Appl Microbiol Biotechnol (2014) 98:831–842 DOI 10.1007/s00253-013-5398-4
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Enhanced production of polyhydroxyalkanoates (PHAs) from beechwood xylan by recombinant Escherichia coli Lucia Salamanca-Cardona & Christopher S. Ashe & Arthur J. Stipanovic & Christopher T. Nomura
Received: 2 October 2013 / Revised: 6 November 2013 / Accepted: 9 November 2013 / Published online: 28 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Microbial conversion of plant biomass to valueadded products is an attractive option to address the impacts of petroleum dependency. In this study, a bacterial system was developed that can hydrolyze xylan and utilize xylan-derived xylose for growth and production of polyhydroxyalkanoates (PHAs). A β-xylosidase and an endoxylanase were engineered into a P(LA-co-3HB)-producing Escherichia coli strain to obtain a xylanolytic strain. Although PHA production yields using xylan as sole carbon source were minimal, when the xylan-based media was supplemented with a single sugar (xylose or arabinose) to permit the accumulation of xylanderived xylose in the media, PHA production yields increased up to 18-fold when compared to xylan-based production, and increased by 37 % when compared to production from single sugar sources alone. 1H-Nuclear magnetic resonance (NMR) analysis shows higher accumulation of xylan-derived xylose in the media when xylan was supplemented with arabinose to prevent xylose uptake by catabolite repression. 1H-NMR, gel permeation chromatography, and differential scanning calorimetry analyses corroborate that the polymers maintain physical properties regardless of the carbon source. This study demonstrates that accumulation of biomass-derived sugars in the media prior to their uptake by microbes is an important Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-5398-4) contains supplementary material, which is available to authorized users. L. Salamanca-Cardona : C. S. Ashe : A. J. Stipanovic : C. T. Nomura (*) Department of Chemistry, State University of New York, College of Environmental Science and Forestry, 318 Jahn Lab, 1 Forestry Dr, Syracuse, NY 13210, USA e-mail: [email protected] C. T. Nomura Center for Applied Microbiology, State University of New York, College of Environmental Science and Forestry, Syracuse 13210 NY, USA
aspect to enhance PHA production when using plant biomass as feedstock. Keywords Polyhydroxyalkanoates . Xylanases . Recombinant Escherichia coli . Xylan . Biomass . Hemicellulose
Introduction Concerns regarding the environmental and economic impacts of petroleum dependency have driven an increased research focus to develop strategies for the production of petroleum-alternative compounds such as bioplastics (polyhydroxyalkanoates, polylactate, polyglycolate) from plant biomass to reduce world dependency on fossil oils. Polyhydroxyalkanoates (PHAs) are a class of polyesters that can be produced natively by various microorganisms as a carbon reservoir during growth under stress conditions (Anderson and Dawes 1990; Sudesh et al. 2000). Poly(3-hydroxyburyrate) (PHB) is the most commonly produced PHA usually requiring only three enzymes: a βketothiolase, an acetoacetyl-CoA reductase, and a PHA synthase for its biosynthesis (Lu et al. 2009). Given the simple machinery required for PHB production, recombinant PHA biosynthesis has flourished in nonnative producers like Escherichia coli due to the ease with which this microorganism can be genetically manipulated (Fidler and Dennis 1992). From this starting point, E. coli has already been modified to produce PHAs with various repeating unit compositions which impart a variety of physical properties and extend the applicability of the bioplastics (Wang et al. 2012; Taguchi et al. 1999; Fukui et al. 1999; Tappel et al. 2012; Tsuge et al. 2003). In 2008, Taguchi et al. reported the production of a new and attractive PHA polymer: poly(lactate-3hydroxybutyrate) [P(LA-co-3HB)] by E. coli (Taguchi et al. 2008). P(LA-co-3HB) polymers exhibit varying degrees of strength, flexibility, and transparency, resulting in improved
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physical properties dependent on their lactic acid (LA) monomer fraction (Yamada et al. 2011). By using xylose as a carbon source instead of glucose, the LA content in P(LA-co-3HB) could be increased 5-fold (Nduko et al. 2012a; Taguchi et al. 2008). These results indicate that xylose has great potential as a carbon substrate for the production of this new class of biopolymers. Xylose is the second most abundant plant biomassderived sugar and the major component of the hemicellulose xylan (Sjostrom 1993). Therefore, plant biomass-derived xylose for PHA production is a promising strategy to achieve lower production costs and better material properties. Production of PHAs from plant biomass has been reported in a variety of wild type and recombinant microorganisms (Pan et al. 2012; Keenan et al. 2006; Nduko et al. 2012b; Reddy et al. 2003). However, these systems require the biomass to be hydrolyzed in a step prior to fermentation through dilute acid or enzymatic hydrolysis to breakdown the polysaccharides and make the sugars available in a form that can be readily taken up by the microorganism (Iranmahboob et al. 2002). The hydrolysis process is often carried out at high temperatures and low pH, conditions which are not only prohibitive for E. coli growth, but also result in the generation of compounds which can be potentially toxic and prevent downstream fermentative processes, thus lowering yields and increasing production costs (Palmqvist and HahnHägerdal 2000a, b; Larsson et al. 1999). Therefore, an approach that couples the hydrolysis and fermentative steps is an attractive strategy for the production of PHAs from plantderived carbon sources like xylose. E. coli cannot natively utilize xylan as a carbon source since it lacks the genes necessary to express the hydrolytic enzymes, xylanases. Based on the chemical composition, two enzymes are required to hydrolyze xylan to its constitutive monomers: endoxylanases and β-xylosidases (Subramaniyan and Prema 2002). Endoxylanases break down the xylopyranose backbone in xylan into oligomer fragments consisting of two or three xylose units (xylobiose and xylotriose, respectively) (Poutanen et al. 1991). For fermentation purposes, the oligomer fragments cannot be utilized by E. coli; thus, xylobiose and xylotriose fragments must be degraded to monomeric sugars by the action of β-xylosidases (Poutanen et al. 1991; John et al. 1979). In this study, E coli LS5218 was genetically engineered to express an endoxylanase (XylB) from Streptomyces coelicolor and a β-xylosidase (XynB) from Bacillus subtilis in order to utilize xylan. This strain was additionally designed to express the PHA synthase from Pseudomonas sp. 61-3 harboring a Ser325Th/Gln481Lys mutation [PhaC1Ps(ST/ QK)], a propionyl- CoA transferase (PCT) from Megasphaera elsdenii , and a β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha under a R. eutropha constitutive promoter (Taguchi et al. 2008) in order to produce PHA polymers. The
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co-expression of the xylanase system and the P(LA-co3HB) system in E. coli resulted in the production of xylan-derived P(LA-co-3HB). Therefore, this study demonstrates for the first time production of PHA polymers by E. coli using xylan as a carbon source.
