Construction and expression of an ethanol production operon in Gram-positive bacteria

Lee A. Talarico, Malgorzata A. Gil, Lorraine P. Yomano, Lonnie O. Ingram and Julie A. Maupin-Furlow

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA

Correspondence
Julie A. Maupin-Furlow
jmaupin{at}ufl.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.


Abbreviations: ADH, alcohol dehydrogenase; PDC, pyruvate decarboxylase; PET, production of ethanol


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pyruvate decarboxylase (PDC, EC 4.1.1.1) is central to homoethanol fermentation and catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. Acetaldehyde generated from this reaction is reduced to ethanol by alcohol dehydrogenase (ADH, EC 1.1.1.1). These two enzymes (PDC and ADH) are sufficient to convert intracellular pools of pyruvate and NADH to ethanol. Currently, the portable production of ethanol (PET) operon used to engineer this pathway in Gram-negative bacteria consists of the pdc and adh genes from the Gram-negative Zymomonas mobilis (Zm) (Ingram et al., 1987, 1998; Ohta et al., 1991a, b). While this strategy has been highly successful in engineering mesophilic Gram-negative bacteria (Ingram et al., 1998, 1999), advanced biocatalysts that withstand low pH, high temperature, high salt, high sugar, high ethanol and various other harsh conditions offer an opportunity to improve the commercial competitiveness of ethanol production (Ingram et al., 1999; Dien et al., 2003). Many of these qualities can be found in Gram-positive bacteria (Gold et al., 1992). Unfortunately, modifying Gram-positive bacteria for ethanol production has had limited success. Ethanol production appears to be limited by the relatively low levels of activities obtained when the Z. mobilis adh and pdc genes are engineered into lactic acid bacteria and Bacillus species (Barbosa & Ingram, 1994; Gold et al., 1996; Hillman et al., 1996; Nichols et al., 2003).

Engineering a Gram-positive host for robust ethanol production has been limited, in part, by the availability of a suitable pool of pdc genes. Although pdc genes are widespread in plants, yeast and fungi, they are absent in animals and rare in bacteria (König, 1998). The Z. mobilis pdc gene has been the workhorse for Gram-negative biocatalysts. Recently, however, the gene sequences encoding functional PDCs from three additional bacteria have become available, including that of the Gram-positive Sarcina ventriculi (Sv), and Gram-negative Acetobacter pasteurianus and Zymobacter palmae (Talarico et al., 2001; Raj et al., 2001, 2002). Expression of these genes in recombinant Escherichia coli results in different levels of PDC protein; in particular, the levels of S. ventriculi PDC are reduced in comparison to the other PDCs (Talarico et al., 2001; Raj et al., 2001, 2002). This deficiency in SvPDC protein is remedied by the inclusion of accessory tRNA genes (Talarico et al., 2001; Raj et al., 2002). Thus, the source of a pdc gene influences the levels of protein that are obtained in recombinant E. coli, which suggests that codon usage needs to be considered when engineering high-level PDC synthesis in Gram-positive hosts.

In this study, we have compared the expression of four different pdc genes in a recombinant Gram-positive bacterium, Bacillus megaterium. Expression was highest for the pdc from S. ventriculi. By coupling the gene to Geobacillus stearothermophilus adh within a single operon, high levels of both enzymes were produced in active forms. Extracts from these cells converted pyruvate to ethanol, demonstrating the engineering of a functional pathway in a Gram-positive host.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
Biochemicals were purchased from Sigma. Other organic and inorganic analytical-grade chemicals were from Fisher Scientific. Restriction enzymes were from New England Biolabs. Oligonucleotides were from Qiagen Operon and Integrated DNA Technologies. Bacillus megaterium Protein Expression System was from MoBiTec. RNase-free water and solutions were from Ambion.

Bacterial strains and media.
Strains and plasmids used in this study are listed in Table 1. E. coli DH5{alpha} was used for routine recombinant DNA experiments. B. megaterium WH320 and derivatives were used for analysis of recombinant PDC protein and pdc-specific mRNA. Strains were grown in Luria–Bertani (LB) medium, unless otherwise indicated. Medium was supplemented with 2 % (w/v) glucose and antibiotics (100 mg ampicillin l–1, 30 mg kanamycin l–1 or 15 mg tetracycline l–1) as needed. All strains were grown at 37 °C and 200 r.p.m. Isolated colonies of B. megaterium were grown overnight in liquid medium and used as a 1·0 % (v/v) inoculum into fresh medium, unless otherwise indicated.


