l protease inhibitor for His-tagged proteins (both from Sigma-Aldrich).

The N-terminally tagged PhaCRe was expressed from the T7 promoterby addition of isopropyl �-D-1-thiogalactopyranoside (IPTG, 1 mM). Toaccumulate chaperone proteins before beginning PhaCRe expression,each culture was supplemented with arabinose (0.5 mg ml�1) and/oranhydrotetracycline (0.2 �g ml�1) depending on the promoter(s) used.The C-terminally tagged PhaCRe was expressed from the tet promoter,which is also induced by anhydrotetracycline. Therefore, in this case onlythose chaperones controlled by the araB promoter were induced frominoculation, while PhaCRe and the remaining chaperones were induced byaddition of anhydrotetracycline (0.2 �g ml�1) at an OD600 of 0.6.

For PHB production, 200-ml cultures were grown in LB medium sup-plemented with glucose (20 g liter�1). Ampicillin (100 �g ml�1) andchloramphenicol (30 �g ml�1) were added as required, and both chaper-one expression and PHB production were induced at the time of inocu-lation by addition of IPTG (1 mM), arabinose (0.5 mg ml�1), and, ifnecessary, anhydrotetracycline (0.2 �g ml�1). Cultures were incubated ateither 30 or 37°C with shaking at 250 rpm for 72 h. Cells were harvested bycentrifugation at 4,000 � g for 15 min, washed once with deionized water,transferred to preweighed polypropylene tubes, freeze-dried, and thenweighed to determine DCW.

Protein purification and analysis. (i) Cobalt affinity purification.Cells were suspended in lysis buffer (2.65 mM NaH2PO4, 47.35 mMNa2HPO4, 300 mM NaCl, 10 mM imidazole, 5% [vol/vol] glycerol and0.05% [wt/vol] Hecameg) (pH 8.0) and ruptured by 5 cycles of sonicationusing a Sonopuls HD2200 sonicator (Bandelin) and MS73 tip, set to a 30-sduration, 10% duty cycle, and 10% power, with 5 min on ice betweencycles. Cell debris was removed by centrifugation at 4°C followed by fil-tration of the supernatant with a cellulose acetate syringe filter with a0.44-�m pore size. Talon His tag purification resin (Clontech) was usedfor cobalt affinity protein purification according to the manufacturer’sinstructions. Lysis buffer was used to equilibrate and wash the column,and purified PhaCRe was eluted using the same buffer containing 100 mMimidazole. Elution fractions containing PhaCRe were combined and con-centrated, and the buffer was replaced with storage buffer (2.65 mMNaH2PO4, 47.35 mM Na2HPO4, 5% [vol/vol] glycerol and 0.05% [wt/vol] Hecameg) (pH 8.0) using centrifugal filters with a molecular masscutoff of 10,000 Da.

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Relevant featuresSource orreference

StrainsE. coli BL21(DE3) E. coli B F� ompT rB

� mB� (�DE3) Novagen

E. coli W3110hns�93 E. coli K12 F� �� rph-1 hns�93 INV(rrnD, rrnE) Chen et al.a

Cupriavidus necator H16 Wild type (ATCC 17699) ATCC

PlasmidspET15phaCRe Expression vector providing N-terminal His6 tag containing phaCRe; T7 promoter; Apr 15pASG1phaCRe Expression vector containing phaCRe with C-terminal His6 tag; tet promoter; Apr This studypGro7 Expression vector for GroEL/GroES; araB promoter; Cmr ClontechpTf16 Expression vector for Tf; araB promoter; Cmr ClontechpG-Tf2 Expression vector for GroEL/GroES/Tf; pzt1 promoter; Cmr ClontechpKJE7 Expression vector for DnaK/DnaJ/GrpE; araB promoter; Cmr ClontechpG-KJE8 Expression vector for GroEL/GroES with pzt1 promoter; DnaK, DnaJ, GrpE with araB

promoter; Cmr

Clontech

pTrcphaCAB Expression vector containing C. necator phaCAB operon; trc promoter; Apr 16a C.-C. Chen, R. Walia, K. J. Mukherjee, and D. K. Summers, submitted for publication.