Materials and methods Media and cultivation conditions All plasmids and strains used in this study are listed in Table 1. E. coli and B. subtilis strains used for cloning were grown on Lennox Broth (LB, DifcoTM) at 37 °C in a rotary shaker at 250 rpm. Carbenicillin (100 μg mL−1) and kanamycin (50 μg mL−1) were supplemented as needed per plasmid requirements. S. coelicolor BAA-471 isolates were selected from cells grown in DifcoTM ISP medium two plates at 30 °C for 48 h. For genomic DNA extraction, isolated colonies were grown in YEME medium (composition per liter: 3 g yeast extract, 5 g Bacto-peptone, 3 g malt extract, 10 g glucose, 170 g sucrose) at 30 °C in a rotary shaker at 250 rpm. Construction of pBBRXBB2 vector for recombinant xylanase production in E. coli Restriction enzymes, CIP, and T4 DNA ligase were purchased from New England Biolabs, Ipswich, USA. All transformations and plasmid manipulations were performed in E. coli JM109 following standard protocols (Sambrook et al. 2001) unless otherwise specified. Genomic DNA was purified from B. subtilis and S. coelicolor with a Wizard genomic DNA purification kit (PROMEGA, Fitchburg, USA). Genes encoding for one endoxylanase (xylB) and one β-xylosidase (xynB) were PCR-amplified from S. coelicolor and B. subtilis, respectively, with the following primers (Integrated DNA Technologies, Coralvile, USA): xylB-F: 5′-CATATGCTGC TCGTCCAGCCGA-3′; xylB-R: 5′-GGATCCCCGCCCGC GCTGCAGGACA-3′; xynB-F: 5′-GCTAGC ATGAAGAT TACCAATCCCG-3′; xynB-R: 5′-GGATCC TTATTTTTCT TTATAACGAAA-3′. The PCR reaction were performed using PrimeSTAR HS polymerase (Clontech, Mountain View, USA) following manufacturer’s instructions in an iCycler thermal cycler (Bio-Rad, Hercules, USA), with 20 % (v /v ) GC-melt (Clontech, Mountain View, USA) supplementing the xylB reaction. The PCR products were gel-purified and cloned into PCR-Blunt cloning vector (Invitrogen, Carlsbad, USA) following manufacturer’s instructions. The ligation products were transformed into One Shot TOP10 chemically competent cells. Successful ligations were confirmed by sequencing (Genewiz, Cambridge, USA). The resulting Blunt-xylB and Blunt-xynB vectors were digested with Nde I and BamH I, and Nhe I and BamH I,
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Table 1 Bacterial strains and plasmids Strain or plasmid
Relevant characteristics
Source or reference
E.coli LS5218 E. coli JM109
fadR601, atoC(Con) recA1 endA1 gyrA96 thi-1 hsdR17 (rK−mK+) supE44 relA1 l-lac [F′ proAB lacIqZDM15] F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λWild type (ATCC Number: BAA-471) Wild type (ATCC number: 23857) pUC ori, Zeor, Kmr, ccdB Broad-host-range vector, lacPOZ′, Kmr pET21 derivative, AvrII site introduced between SacI and EcoRI sites of polylinker, Ampr pTV118N derivative, M. elsdenii pct, Pseudomonas sp. 61-3 phaC1(ST/QK), R. eutropha phaB, phaA, Ampr pBBR1-MCS2 derivative, B. subtilis xynB, S. coelicolor xlnB, Kmr
Coli Genetic Stock Center Promega
E. coli TOP10TM S. coelicolor B. subtilis pCR-Blunt pBBR1-MCS2 pKH22 pTV118NpctphaC1(ST/QK)AB pBBRXBB2
respectively, and ligated into the respective sites of the pKH22 vector. The pKH-xylB was digested with XbaI and AvrII and ligated into pKH-xynB cut with XbaI to form pKHXBB2. The xylB-xynB fragment was removed by digesting with XbaI and AvrII and inserted into the XbaI site of pBBR1MCS2 to obtain pBBRXBB2. Correct orientation of the construct was confirmed by restriction enzyme digest with BamHI. For cloning of DNA fragments with single restriction sites, digested DNAs interest were treated with Calf Intestine Phosphatase prior to gel purification, and T4 DNA ligase was used for ligations.