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Table 1. Strains, plasmids and primers

 
Protoplast formation and transformation of B. megaterium.
B. megaterium WH320 was grown to exponential phase to an OD600 of 0·6 (1 cm path length), and protoplasts were generated according to the method of Puyet et al. (1987), with the following modifications. Cells were treated with lysozyme (10 µg ml–1) for 20 min. Protoplasts were stored at –70 °C and transformed according to MoBiTec.

DNA isolation and cloning.
Plasmid DNA was isolated and purified from E. coli using the QIAprep Spin Miniprep Kit (Qiagen). DNA was eluted from 0·8 % (w/v) SeaKem GTG agarose (Cambrex Corp.) gels using the QIAquick gel elution kit (Qiagen). Similar strategies were used to generate the B. megaterium expression plasmids pJAM420, pJAM430, pJAM432 and pJAM435 (Fig. 1; Table 1). As an example, plasmid pJAM420 was constructed as follows. A BspHI–XhoI DNA fragment with the complete pdc gene of S. ventriculi strain Goodsir (ATCC 55887) was generated by PCR amplification from plasmid pJAM410 and cloned into the NcoI and XhoI sites of plasmid pET21d (Talarico et al., 2001). The 1·9 kb XbaI–BspEI DNA fragment of the resulting pJAM419 was ligated into the SpeI and XmaI sites of plasmid pWH1520. This resulted in a pWH1520-based expression plasmid (pJAM420) that carried the pdc gene, along with the Shine–Dalgarno site and T7 transcriptional terminator of the original pET21d vector. The pdc gene was positioned to interrupt the B. megaterium xylA gene of plasmid pWH1520 and to generate a stop codon within xylA (xylA'). The Shine–Dalgarno site, originally from pET21d and upstream of the inserted pdc, was positioned directly downstream of the xylA' stop codon. This allowed for translational coupling of xylA' and pdc, in which the ribosomes would terminate at the stop codon of xylA' and reinitiate at the start codon of pdc. Sources of the pdc genes of plasmids pJAM430, pJAM432 and pJAM435 originated from A. pasteurianus strain NCIB 8619 (ATCC 12874) (Raj et al., 2001), Z. mobilis strain CP4 (Conway et al., 1987) and PDC1 of Saccharomyces cerevisiae (Wei et al., 2002) (Table 1). The fidelity of all cloned PCR products was confirmed by DNA sequencing by the Sanger dideoxy method (Sanger et al., 1977) using a LICOR sequencer (DNA Sequencing Facility, Department of Microbiology and Cell Science, University of Florida).



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Fig. 1. Strategy used to construct plasmids for expression of S. ventriculi pdc in recombinant B. megaterium. A similar approach was used to generate plasmids for expression of Z. mobilis, A. pasteurianus, and Sac. cerevisiae pdc genes in B. megaterium. Abbreviations: Apr, ampicillin resistance; Tcr, tetracycline resistance; pT7 and T7T, T7 polymerase promoter and terminator, respectively; lacI, gene encoding lactose operon repressor; lacO, lactose operon operator; f1ori, filamentous bacteriophage f1 origin of replication; pBR ori, E. coli plasmid pBR322 origin of replication; pBC16 ori, Bacillus cereus plasmid pBC16 origin of replication; Pxyl, xylA promoter; xylR, gene encoding xylose repressor.

 
A Gram-positive PET operon was constructed as follows. The HindIII–MfeI fragment of pLOI1742 containing the adh gene from G. stearothermophilus (Gsadh) strain XL-65-6 was blunt-end ligated into the BlpI site of pJAM420 using Vent DNA Polymerase (New England Biolabs) (Table 1). This resulted in plasmid pJAM423, which was designed to couple the translation of Svpdc to Gsadh, with the xylA' promoter upstream of Svpdc and the T7 terminator following Gsadh.

Recombinant protein synthesis, protein electrophoresis and enzyme assays.
PDC and ADH proteins were synthesized in recombinant B. megaterium using the expression plasmids described above. Cells were grown to an OD600 of 0·3 (exponential phase), and transcription was induced from the xylA' promoter by addition of 0·5 % (w/v) xylose for 3 h. Cells were harvested by centrifugation (5000 g, 10 min, 4 °C) and stored at –80 °C. For PDC activity assays, cell pellets (0·5 g) were thawed in 6 vols (wet w/v) of 50 mM sodium phosphate buffer, pH 6·5, containing 1 mM MgSO4 and 1 mM thiamine pyrophosphate (TPP) (Buffer A). Cells were passed through a French pressure cell at 20 000 p.s.i. (138 MPa). For ethanol production and ADH activity assays, cells (from 100 ml culture) were resuspended in 3 ml Buffer A and lysed by sonication. Debris was removed by centrifugation (16 000 g, 20 min, 4 °C).