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(ii) PhaCRe activity assay. The final protein concentration of eachsample was determined by measuring the absorbance at 280 nm (A280)with a Nanodrop spectrophotometer (Fisher Scientific). The molar ex-tinction coefficient was taken to be 162,000 M�1 cm�1 (17). Enzymeactivity was determined by measuring the decrease in A236 resulting fromthe cleavage of the thioester bond with an extinction coefficient of 4,500M�1 cm�1 (18). The reaction mixture consisted of 50 mM phosphatebuffer, 200 �M (DL)-3-hydroxybutyryl-CoA (Sigma-Aldrich), 4 �g Pha-CRe, and double-distilled water (ddH2O) with a total volume of 400 �l.One enzyme unit was defined as the amount of enzyme that catalyzed therelease of 1 �mol CoA per min.

(iii) Polyacrylamide gel electrophoresis and Western blotting. Thepurity of PhaCRe was checked by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). A 20-�l portion of each sample of puri-fied PhaCRe was mixed with 10 �l of 3� SDS-PAGE buffer and incubatedat 95°C for 3 min before being loaded onto a 10% polyacrylamide gel inTris-glycine buffer. The loading volume was adjusted so that 1.22 �gprotein was loaded per sample. Gels were stained with PageBlue protein-staining solution (Fermentas).

For Western blotting, 10 ml samples of cell culture were taken prior toharvesting. The cells were recovered by centrifugation and washed once indeionized H2O. The cell pellet was resuspended in 500 �l BugBuster celllysis buffer (Novagen) with 250 �l of Benzonase nuclease (Sigma-Aldrich)and shaken at room temperature for 30 min. The lysis mixture was cen-trifuged at 17,000 � g for 30 min at 4°C. The supernatant constituted thesoluble protein fraction, and the pellet (the insoluble fraction) was dis-solved in 500 �l of 1% (wt/vol) SDS. The proteins were separated bySDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) mem-brane using a Trans-Blot semidry blotting system (Bio-Rad). His6-taggedPhaCRe was specifically stained using the HisProbe HRP conjugate kit andmetal-enhanced DAB substrate (Fisher Scientific). Densitometry analysiswas performed using the gel analysis function of ImageJ (National Insti-tutes of Health).

(iv) Analysis of PHB production. Approximately 20 mg of dried cellswere used for methanolysis to convert PHB into its methyl ester by mixingwith 2 ml each of chloroform and methanol-sulfuric acid (85:15 vol/vol)and heating at 100°C for 140 min (19). After addition of 1 ml ddH2O toinduce phase separation, the filtered lower chloroform layer was subjectedto gas chromatography (GC) analysis (Shimadzu GC 2014), with methylbenzoate (0.05%) as an internal standard, in order to calculate the amountof accumulated PHB.

PHB was extracted from dried cells by stirring with 50 ml chloroformper 100 mg PHB for 72 h. After filtering to remove cell debris, the polymerwas precipitated by slowly dropping the chloroform solution into meth-anol and recovered by a further filtration step. The purified polymer wasthen redissolved in chloroform at a concentration of 1 mg ml�1 for gelpermeation chromatography (GPC) to determine the molecular weight.The GPC analysis was conducted at 40°C using a Shimadzu 10A GPCsystem with Shodex K-806 M and K-802 columns and a 10A refractiveindex detector. Chloroform was used as the eluent at a flow rate of 0.8 mlmin�1. Polystyrene standards (Mp [peak molecular mass] � 1.3 � 103 to7.5 � 106) with low polydispersity were used to produce a calibrationcurve.

RESULTSProduction and purification of PhaCRe. To test the productivityand activity of PhaCRe, with or without coexpression of chaperoneproteins, N-terminally His6-tagged PhaCRe was purified from200-ml shake flask cultures of E. coli BL21(DE3) containingpET15phaCRe, by cobalt affinity chromatography. It has been re-ported that modifications to the C terminus of PhaCRe can beintroduced without affecting enzyme activity only if the hydro-phobic environment surrounding the C terminus is maintained(20). To test whether the coexpression of chaperones could pre-vent the reduction in PhaCRe activity caused by a C-terminal His6

tag, the experiment was repeated using pASG1phaCRe to express aC-terminally tagged protein.