Production of PHA in E. coli LS5218 The plasmids pTV118NpctphaC1(ST/QK)AB (referred to as pTVSTQKAB from here on) and pBBRXBB2 were transformed into electrocompetent E. coli LS5218. Successful transformants were selected by growth on LB plates with kanamycin and carbenicillin and by the formation of clear halos on plates containing xylan and isopropyl β-D -1-thiogalactopyranoside (IPTG, 0.1 mM) (Teather and Wood 1982) as a qualitative measure of extracellular xylanase activity. Single colonies were inoculated into 50 mL LB media and grown overnight at 30 °C at 250 rpm in a rotary shaker. One milliliter of overnight culture was transferred into 100 mL of LB supplemented with a single sugar substrate (20 g L−1), or beechwood xylan (10 g L−1), or a combination of both a single sugar substrate (20 g L−1) and xylan (10 g L−1). Kanamycin and carbenicillin were used for plasmid maintenance in all studies. IPTG was added to all experiments to a final concentration of 0.1 mM at an optical density (OD) of 0.6–0.7 at 600 nm. Growth was monitored over the course of 48 h by measuring OD600 at time intervals of 12 h after inoculation in a Genesys 10uv spectrophotometer (Thermo Electron Corporation, Waltham, USA). The single sugar substrates
Invitrogen ATCC ATCC Invitrogen Kovach et al. (1995) Lundgren and Boddy (2007) Taguchi et al. (2008) This study
used were D -xylose (Acros, Waltham, USA) and L -arabinose (Sigma, Saint Louis, USA). Beechwood xylan was purchased from Sigma, Saint Louis, USA. All PHA production experiments were done in triplicate in 500 mL baffled flasks incubated at 30 °C for 48 h in a rotary shaker at 250 rpm. After incubation, the cells were harvested by centrifugation at 4, 000×g for 15 min. The cells were resuspended and washed twice in Nanopure water (Barnstead, Waltham, USA), followed by lyophilization for a minimum of 24 h. Extraction of PHA Dried cells were resuspended in methanol and mixed by vigorous stirring at room temperature for 5–10 min, followed by centrifugation at 4,000×g for 15 min. The samples were washed with Nanopure water and freeze-dried by lyophilization for a minimum of 16 h. Polymers were extracted by soxhlet extraction with 100 mL of refluxing chloroform at 95 °C for 5 h. After refluxing, the samples were cooled to room temperature and the chloroform was completely removed by evaporation in a rotary evaporator. The polymer was washed twice with ice-cold methanol and redissolved in 5–6 mL of chloroform and cast on a glass dish. Gas chromatography analysis PHA production yields and relative monomer composition were determined by gas chromatography (GC). A 15–20-mg sample of dried cells were treated with 2 mL of chloroform and 2 mL of methanol/sulfuric acid solution (85:15) and incubated for 140 min at 100 °C. After the incubation period, the samples were cooled to room temperature and 1 mL of Nanopure water was added, followed by vortexing and separation of the aqueous and organic layers by centrifugation at 750 rpm for 2 min. The organic layer was filtered through a
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0.45-μm polytetrafluoroethylene (PTFE) syringe filter (Restek, Bellefonte, USA). A 0.5-mL portion of filtered sample was mixed with 0.5 mL of caprylic acid (0.1 g L−1) as an internal standard. The parameters for GC analysis were as follows: 1 μL injection volume, 30 m Rtx ®-5 column (Restek, Bellefonte, USA, 0.25 mm id and 0.25 μm df), 45 °C column temperature and a column oven heating profile of 45 °C initial temperature, hold for 7 min, ramp up to 100 °C at 20 °C min−1, ramp up to 200 °C at 5 °C min−1, and increase to final temperature of 280 °C at 30 °C min−1 and hold for 2 min. Analysis was carried by split injection in a GC2010 gas chromatograph with a flame ionization detector (Shimadzu, Kyoto, Japan). Nuclear magnetic resonance analysis Approximately 20 mg of purified polymer was dissolved in 1 mL of deuterated chloroform (Cambridge Isotope Laboratories, Cambridge, USA) and passed through a glass– wool plug to remove impurities. The sample was analyzed at 30 °C in a Bruker AVANCE III 600 spectrometer (600 MHz 1H frequency) equipped with a 5-mm IXI z-gradient probe. Data and spectra were processed in TOPSIN v1.3 from Bruker BioSpin, Billerica, USA. Sugar concentrations in the media were determined using the same parameters. The samples were prepared by collecting approximately 2 mL of media every 12 h, separating the cells by centrifugation and filtering the supernatant through a 0.45-μm PES syringe filter. Glucosamine dissolved in deuterium oxide (Acros, Waltham, USA) was used as a reference compound and added to the samples in a 1:9 ratio to a final concentration of 10 g L−1 (Kiemle et al. 2004) Gel permeation chromatography analysis Determination of weight-average molecular weight (M w), number-average molecular weight (M n), and polydispersity (Đ M =M w /M n) of polymers produced was by gel permeation chromatography (GPC). Samples (∼0.7 mg) of polymer were dissolved in 1 mL of chloroform and filtered through a 0.45-μm PTFE syringe filter into GPC vials. The PGC analysis parameters were as follows: 50 μL injection volume, styrenedivinylbenzene 8×300 mm with 5 μm porosity column (Polymer Standards Service), 40 °C column oven temperature, chloroform mobile phase at 1 mL min−1 flow rate. The analysis was carried in a LC-20 AD Liquid Chromatography with a RID-10A refractive index detector (Shimadzu, Kyoto, Japan).
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and heated to 200 °C at 10 °C min−1, then cooled to −80 °C at 5 °C min−1, followed by temperature increase to 200 °C at 10 °C min−1. Analysis was carried out under nitrogen atmosphere with a flow rate of 50 mL min−1 in a DSC Q200 differential scanning calorimeter, New Castle, USA. Data were selected from the second heating cycle (T g and T m) and the cooling cycle (T c). Determination of xylanase specific activity E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 was inoculated into 50 mL of LB media and grown overnight at 30 °C at 250 rpm in a rotary shaker. One milliliter of overnight culture was transferred into 100 mL of LB supplemented with kanamycin, carbanecilin, and IPTG. At 12 h intervals, 5 mL of liquid culture was removed and placed on ice for 5 min. After cooling, the sample was centrifuged and the supernatant passed through a 0.2-μm PES sterile syringe filter. The filtered supernatant was supplemented with leupeptin and pepstatin-A to a final concentration of 2 μg mL−1 and injected into slyde-a-lyzer dialysis cassettes (Pierce, Waltham, USA) with a 10,000 molecular weight cutoff. The protein samples were dialyzed overnight at 4 °C with constant stirring in phosphate buffered saline solution at a pH of 6.96. The buffer solution was exchanged twice. After overnight dialysis, protein concentrations were determined using the Bradford assay kit (Thermo Scientific, Waltham, USA) following manufacturer’s instructions. The protein solutions were then aliquoted and diluted with F1 salts to five different concentrations following a linear trend (i.e., 80, 60, 40, 20 % of initial concentration). Xylanase activity in total protein was measured by the dinitrosalicylic acid (DNS) method in 96-well plates at 30 °C with a reaction time of 30 min and a reaction volume of 60 μL (30 μL protein solution and 30 μL of 1 % commercial xylan in F1 salts). At the end of the reaction, an equal volume of DNS reagent was added and the mixture was incubated at 95 °C for 5 min (Xiao et al. 2005) in an iCycler thermal cycler (BioRad, Hercules, USA). Development of color was recorded at 532 nm with a Synergy HT microplate reader (BIO-TEK, Winooski, USA). Pure xylose standard curves were generated for each time point. Protein activity was defined as micromole of xylose per minute per milligram of total protein. Composition of F1 salts per liter: 3 g KH2PO4, 6.62 g K2HPO4, 4 g (NH4)2SO4, 0.15 g MgSO4 (Lundgren and Boddy 2007). Composition of DNS reagent per liter was as follows: 14 g DNS, 2.8 g phenol, 0.7 g Na2SO3, 280 g KNaC4H4O6·4H2O, 14 g NaOH.
Differential scanning calorimetry analysis
Results
Glass transition (T g), melting (T m), and crystalinization (T c) temperatures of polymers produced were determined by differential scanning calorimetry (DSC). Samples (∼7 mg) of polymer were loaded onto a TzeroTM aluminum pans (TA Instruments)
Xylan hydrolysis by recombinant E. coli LS5218 Native E. coli cannot utilize xylan as carbon source because it lacks the necessary genes to express xylan-degrading
Appl Microbiol Biotechnol (2014) 98:831–842
enzymes. In order to develop an E. coli strain capable of using xylan as carbon source for growth and PHA production, an endoxylanase gene (xylB ) from S. coelicolor and a βxylosidase gene (xynB) from B. subtilis were cloned into the pBBR1MCS-2 expression vector under the control of the lac promoter. The resulting pBBRXBB2 plasmid was transformed into E. coli LS5218 and preliminary xylan degradation by the recombinant cells was qualitatively confirmed by the formation of clear halos surrounding individual colonies grown on LB plates supplemented with xylan and IPTG. Initially, the appearance of halos was checked for at 30 and 37 °C. The colonies grown at 37 °C showed slightly larger and more defined halos than colonies grown at 30 °C (Fig. S1, supplementary materials). The two xylanases selected for this study have been previously characterized (Jordan et al. 2013; Biely, et al. 1993). However, their synergistic activity when produced by recombinant E. coli could not be assumed to be reflected by the sum of the independent activities. Therefore, the specific total xylanase activity in this system was determined at intervals of 12 h post-inoculation under relevant conditions for optimized PHA production by recombinant E coli LS5218. Since xylanases are secreted proteins, total extracellular protein was assayed using beechwood xylan as a substrate and xylose conversion was quantified by the DNS method as previously described (Xiao et al. 2005). The total specific activity was defined as micromole of xylose converted per minute per milligram of total protein and quantified as the slope of micromole per minute (IU) over milligram of total protein as shown in Fig. 1. Total extracellular protein and xylanase-specific activities at each time interval are shown in Table S1 in supplementary materials. Total protein production increased throughout the growth period; however, protein concentrations were constant between 24 and 36 h. Xylanase
Fig. 1 Specific xylanase activity from E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 grown at 30 °C. Activity was determined at 12 h (circle), 24 h (diamond), 36 h (triangle), and 48 h (square) as the amount of xylose conversion per minute of reaction over total protein concentration (IU per milligram)
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activities remained constant throughout the growth period with measured activities of 6.7 IU mg − 1 at 12 h, 6.2 IU mg−1 at 24 h, 6.7 IU mg−1 at 36 h, and 7.5 IU mg−1 at 48 h. These results demonstrate that functional xylanases are being secreted by recombinant E. coli. PHA production from xylan by E. coli LS5218 In order to obtain a strain that can produce polyhydroxyalkanoates using xylan as a carbon source, we transformed the pTVSTQKAB plasmid into E. coli LS5218 harboring pBBRXBB. E. coli LS5218 was selected for this study because the mutation in the AtoC regulator makes it constitutively expressed which leads to better recycling and uptake of acetate (Spratt et al. 1981). Upon autoclaving, the acetate groups present in xylan are cleaved and acetate concentration in the media increases (Klinke et al. 2004). Therefore, a bacterial strain that can better recycle and uptake acetate is desirable. Furthermore, furfural-derived compounds are another potential byproduct of xylan autoclaving (Klinke et al. 2004) and E. coli LS5218 has been shown to be resistant to inhibition by furfural-derived compounds during PHA production (Nduko et al. 2012b). The plasmid pTVSTQKAB harbors three PHA-producing enzymes, a β-ketothiolase (PhaA), an acetoacetyl-coA reductase (PhaB), and an engineered PHA synthase from Pseudomonas sp. 61-3 harboring a Ser325Th/Gln481Lys mutation [PhaC1Ps(ST/QK)] (Nomura and Taguchi 2007; Takase et al. 2003); it also encodes for the gene for a PCT which grants the system the ability to incorporate lactic acid monomers into PHA to make P(LA-co-3HB). Production of P(LA-co -3HB) using xylan as a carbon source was first attempted by growing the cells in LB supplemented with 1 % (w /v ) xylan at 37 °C for 3 days. This temperature was selected based on the higher activity of the xylanases at 37 °C. Cell mass and PHA yields were minimal reaching 2.1 and 0.025 g L−1, respectively. Based on these results, two control experiments were performed to elucidate the factors affecting production: the cells were grown under the same conditions using xylose or glucose as a substrate for PHA production. As shown in Table 2, this again resulted in low PHA yields suggesting that PHA production at 37 °C is far from optimal. This is suggested to be a result of protein misfolding of the engineered STQK PHA synthase that occurs at 37 °C (K. Matsumoto, personal communication). In order to increase polymer yields, the cells were grown in LB supplemented with 1 % xylan (w /v) or 2 % xylose (w/v) as a control at 30 °C for 48 h. Surprisingly, the yields did not increase significantly for the cells grown in xylan, with cell mass reaching 2.1 g L−1 and a PHA yield of 0.03 g L−1. The xylose control resulted in cell mass and PHA yields of 7.0 and 3.3 g L−1, respectively. All results, including PHA content and composition are summarized on Table 3.