Growth of each culture was quantified by measuring the aver-age final OD600 (Table 2). If necessary, cultures were diluted 10-fold with LB medium to bring the optical density within the linearrange of the spectrophotometer. Growth was not affected by ex-pression of GroEL/GroES (pGro7) or Tf (pTf16), but was substan-tially reduced by expression of GroEL/GroES/Tf (pG-Tf2), DnaK/DnaJ/GrpE (pKJE7) and GroEL/GroES/DnaK/DnaJ/GrpE (pG-KJE8). Chaperone proteins were efficiently produced at highconcentration, as determined by SDS-PAGE analysis (data notshown), and so were assumed to be saturating.

Table 2 also shows the average amount of soluble PhaCRe re-covered per liter of bacterial culture. The N-terminally His6-

TABLE 2 Effect of chaperone co-expression on yields of soluble PhaCRea

Plasmid content Chaperone(s) expressed Final OD600b

Protein recovered(mg liter�1)

Specific productivity(mg liter�1 OD unit�1)

pET15phaCRec

Alone 4.77 � 1.7 14.55 � 6.2 3.55 � 2.7pGro7 GroEL/GroES 4.89 � 1.4 44.37 � 9.4 9.99 � 5.2pTf16 Tf 4.73 � 1.2 36.14 � 9.5 8.20 � 4.0pG-Tf2 GroEL/GroES/Tf 0.74 � 0.3 13.44 � 5.5 23.13 � 20.1pKJE7 DnaK/DnaJ/GrpE 1.65 � 0.5 10.10 � 4.0 7.10 � 4.7pG-KJE8 GroEL/GroES/DnaK/DnaJ/GrpE 2.00 � 0.9 15.84 � 5.9 8.71 � 4.6

pASG1phaCRed

Alone 5.17 � 1.4 1.77 � 1.1 0.32 � 0.1pGro7 GroEL/GroES 5.20 � 0.2 13.40 � 2.3 2.57 � 0.4pTf16 Tf 4.93 � 0.3 17.67 � 3.5 3.58 � 0.6pG-Tf2 GroEL/GroES/Tf 0.48 � 0.1 8.00 � 4.1 16.70 � 8.7pKJE7 DnaK/DnaJ/GrpE 1.38 � 0.8 1.68 � 0.9 1.22 � 0.1pG-KJE8 GroEL/GroES/DnaK/DnaJ/GrpE 3.49 � 1.1 10.35 � 6.7 2.78 � 0.9

a All values are averages for cultures grown in triplicate � standard deviations.b Optical density was measured at 600 nm immediately before cells were harvested.c N-terminally His6 tagged.d C-terminally His6 tagged.

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tagged version of PhaCRe (pET15phaCRe) was recovered in muchlarger quantities than the C-terminally His6-tagged version(pASG1phaCRe). Within the set of strains expressing the N-termi-nally His6-tagged version, coexpression with GroEL/GroES andwith Tf resulted in approximately 3-fold increases in soluble pro-tein, with 44.37 mg liter�1 and 36.14 mg liter�1, respectively, be-ing recovered, compared to 14.55 mg liter�1 for PhaCRe alone.The other chaperone systems resulted in either a small increase(GroEL/GroES/DnaK/DnaJ/GrpE) or a small decrease (GroEL/GroES/Tf and DnaK/DnaJ/GrpE) compared to the control.

A similar pattern was seen for the set of C-terminally taggedPhaCRe strains. GroEL/GroES (13.40 mg liter�1) and Tf (17.67 mgliter�1) resulted in 8- to 10-fold increases compared to the control(1.77 mg liter�1). GroEL/GroES/Tf and GroEL/GroES/DnaK/DnaJ/GrpE coexpression resulted in 5- to 6-fold increases, to 8.00mg liter�1 and 10.35 mg liter�1, respectively.

To allow for the differences in final culture density, the specificproductivity of PhaCRe (mg protein per liter per OD600 unit) wasalso calculated (Table 2). This confirms that the production ofPhaCRe is far more efficient with an N-terminal His6 tag (3.55 mgliter�1 OD unit�1) than a C-terminal His6 tag (0.32 mg liter�1 ODunit�1). Interestingly, despite causing a serious reduction ingrowth, coexpression with GroEL/GroES/Tf resulted in by far thelargest specific productivity for both N-terminally (23.13 mg li-ter�1 OD unit�1) and C-terminally (16.70 mg liter�1 OD unit�1)tagged synthase.

A one-step purification protocol was used to reduce samplelosses. Therefore, some coeluting proteins remained in the finalPhaCRe solutions. To assess the degree of contamination fromcoeluting proteins, samples (approximately 1.2 �g) of PhaCRe so-lution from either pET15phaCRe or pASG1phaCRe with or with-out the coexpression of chaperones were electrophoresed througha 10% polyacrylamide gel (Fig. 1). Individual protein bands wereexcised and identified by trypsin digestion followed by mass spec-trometry to produce a protein fingerprint.

The major band in each sample was identified as PhaCRe,which comprised 76 to 90% of the protein in each sample as de-termined by densitometry using ImageJ software (Fig. 1). Addi-

tional bands were observed, particularly in the samples with coex-pression of DnaK/DnaJ/GrpE. The most abundant of these was an56-kDa digestion product of PhaCRe (10 to 24% of total pro-tein), which most likely resulted from cleavage of 90 to 100 aminoacids from the end of the protein. Low concentrations of the DnaKand DnaJ chaperones were also detected in the samples where theywere overexpressed. Two additional proteins, which were identi-fied as a putative ligase and SlyD (a peptidyl-prolyl cis-transisomerase similar to trigger factor), were detected in samples ofPhaCRe expressed from pASG1phaCRe but not in samples frompET15phaCRe. None of the copurified proteins, including thePhaCRe degradation product, are expected to display PHA poly-merase activity.

Using estimated absorption coefficients generated by the on-line ProtParam program (web.expasy.org/protparam), we deter-mined the percentage contribution to the A280 of PhaCRe in eachsample (data not shown). Due to the relatively high molecularweight and extinction coefficient of PhaCRe, the coeluted proteincontributed disproportionately less to the estimate of protein con-centration than PhaCRe. Therefore, while the average purity ofPhaCRe (in the case of the C-terminally tagged protein, whichcontained more coeluted proteins) was 82%, it contributed to-ward an average of 89% of the absorbance. This indicates that theaverage error attributed to impurities following a one-step purifi-cation procedure is approximately 11%.

Effect of chaperones on PhaCRe solubility. To find outwhether the increased yield of soluble PhaCRe when coexpressedwith chaperones was due to a net increase in PhaCRe synthesis orto an increase in the proportion of soluble protein, soluble andinsoluble protein fractions from cells containing pET15phaCRe

alone or with each of the chaperone systems were compared bySDS-PAGE. Because DnaK (70 kDa), GroEL (60 kDa), and triggerfactor (56 kDa) all have sizes similar to that of PhaCRe, they ob-scured the PhaCRe band on an SDS-PAGE gel. Therefore, the Pha-CRe was selectively visualized by Western blotting (Fig. 2). Theloading volume in each lane was normalized according to OD600.

With the exception of protein from cells expressing GroEL/GroES/Tf, which grew very poorly, making recovery of protein

FIG 1 SDS-PAGE gels stained with Coomassie blue, showing PhaCRe purified by cobalt affinity chromatography after expression in BL21(DE3)/pET15phaCRe

(a) or BL21(DE3)/pASG1phaCRe (b) with and without chaperone protein coexpression. Bands were identified by digestion with trypsin followed by massspectrometry fingerprinting. M, prestained molecular mass markers; lane 1, PhaCRe only; lanes 2 to 6, coexpression from pGro7, pTf16, pG-Tf2, pKJE7, andpG-KJE8, respectively. PhaCRe purities shown in the figure were calculated from densitometry analysis.

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difficult, the intensities of the bands for the insoluble fractionswere similar (ranging from 1.3 to 2.8 times the intensity of thesoluble control). However, the intensity of the bands for the sol-uble fractions increased when chaperone proteins were coex-pressed. The two best-performing systems from the purificationexperiment, GroEL/GroES and Tf, had 1.6- and 2.0-fold moresoluble PhaCRe than the control. However, the relative propor-tions of PhaCRe in these two strains were 0.57 and 0.80, respec-tively. This is larger than that of PhaCRe expressed without chap-erones (0.4) but smaller than those for the other three strains. Thissuggests that the other chaperone combinations were more effi-cient at solubilizing the PhaCRe that they produced but either pro-duced less PhaCRe per cell or resulted in less cell growth.