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Table 2 PHA accumulation in E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 grown at 37 °C Sample
1 2 3
Carbon source
Xylan Xylose Glucose
CDW (g L−1)
2.1±0.0 2.8±0.4 3.0±0.1
PHA (wt%)
PHA composition (mol %)
1.1±0.1 1.9±0.2 1.8±0.0
2HP
3HB
ND ND ND
100 100 100
Total polymer yield (g L−1)
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Enhanced production of polyhydroxyalkanoates (PHAs) from beechwood xylan by recombinant Escherichia coli Lucia Salamanca-Cardona & Christopher S. Ashe & Arthur J. Stipanovic & Christopher T. Nomura
Received: 2 October 2013 / Revised: 6 November 2013 / Accepted: 9 November 2013 / Published online: 28 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Microbial conversion of plant biomass to valueadded products is an attractive option to address the impacts of petroleum dependency. In this study, a bacterial system was developed that can hydrolyze xylan and utilize xylan-derived xylose for growth and production of polyhydroxyalkanoates (PHAs). A β-xylosidase and an endoxylanase were engineered into a P(LA-co-3HB)-producing Escherichia coli strain to obtain a xylanolytic strain. Although PHA production yields using xylan as sole carbon source were minimal, when the xylan-based media was supplemented with a single sugar (xylose or arabinose) to permit the accumulation of xylanderived xylose in the media, PHA production yields increased up to 18-fold when compared to xylan-based production, and increased by 37 % when compared to production from single sugar sources alone. 1H-Nuclear magnetic resonance (NMR) analysis shows higher accumulation of xylan-derived xylose in the media when xylan was supplemented with arabinose to prevent xylose uptake by catabolite repression. 1H-NMR, gel permeation chromatography, and differential scanning calorimetry analyses corroborate that the polymers maintain physical properties regardless of the carbon source. This study demonstrates that accumulation of biomass-derived sugars in the media prior to their uptake by microbes is an important Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-5398-4) contains supplementary material, which is available to authorized users. L. Salamanca-Cardona : C. S. Ashe : A. J. Stipanovic : C. T. Nomura (*) Department of Chemistry, State University of New York, College of Environmental Science and Forestry, 318 Jahn Lab, 1 Forestry Dr, Syracuse, NY 13210, USA e-mail: [email protected] C. T. Nomura Center for Applied Microbiology, State University of New York, College of Environmental Science and Forestry, Syracuse 13210 NY, USA
aspect to enhance PHA production when using plant biomass as feedstock. Keywords Polyhydroxyalkanoates . Xylanases . Recombinant Escherichia coli . Xylan . Biomass . Hemicellulose
Introduction Concerns regarding the environmental and economic impacts of petroleum dependency have driven an increased research focus to develop strategies for the production of petroleum-alternative compounds such as bioplastics (polyhydroxyalkanoates, polylactate, polyglycolate) from plant biomass to reduce world dependency on fossil oils. Polyhydroxyalkanoates (PHAs) are a class of polyesters that can be produced natively by various microorganisms as a carbon reservoir during growth under stress conditions (Anderson and Dawes 1990; Sudesh et al. 2000). Poly(3-hydroxyburyrate) (PHB) is the most commonly produced PHA usually requiring only three enzymes: a βketothiolase, an acetoacetyl-CoA reductase, and a PHA synthase for its biosynthesis (Lu et al. 2009). Given the simple machinery required for PHB production, recombinant PHA biosynthesis has flourished in nonnative producers like Escherichia coli due to the ease with which this microorganism can be genetically manipulated (Fidler and Dennis 1992). From this starting point, E. coli has already been modified to produce PHAs with various repeating unit compositions which impart a variety of physical properties and extend the applicability of the bioplastics (Wang et al. 2012; Taguchi et al. 1999; Fukui et al. 1999; Tappel et al. 2012; Tsuge et al. 2003). In 2008, Taguchi et al. reported the production of a new and attractive PHA polymer: poly(lactate-3hydroxybutyrate) [P(LA-co-3HB)] by E. coli (Taguchi et al. 2008). P(LA-co-3HB) polymers exhibit varying degrees of strength, flexibility, and transparency, resulting in improved
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physical properties dependent on their lactic acid (LA) monomer fraction (Yamada et al. 2011). By using xylose as a carbon source instead of glucose, the LA content in P(LA-co-3HB) could be increased 5-fold (Nduko et al. 2012a; Taguchi et al. 2008). These results indicate that xylose has great potential as a carbon substrate for the production of this new class of biopolymers. Xylose is the second most abundant plant biomassderived sugar and the major component of the hemicellulose xylan (Sjostrom 1993). Therefore, plant biomass-derived xylose for PHA production is a promising strategy to achieve lower production costs and better material properties. Production of PHAs from plant biomass has been reported in a variety of wild type and recombinant microorganisms (Pan et al. 2012; Keenan et al. 2006; Nduko et al. 2012b; Reddy et al. 2003). However, these systems require the biomass to be hydrolyzed in a step prior to fermentation through dilute acid or enzymatic hydrolysis to breakdown the polysaccharides and make the sugars available in a form that can be readily taken up by the microorganism (Iranmahboob et al. 2002). The hydrolysis process is often carried out at high temperatures and low pH, conditions which are not only prohibitive for E. coli growth, but also result in the generation of compounds which can be potentially toxic and prevent downstream fermentative processes, thus lowering yields and increasing production costs (Palmqvist and HahnHägerdal 2000a, b; Larsson et al. 1999). Therefore, an approach that couples the hydrolysis and fermentative steps is an attractive strategy for the production of PHAs from plantderived carbon sources like xylose. E. coli cannot natively utilize xylan as a carbon source since it lacks the genes necessary to express the hydrolytic enzymes, xylanases. Based on the chemical composition, two enzymes are required to hydrolyze xylan to its constitutive monomers: endoxylanases and β-xylosidases (Subramaniyan and Prema 2002). Endoxylanases break down the xylopyranose backbone in xylan into oligomer fragments consisting of two or three xylose units (xylobiose and xylotriose, respectively) (Poutanen et al. 1991). For fermentation purposes, the oligomer fragments cannot be utilized by E. coli; thus, xylobiose and xylotriose fragments must be degraded to monomeric sugars by the action of β-xylosidases (Poutanen et al. 1991; John et al. 1979). In this study, E coli LS5218 was genetically engineered to express an endoxylanase (XylB) from Streptomyces coelicolor and a β-xylosidase (XynB) from Bacillus subtilis in order to utilize xylan. This strain was additionally designed to express the PHA synthase from Pseudomonas sp. 61-3 harboring a Ser325Th/Gln481Lys mutation [PhaC1Ps(ST/ QK)], a propionyl- CoA transferase (PCT) from Megasphaera elsdenii , and a β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha under a R. eutropha constitutive promoter (Taguchi et al. 2008) in order to produce PHA polymers. The
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co-expression of the xylanase system and the P(LA-co3HB) system in E. coli resulted in the production of xylan-derived P(LA-co-3HB). Therefore, this study demonstrates for the first time production of PHA polymers by E. coli using xylan as a carbon source.