These values are smaller than the relative increases in solubleprotein recovered during protein purification, possibly becauseantibody binding was inhibited by the large quantities of chaper-one proteins present in the Western blots. Therefore, these resultsshould be considered only as a semiquantitative indication of therelative amounts of PhaCRe in each fraction. As such, they suggestthat chaperones successfully increased both the total amount ofprotein produced and its solubility, although substantial amountsof protein remained in the insoluble fraction.

Optimization of inducer concentrations. The most successfulcoexpression combination in the initial study was N-terminallytagged PhaCRe with GroEL/GroES, which resulted in a 3-fold in-crease in soluble protein recovered (Table 2). To investigatewhether further increases could be achieved, cultures ofBL21(DE3) containing pET15phaCRe and pGro7 were grown asbefore with 0.1, 0.5, or 1.0 mM IPTG and 0, 0.25, 0.5, 1.0, 2.5, or5.0 mg ml�1 arabinose. The results are displayed in Table 3.

Lower concentrations of IPTG resulted in larger quantities ofPhaCRe, suggesting that gentle induction is necessary to allow timefor the protein to fold (Table 3). Increasing the concentration ofarabinose also increased PhaCRe production. Consequently, boththe largest amount of PhaCRe (82.37 mg liter�1) and the highestspecific productivity (15.43 mg liter�1 OD unit�1) were achievedwith 0.1 mM IPTG and 5.0 mg ml�1 arabinose. This correspondsto a 5.7-fold increase compared to production from pET15phaConly. Gentle expression of PhaCRe combined with coexpression oflarge amounts of GroEL/GroES is therefore the best strategy toincrease soluble PhaCRe production.

Influence of chaperone coexpression on PhaC activity. Tocalculate the specific activity of our PhaCRe preparations, the total

yield of PhaCRe protein from each 200-ml culture was calculatedfrom the final volume and concentration of purified protein solu-tion after desalting. Activity assays were performed in triplicate foreach sample by measuring the decrease in absorbance at 236 nm,corresponding to the 3HB-CoA thioester bond that is broken dur-ing polymerization. Analyses for statistical significance were per-formed using two-tailed t tests assuming unequal sample variance.

The average specific activity of N-terminally His6-taggedPhaCRe produced without chaperone protein coexpression was12.1 U mg�1 (Fig. 3). The cultures in which protein recovery wasincreased showed no significant change in specific activity (P �0.05). However, the specific activity was significantly reduced(P � 0.01) by coexpression of GroEL/GroES/Tf (9.0 U mg�1) andDnaK/DnaJ/GrpE (8.8 U mg�1). When the specific activity wasmultiplied by the protein productivity to give the average totalyield of enzyme (U liter�1), GroEL/GroES and Tf again resulted insignificant (P � 0.01) increases (586.3 and 439.7 U liter�1, respec-tively) compared to PhaCRe alone (168.7 U liter�1). Only DnaK/DnaJ/GrpE significantly (P � 0.01) decreased the yield (82.4 Uliter�1). Thus, the most influential effect of the GroEL/GroES and Tfsystems (plasmids pGro7 and pTf16) was to increase the amount ofsoluble protein produced, rather than increasing its specific activity.

The specific activity of C-terminally tagged PhaCRe (expressedfrom pASG1phaCRe) was significantly lower (P � 0.01) than thatof the N-terminally tagged protein (Fig. 3b), so chaperones werenot able to rectify the misfolding of PhaCRe caused by His6 tagaddition to the C terminus. Compared to an average specific ac-tivity of 1.7 U mg�1 for protein produced in cells containingpASG1phaCRe alone, chaperone coexpression either showed nochange (P � 0.05) or resulted in a decrease (P � 0.01). Coexpres-sion with GroEL/GroES or Tf both resulted in average specificactivities of 1.7 U mg�1. The other three chaperone combinationsall resulted in decreased specific activities, with the smallest being0.9 U mg�1 for DnaK/DnaJ/GrpE. As seen for the N-terminallyHis6-tagged PhaCRe, the differences between the yields of purifiedprotein were more important than the variation in specific activ-ity. The greatest yield was 30.2 U liter�1 for Tf coexpression, whichrepresents a 10-fold increase over the yield of 3.0 U liter�1 whenPhaCRe was expressed alone. Coexpression with every chaperonecombination except DnaK/DnaJ/GrpE resulted in a significant in-crease (P � 0.01) in protein yield compared to PhaCRe alone.