Materials and methods Media and cultivation conditions All plasmids and strains used in this study are listed in Table 1. E. coli and B. subtilis strains used for cloning were grown on Lennox Broth (LB, DifcoTM) at 37 °C in a rotary shaker at 250 rpm. Carbenicillin (100 μg mL−1) and kanamycin (50 μg mL−1) were supplemented as needed per plasmid requirements. S. coelicolor BAA-471 isolates were selected from cells grown in DifcoTM ISP medium two plates at 30 °C for 48 h. For genomic DNA extraction, isolated colonies were grown in YEME medium (composition per liter: 3 g yeast extract, 5 g Bacto-peptone, 3 g malt extract, 10 g glucose, 170 g sucrose) at 30 °C in a rotary shaker at 250 rpm. Construction of pBBRXBB2 vector for recombinant xylanase production in E. coli Restriction enzymes, CIP, and T4 DNA ligase were purchased from New England Biolabs, Ipswich, USA. All transformations and plasmid manipulations were performed in E. coli JM109 following standard protocols (Sambrook et al. 2001) unless otherwise specified. Genomic DNA was purified from B. subtilis and S. coelicolor with a Wizard genomic DNA purification kit (PROMEGA, Fitchburg, USA). Genes encoding for one endoxylanase (xylB) and one β-xylosidase (xynB) were PCR-amplified from S. coelicolor and B. subtilis, respectively, with the following primers (Integrated DNA Technologies, Coralvile, USA): xylB-F: 5′-CATATGCTGC TCGTCCAGCCGA-3′; xylB-R: 5′-GGATCCCCGCCCGC GCTGCAGGACA-3′; xynB-F: 5′-GCTAGC ATGAAGAT TACCAATCCCG-3′; xynB-R: 5′-GGATCC TTATTTTTCT TTATAACGAAA-3′. The PCR reaction were performed using PrimeSTAR HS polymerase (Clontech, Mountain View, USA) following manufacturer’s instructions in an iCycler thermal cycler (Bio-Rad, Hercules, USA), with 20 % (v /v ) GC-melt (Clontech, Mountain View, USA) supplementing the xylB reaction. The PCR products were gel-purified and cloned into PCR-Blunt cloning vector (Invitrogen, Carlsbad, USA) following manufacturer’s instructions. The ligation products were transformed into One Shot TOP10 chemically competent cells. Successful ligations were confirmed by sequencing (Genewiz, Cambridge, USA). The resulting Blunt-xylB and Blunt-xynB vectors were digested with Nde I and BamH I, and Nhe I and BamH I,
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Table 1 Bacterial strains and plasmids Strain or plasmid
Relevant characteristics
Source or reference
E.coli LS5218 E. coli JM109
fadR601, atoC(Con) recA1 endA1 gyrA96 thi-1 hsdR17 (rK−mK+) supE44 relA1 l-lac [F′ proAB lacIqZDM15] F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λWild type (ATCC Number: BAA-471) Wild type (ATCC number: 23857) pUC ori, Zeor, Kmr, ccdB Broad-host-range vector, lacPOZ′, Kmr pET21 derivative, AvrII site introduced between SacI and EcoRI sites of polylinker, Ampr pTV118N derivative, M. elsdenii pct, Pseudomonas sp. 61-3 phaC1(ST/QK), R. eutropha phaB, phaA, Ampr pBBR1-MCS2 derivative, B. subtilis xynB, S. coelicolor xlnB, Kmr
Coli Genetic Stock Center Promega
E. coli TOP10TM S. coelicolor B. subtilis pCR-Blunt pBBR1-MCS2 pKH22 pTV118NpctphaC1(ST/QK)AB pBBRXBB2
respectively, and ligated into the respective sites of the pKH22 vector. The pKH-xylB was digested with XbaI and AvrII and ligated into pKH-xynB cut with XbaI to form pKHXBB2. The xylB-xynB fragment was removed by digesting with XbaI and AvrII and inserted into the XbaI site of pBBR1MCS2 to obtain pBBRXBB2. Correct orientation of the construct was confirmed by restriction enzyme digest with BamHI. For cloning of DNA fragments with single restriction sites, digested DNAs interest were treated with Calf Intestine Phosphatase prior to gel purification, and T4 DNA ligase was used for ligations.
Production of PHA in E. coli LS5218 The plasmids pTV118NpctphaC1(ST/QK)AB (referred to as pTVSTQKAB from here on) and pBBRXBB2 were transformed into electrocompetent E. coli LS5218. Successful transformants were selected by growth on LB plates with kanamycin and carbenicillin and by the formation of clear halos on plates containing xylan and isopropyl β-D -1-thiogalactopyranoside (IPTG, 0.1 mM) (Teather and Wood 1982) as a qualitative measure of extracellular xylanase activity. Single colonies were inoculated into 50 mL LB media and grown overnight at 30 °C at 250 rpm in a rotary shaker. One milliliter of overnight culture was transferred into 100 mL of LB supplemented with a single sugar substrate (20 g L−1), or beechwood xylan (10 g L−1), or a combination of both a single sugar substrate (20 g L−1) and xylan (10 g L−1). Kanamycin and carbenicillin were used for plasmid maintenance in all studies. IPTG was added to all experiments to a final concentration of 0.1 mM at an optical density (OD) of 0.6–0.7 at 600 nm. Growth was monitored over the course of 48 h by measuring OD600 at time intervals of 12 h after inoculation in a Genesys 10uv spectrophotometer (Thermo Electron Corporation, Waltham, USA). The single sugar substrates
Invitrogen ATCC ATCC Invitrogen Kovach et al. (1995) Lundgren and Boddy (2007) Taguchi et al. (2008) This study
used were D -xylose (Acros, Waltham, USA) and L -arabinose (Sigma, Saint Louis, USA). Beechwood xylan was purchased from Sigma, Saint Louis, USA. All PHA production experiments were done in triplicate in 500 mL baffled flasks incubated at 30 °C for 48 h in a rotary shaker at 250 rpm. After incubation, the cells were harvested by centrifugation at 4, 000×g for 15 min. The cells were resuspended and washed twice in Nanopure water (Barnstead, Waltham, USA), followed by lyophilization for a minimum of 24 h. Extraction of PHA Dried cells were resuspended in methanol and mixed by vigorous stirring at room temperature for 5–10 min, followed by centrifugation at 4,000×g for 15 min. The samples were washed with Nanopure water and freeze-dried by lyophilization for a minimum of 16 h. Polymers were extracted by soxhlet extraction with 100 mL of refluxing chloroform at 95 °C for 5 h. After refluxing, the samples were cooled to room temperature and the chloroform was completely removed by evaporation in a rotary evaporator. The polymer was washed twice with ice-cold methanol and redissolved in 5–6 mL of chloroform and cast on a glass dish. Gas chromatography analysis PHA production yields and relative monomer composition were determined by gas chromatography (GC). A 15–20-mg sample of dried cells were treated with 2 mL of chloroform and 2 mL of methanol/sulfuric acid solution (85:15) and incubated for 140 min at 100 °C. After the incubation period, the samples were cooled to room temperature and 1 mL of Nanopure water was added, followed by vortexing and separation of the aqueous and organic layers by centrifugation at 750 rpm for 2 min. The organic layer was filtered through a
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0.45-μm polytetrafluoroethylene (PTFE) syringe filter (Restek, Bellefonte, USA). A 0.5-mL portion of filtered sample was mixed with 0.5 mL of caprylic acid (0.1 g L−1) as an internal standard. The parameters for GC analysis were as follows: 1 μL injection volume, 30 m Rtx ®-5 column (Restek, Bellefonte, USA, 0.25 mm id and 0.25 μm df), 45 °C column temperature and a column oven heating profile of 45 °C initial temperature, hold for 7 min, ramp up to 100 °C at 20 °C min−1, ramp up to 200 °C at 5 °C min−1, and increase to final temperature of 280 °C at 30 °C min−1 and hold for 2 min. Analysis was carried by split injection in a GC2010 gas chromatograph with a flame ionization detector (Shimadzu, Kyoto, Japan). Nuclear magnetic resonance analysis Approximately 20 mg of purified polymer was dissolved in 1 mL of deuterated chloroform (Cambridge Isotope Laboratories, Cambridge, USA) and passed through a glass– wool plug to remove impurities. The sample was analyzed at 30 °C in a Bruker AVANCE III 600 spectrometer (600 MHz 1H frequency) equipped with a 5-mm IXI z-gradient probe. Data and spectra were processed in TOPSIN v1.3 from Bruker BioSpin, Billerica, USA. Sugar concentrations in the media were determined using the same parameters. The samples were prepared by collecting approximately 2 mL of media every 12 h, separating the cells by centrifugation and filtering the supernatant through a 0.45-μm PES syringe filter. Glucosamine dissolved in deuterium oxide (Acros, Waltham, USA) was used as a reference compound and added to the samples in a 1:9 ratio to a final concentration of 10 g L−1 (Kiemle et al. 2004) Gel permeation chromatography analysis Determination of weight-average molecular weight (M w), number-average molecular weight (M n), and polydispersity (Đ M =M w /M n) of polymers produced was by gel permeation chromatography (GPC). Samples (∼0.7 mg) of polymer were dissolved in 1 mL of chloroform and filtered through a 0.45-μm PTFE syringe filter into GPC vials. The PGC analysis parameters were as follows: 50 μL injection volume, styrenedivinylbenzene 8×300 mm with 5 μm porosity column (Polymer Standards Service), 40 °C column oven temperature, chloroform mobile phase at 1 mL min−1 flow rate. The analysis was carried in a LC-20 AD Liquid Chromatography with a RID-10A refractive index detector (Shimadzu, Kyoto, Japan).
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and heated to 200 °C at 10 °C min−1, then cooled to −80 °C at 5 °C min−1, followed by temperature increase to 200 °C at 10 °C min−1. Analysis was carried out under nitrogen atmosphere with a flow rate of 50 mL min−1 in a DSC Q200 differential scanning calorimeter, New Castle, USA. Data were selected from the second heating cycle (T g and T m) and the cooling cycle (T c). Determination of xylanase specific activity E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 was inoculated into 50 mL of LB media and grown overnight at 30 °C at 250 rpm in a rotary shaker. One milliliter of overnight culture was transferred into 100 mL of LB supplemented with kanamycin, carbanecilin, and IPTG. At 12 h intervals, 5 mL of liquid culture was removed and placed on ice for 5 min. After cooling, the sample was centrifuged and the supernatant passed through a 0.2-μm PES sterile syringe filter. The filtered supernatant was supplemented with leupeptin and pepstatin-A to a final concentration of 2 μg mL−1 and injected into slyde-a-lyzer dialysis cassettes (Pierce, Waltham, USA) with a 10,000 molecular weight cutoff. The protein samples were dialyzed overnight at 4 °C with constant stirring in phosphate buffered saline solution at a pH of 6.96. The buffer solution was exchanged twice. After overnight dialysis, protein concentrations were determined using the Bradford assay kit (Thermo Scientific, Waltham, USA) following manufacturer’s instructions. The protein solutions were then aliquoted and diluted with F1 salts to five different concentrations following a linear trend (i.e., 80, 60, 40, 20 % of initial concentration). Xylanase activity in total protein was measured by the dinitrosalicylic acid (DNS) method in 96-well plates at 30 °C with a reaction time of 30 min and a reaction volume of 60 μL (30 μL protein solution and 30 μL of 1 % commercial xylan in F1 salts). At the end of the reaction, an equal volume of DNS reagent was added and the mixture was incubated at 95 °C for 5 min (Xiao et al. 2005) in an iCycler thermal cycler (BioRad, Hercules, USA). Development of color was recorded at 532 nm with a Synergy HT microplate reader (BIO-TEK, Winooski, USA). Pure xylose standard curves were generated for each time point. Protein activity was defined as micromole of xylose per minute per milligram of total protein. Composition of F1 salts per liter: 3 g KH2PO4, 6.62 g K2HPO4, 4 g (NH4)2SO4, 0.15 g MgSO4 (Lundgren and Boddy 2007). Composition of DNS reagent per liter was as follows: 14 g DNS, 2.8 g phenol, 0.7 g Na2SO3, 280 g KNaC4H4O6·4H2O, 14 g NaOH.