Effect of chaperones on PHB production. In E. coli, the mo-lecular weight of PHB varies in inverse proportion to the amountof PhaCRe produced (21). As an initial investigation into the ef-fects of improved PhaCRe production (due to chaperone coex-

TABLE 3 Optimization of PhaCRe production by varying inducerconcentration in BL21(DE3) containing pET15phaCRe and pGro7

Arabinoseconcn(mg/ml)

Protein recovery(mg liter�1) at IPTGconcn (mM) of:

Specific productivity(mg liter�1 OD unit�1) atIPTG concn (mM) of:

0.1 0.5 1.0 0.1 0.5 1.0

0 12.71 10.43 13.89 2.93 2.35 2.670.25 51.44 49.48 49.90 10.03 10.48 8.090.50 66.53 51.12 52.04 10.35 10.08 7.951.00 81.65 50.33 59.89 14.49 10.16 9.902.50 78.92 58.94 66.90 14.47 12.38 11.545.00 82.37 59.88 64.60 15.43 10.97 11.71

FIG 2 Distribution of PhaCRe between the insoluble (I) and soluble (S) pro-tein fractions. PhaCRe was expressed from BL21(DE3) containingpET15phaCRe on its own or coexpressed with chaperone proteins. The relativedensities were calculated using ImageJ software. Density relative to PhaCRe isthe ratio of the density of each band to the soluble fraction of PhaCRe alone(bold). Relative densities are the ratios of soluble to insoluble fractions withineach sample. pGro7, GroEL/GroES; pTf16, Tf; pG-Tf2, GroEL/GroES/Tf;pKJE7, DnaK/DnaJ/GrpE; pG-KJE8, GroEL/GroES/DnaK/DnaJ/GrpE.

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pression) on PHB biosynthesis, E. coli W3110hns�93-1 carryingplasmid pTrcphaCAB alone and equivalent strains carrying eachof the five chaperone expression plasmids were grown for 72 h inLB medium supplemented with glucose (20 g liter�1) at either 30or 37°C. The long culture time was chosen to allow sufficient timefor PHB accumulation, even if chaperone coexpression negativelyinfluenced cell growth, as for PhaCRe production.

In every case, growth (determined by measuring DCW) wasbetter at 30°C than at 37°C (Table 4). As seen in the PhaCRe pro-duction experiments, chaperone protein coexpression sometimes in-hibited bacterial growth, and this was particularly evident when cellscontained the pGro7 or pG-Tf2 chaperone expression plasmids.

There were large variations in the amount of PHB produced byeach culture, both between temperatures and between chaperoneexpression systems. In the absence of chaperone coexpression,PHB accumulated to 65% DCW at 30°C and to 71% DCW at37°C. In almost every case, coexpression of chaperone proteinssubstantially reduced the yield of PHB. The biggest reductions

were for GroEL/GroES and GroEL/GroES/Tf, with the smallestyields being for GroEL/GroES/Tf coexpression (3% and 1% at 30and 37°C, respectively).

The number-averaged molecular weights (Mn) were generallydecreased by chaperone coexpression (Table 4). At 30°C, Tf andGroEL/GroES/DnaK/DnaJ/GrpE reduced the Mn of PHB by21.8% and 37.2%, respectively. At 37°C the largest reductionswere with GroEL/GroES and GroEL/GroES/DnaK/DnaJ/GrpE(37.6% and 31.6%). Additionally, Mn for each sample was larger at37°C than at 30°C. Polydispersities (defined as the ratio of Mw toMn) were not significantly affected by chaperone protein coex-pression. Due to poor growth and low PHB yield, molecularweight data were not gathered for GroEL/GroES/Tf.

DISCUSSION

Chaperone protein coexpression was found to assist in the pro-duction of soluble PhaCRe in BL21(DE3). The most significantimprovements were seen with the production of N-terminally

FIG 3 Average specific activities (hatched bars) and total yield per liter (crosshatched bars) for PhaCRe produced from pET15phaCRe (N-terminal His6 tag) (a)and pASG1phaCRe (C-terminal His6 tag) (b) with and without coexpression of chaperone proteins. Error bars represent the standard deviations of results fromcultures grown in triplicate, with three replicates of each assay. Values that are significantly different (P � 0.01) from that for PhaCRe only are indicated byasterisks.

TABLE 4 Production of PHB during overexpression of molecular chaperone proteins

Temp Protein(s) expressedDCW(g liter�1)

PHB content(% DCW) Mn (106) Mw (106) Mw/Mn

30°C PhaCABAlone 6.37 65 1.37 3.09 2.3GroEL/GroES 2.12 27 1.29 2.78 2.2Tf 6.85 63 1.07 2.18 2.0GroEL/GroES/Tf 2.24 3 NDa ND NDDnaK/DnaJ/GrpE 7.47 49 1.42 3.00 2.1GroEL/GroES/DnaK/DnaJ/GrpE 7.76 59 0.86 1.93 2.2

37°C PhaCABAlone 2.46 71 2.34 4.46 1.9GroEL/GroES 0.93 13 1.46 2.75 1.9Tf 2.06 70 2.07 4.71 2.3GroEL/GroES/Tf 1.08 1 ND ND NDDnaK/DnaJ/GrpE 2.20 44 2.42 4.66 1.9GroEL/GroES/DnaK/DnaJ/GrpE 2.53 28 1.60 3.35 2.1

a ND, not determined.

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tagged PhaCRe and chaperone expression was not able to restorereduced activity caused by a C-terminal His6 tag. Coexpression ofthe GroEL/GroES operon (pGro7) was the most successful strat-egy, increasing soluble PhaCRe recovery 5.7-fold. Enzyme activitywas largely unaffected by chaperone protein coexpression, sug-gesting that soluble PhaCRe is correctly folded in E. coli. The in-crease in yield was due to substantial increases in the quantity ofsoluble, active protein that could be recovered by cobalt affinitychromatography. Therefore, the chaperone proteins assisted thefolding of polypeptide chains that would otherwise have accumu-lated in inclusion bodies or be targeted for protein degradation.

As shown in Fig. 2, considerable quantities of insoluble PhaCRe

remained in each strain, although the fraction of soluble proteinwas increased by coexpressing chaperones. This suggests thatchaperone proteins are able to allow greater quantities of PhaCRe

to be produced in total, either by increasing production or byreducing the amount of protein that is targeted for protease deg-radation.

Optimization of the expression strategy resulted in even higheryields of soluble PhaCRe (Table 3). The most successful strategywas to use gentle induction of PhaCRe expression (0.1 mM IPTG)but high-level expression of the GroEL/GroES system (5 mg ml�1

arabinose). This ensures a plentiful supply of chaperones to pre-vent aggregation and assist folding of each PhaCRe polypeptide assoon as it is produced. Further improvements may be possible bycombining chaperone coexpression with other strategies, such asexpression at reduced temperature, use of longer culture times, orexpression in fermentor cultures.

Reduced bacterial growth, a known side effect of chaperoneprotein coexpression (7), contributed to the difference in totalprotein yields between the strains used in this study. Another po-tential cause of these variations is plasmid instability. We at-tempted to quantify the magnitude of this effect by plating sam-ples of each culture on antibiotic-free and antibiotic-containingLB agar plates, prior to induction (OD600 � 0.6) and after the 6-hproduction period, and comparing the number of colonies (datanot shown). There was no evidence for plasmid instability prior toinduction. However, fewer colonies grew with or without antibi-otics following protein production, indicating a loss of colony-forming ability during expression, as suggested by others (22).This is in keeping with the observation that the high specific pro-ductivity of strains containing pG-Tf2 appeared to be deleteriousto growth and suggests that most cells retain the plasmids duringprotein accumulation. Modulation of the level of chaperone ex-pression could potentially reduce the negative effects on bacterialgrowth while maintaining the beneficial effects on protein folding,although this effect was not seen in our optimization experiment.

There were differences between the specific activities of PhaCRe

expressed with different chaperone systems. It is likely that muchof the difference in specific activities is due to variations in therelative amounts of contaminating proteins in each sample. Inparticular, coexpression from pKJE7 had a larger number of coe-luting proteins than other samples and also had a low specificactivity (Fig. 1 and 3). The contaminating proteins were identifiedas a mixture of chaperones (DnaK/J), SlyD, and degradationproducts of PhaCRe. Both DnaK and DnaJ, as well as SlyD, arecommon contaminants of His6 tag-purified proteins due to natu-rally occurring oligo(His) sequences in their primary structure(23). Therefore, it is not surprising to find traces of them, partic-ularly in a strain overexpressing DnaK and DnaJ proteins.