Differential scanning calorimetry analysis
Results
Glass transition (T g), melting (T m), and crystalinization (T c) temperatures of polymers produced were determined by differential scanning calorimetry (DSC). Samples (∼7 mg) of polymer were loaded onto a TzeroTM aluminum pans (TA Instruments)
Xylan hydrolysis by recombinant E. coli LS5218 Native E. coli cannot utilize xylan as carbon source because it lacks the necessary genes to express xylan-degrading
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enzymes. In order to develop an E. coli strain capable of using xylan as carbon source for growth and PHA production, an endoxylanase gene (xylB ) from S. coelicolor and a βxylosidase gene (xynB) from B. subtilis were cloned into the pBBR1MCS-2 expression vector under the control of the lac promoter. The resulting pBBRXBB2 plasmid was transformed into E. coli LS5218 and preliminary xylan degradation by the recombinant cells was qualitatively confirmed by the formation of clear halos surrounding individual colonies grown on LB plates supplemented with xylan and IPTG. Initially, the appearance of halos was checked for at 30 and 37 °C. The colonies grown at 37 °C showed slightly larger and more defined halos than colonies grown at 30 °C (Fig. S1, supplementary materials). The two xylanases selected for this study have been previously characterized (Jordan et al. 2013; Biely, et al. 1993). However, their synergistic activity when produced by recombinant E. coli could not be assumed to be reflected by the sum of the independent activities. Therefore, the specific total xylanase activity in this system was determined at intervals of 12 h post-inoculation under relevant conditions for optimized PHA production by recombinant E coli LS5218. Since xylanases are secreted proteins, total extracellular protein was assayed using beechwood xylan as a substrate and xylose conversion was quantified by the DNS method as previously described (Xiao et al. 2005). The total specific activity was defined as micromole of xylose converted per minute per milligram of total protein and quantified as the slope of micromole per minute (IU) over milligram of total protein as shown in Fig. 1. Total extracellular protein and xylanase-specific activities at each time interval are shown in Table S1 in supplementary materials. Total protein production increased throughout the growth period; however, protein concentrations were constant between 24 and 36 h. Xylanase
Fig. 1 Specific xylanase activity from E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 grown at 30 °C. Activity was determined at 12 h (circle), 24 h (diamond), 36 h (triangle), and 48 h (square) as the amount of xylose conversion per minute of reaction over total protein concentration (IU per milligram)
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activities remained constant throughout the growth period with measured activities of 6.7 IU mg − 1 at 12 h, 6.2 IU mg−1 at 24 h, 6.7 IU mg−1 at 36 h, and 7.5 IU mg−1 at 48 h. These results demonstrate that functional xylanases are being secreted by recombinant E. coli. PHA production from xylan by E. coli LS5218 In order to obtain a strain that can produce polyhydroxyalkanoates using xylan as a carbon source, we transformed the pTVSTQKAB plasmid into E. coli LS5218 harboring pBBRXBB. E. coli LS5218 was selected for this study because the mutation in the AtoC regulator makes it constitutively expressed which leads to better recycling and uptake of acetate (Spratt et al. 1981). Upon autoclaving, the acetate groups present in xylan are cleaved and acetate concentration in the media increases (Klinke et al. 2004). Therefore, a bacterial strain that can better recycle and uptake acetate is desirable. Furthermore, furfural-derived compounds are another potential byproduct of xylan autoclaving (Klinke et al. 2004) and E. coli LS5218 has been shown to be resistant to inhibition by furfural-derived compounds during PHA production (Nduko et al. 2012b). The plasmid pTVSTQKAB harbors three PHA-producing enzymes, a β-ketothiolase (PhaA), an acetoacetyl-coA reductase (PhaB), and an engineered PHA synthase from Pseudomonas sp. 61-3 harboring a Ser325Th/Gln481Lys mutation [PhaC1Ps(ST/QK)] (Nomura and Taguchi 2007; Takase et al. 2003); it also encodes for the gene for a PCT which grants the system the ability to incorporate lactic acid monomers into PHA to make P(LA-co-3HB). Production of P(LA-co -3HB) using xylan as a carbon source was first attempted by growing the cells in LB supplemented with 1 % (w /v ) xylan at 37 °C for 3 days. This temperature was selected based on the higher activity of the xylanases at 37 °C. Cell mass and PHA yields were minimal reaching 2.1 and 0.025 g L−1, respectively. Based on these results, two control experiments were performed to elucidate the factors affecting production: the cells were grown under the same conditions using xylose or glucose as a substrate for PHA production. As shown in Table 2, this again resulted in low PHA yields suggesting that PHA production at 37 °C is far from optimal. This is suggested to be a result of protein misfolding of the engineered STQK PHA synthase that occurs at 37 °C (K. Matsumoto, personal communication). In order to increase polymer yields, the cells were grown in LB supplemented with 1 % xylan (w /v) or 2 % xylose (w/v) as a control at 30 °C for 48 h. Surprisingly, the yields did not increase significantly for the cells grown in xylan, with cell mass reaching 2.1 g L−1 and a PHA yield of 0.03 g L−1. The xylose control resulted in cell mass and PHA yields of 7.0 and 3.3 g L−1, respectively. All results, including PHA content and composition are summarized on Table 3.
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Table 2 PHA accumulation in E. coli LS5218 harboring pTVSTQKAB and pBBRXBB2 grown at 37 °C Sample
1 2 3
Carbon source
Xylan Xylose Glucose
CDW (g L−1)
2.1±0.0 2.8±0.4 3.0±0.1
PHA (wt%)
PHA composition (mol %)
1.1±0.1 1.9±0.2 1.8±0.0
2HP
3HB
ND ND ND
100 100 100
Total polymer yield (g L−1)