The specific activity of PhaCRe has previously been reported tobe 40 U mg�1, which is substantially higher than the activitiesreported here (17). The protein preparation in the previous reportinvolved an extra purification step using size exclusion chroma-tography to remove soluble aggregations of PhaCRe. Althoughsuch a procedure would be expected to increase the specific activ-ities in this case (and to reduce variance), absolute specific activityhad far less influence on relative yields between cultures than theincrease in total soluble protein. Therefore, the conclusions fol-lowing a one-step purification procedure remain valid.

The C-terminally tagged protein produced from pASG1phaCRe

had much lower specific activities than N-terminally tagged pro-tein from pET15phaCRe and was also produced in lower quanti-ties. This suggests that chaperone proteins were not able to rectifythe misfolding of PhaCRe caused by the addition of a His6 tag to theC terminus. It is not clear whether the lower quantity of proteinwas due to differences in the expression signals and/or copy num-ber between the plasmids, increased accumulation in inclusionbodies, or recognition of the badly folded protein and targeting fordegradation. However, since chaperone proteins were still able toincrease the amount of soluble PhaCRe, it is evident that they playa part in the folding mechanism and are at least partially successfulin preventing the aggregation of badly folded protein.

Chaperone protein coexpression generally resulted in a de-crease in both the number-averaged and weight-averaged molec-ular weights of PHB produced in E. coli, particularly for theGroEL/GroES, Tf, and GroEL/GroES/DnaK/DnaJ/GrpE systems.This can be attributed at least in part to the production of a largernumber of active PhaCRe molecules. These compete for the avail-able monomeric units and catalyze the production of a largernumber of shorter chains than when only the PHB operon is ex-pressed. Consistent with this, pKJE7 caused a small increase in Mn

and was also responsible for a reduction in PhaCRe productivity.Several studies have shown that the addition of exogenous hy-

droxylated molecules can usefully alter the molecular weights ofPHAs generated in vivo. This is thought to occur by transmem-brane migration of the agents to assist in chain termination of thepolymer (24, 25). Our results point to the use of chaperone-me-diated alteration of the monomer-enzyme balance as an alterna-tive route to altering the molecular weight distribution of PHAs.Modulation of PhaC activity has previously been used to vary themolecular weight and yield of PHA produced in E. coli (26) and P.putida (27). Our results agree with the previous finding that PHAmolecular weights are inversely related to PhaC activity. Whileproduct control by an exogenous agent can be toxicity limited, theexpression of the endogenous chaperone could be more flexiblytuned to control the molecular weight of the resulting polymer inorder to suit a range of applications.

PHB production relies on an interconnected web of relatedbiochemical pathways, including cellular respiration, which pro-vides the acetyl-CoA that is converted into the monomeric unit forPHB. Since chaperone proteins are actively involved in the foldingof the majority of bacterial proteins, it is very likely that overex-pression of any of the systems described in this study will simul-taneously also affect the levels of the PhaA and PhaB proteins aswell as many more diverse native proteins. This hinders a simpleinterpretation of the results but suggests that the large variationsin bacterial growth and PHB accumulation could be due to imbal-ances in the core biochemical pathways of the cells. Further stud-ies are required to fully understand the mechanism of PHB mo-

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lecular weight reduction during chaperone coexpression and howthis could be translated into a commercially relevant productionprocess.

Chaperone protein coexpression is an effective method for in-creasing the yield of PhaCRe. By using a widely available set ofexpression plasmids, this study has demonstrated increases in to-tal enzyme yield of up to 6 times without the need for costly andtime-consuming processes such as expression at reduced temper-ature or in vitro protein refolding. This will facilitate the laborato-ry-scale study of the enzyme for the purposes of elucidating itsstructure and catalytic mechanism as well as developing in vitroPHB production methods. The method should also be easilytransferrable to the study of other PHA synthases.

ACKNOWLEDGMENT

N.M.T. is funded by the UK Engineering and Physical Sciences ResearchCouncil, grant number EP/P504120/1.

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