PDC activity was assayed by monitoring the pyruvic acid-dependent oxidation of NADH with ADH as a coupling enzyme at pH 6·5, as previously described (Conway et al., 1987). Cell lysate (10 µl) was added to a final volume of 1 ml containing 0·15 mM NADH, 0·1 mM TPP, 50 mM pyruvate and 10 U ADH in 50 mM K-MES buffer, pH 6·5, with 5 mM MgCl2. Control reactions without added ADH were performed to correct for NADH-oxidizing enzymes, such as lactate dehydrogenase, in cell lysate. ADH was assayed by monitoring the ethanol-dependent reduction of NAD+, as described elsewhere (Dekker, 1977). Cell lysate (10–30 µl) was added to a final volume of 1 ml containing 333 mM ethanol and 8·3 mM NAD+ in 50 mM sodium phosphate buffer, pH 6·5. NADH oxidation/NAD+ reduction was monitored in a 1 cm path length cuvette at 340 nm over a 5 min period using a SmartSpec 300 spectrophotometer (Bio-Rad). One unit of PDC/ADH activity is defined as the generation of 1 µmol NAD+/NADH per min under the conditions specified. Protein concentration was determined using Bio-Rad Protein assay dye with BSA as standard.

Ethanol production was monitored by gas chromatography as previously described (Beall et al., 1991). Cell lysate (1·25 mg protein) was added to a final volume of 1 ml containing 40 mM NADH and carbon substrate in Buffer A and incubated in screw cap tubes (37 °C, 15 h). Carbon substrates included 150 mM pyruvate, 2 % (w/v) xylose and 2 % (w/v) glucose. The concentration of ethanol in aliquots of the reactions was determined by comparison to ethanol standards (0·1–10 g l–1) with 1 % (v/v) 1-propanol as the internal standard. Formaldehyde was included at 0·05 % (v/v) to minimize bacterial growth in the standards.

Protein molecular masses were analysed by reducing and denaturing SDS-PAGE using 12 % polyacrylamide gels that were stained by heating in the presence of Coomassie blue R-250 (Wong, 2000). Molecular mass standards were phosphorylase b (97·4 kDa), serum albumin (66·2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21·5 kDa) and lysozyme (14·4 kDa).

RNA isolation and quantification.
B. megaterium strains expressing the various pdc genes were grown in triplicate to an OD600 of 0·3, and recombinant transcription was induced for 15 min as described above. Total RNA was isolated using the RNeasy miniprep kit. Samples were treated with lysozyme and On-column DNase, as recommended by the supplier (Qiagen). Removal of DNA was confirmed by PCR using Jumpstart Taq Readymix in the absence of reverse transcriptase (Sigma). Quality and quantity of RNA were assessed by 0·8 % (w/v) agarose gel electrophoresis and absorbance at 260 nm, respectively.

Total RNA (100 pg) served as template with the primers listed in Table 1 for quantitative real-time reverse transcriptase PCR (RT-PCR). Transcripts synthesized in vitro were used to generate standard curves of absolute copy number for each RT-PCR experiment. In vitro transcripts were generated from the E. coli pdc-expression vectors (pJAM419, pJAM429, pJAM431 and pScPDC1) with the MAXIscript T7 in vitro transcription kit (Ambion). Nuc-Away spin columns (Ambion) were used to remove unincorporated nucleotides. RT-PCR was performed using the QuantiTect SYBR Green 1-step RT-PCR kit (Qiagen) with an Icycler thermal cycler (Bio-Rad). Data with PCR efficiencies of 90–100 % were analysed using the Icycler software version 3.0.6070 (Bio-Rad) and Microsoft Excel.

Pulse–chase.
Recombinant B. megaterium strains expressing Svpdc and Zmpdc genes were grown in minimal medium (10 g sucrose, 2·5 g K2HPO4, 2·5 g KH2PO4, 1·0 g (NH4)2HPO4, 0·2 g MgSO4.7H2O, 10 mg FeSO4.7H2O, 7 mg MnSO4.H2O in 985 ml deionized H2O at pH 7·0) supplemented with tetracycline (MM Tet) using a 1 % (v/v) inoculum. Cells were grown to an OD600 of 0·3 and recombinant gene transcription was induced for 15 min with 0·5 % xylose. Cells were harvested by centrifugation (5000 g, 10 min, 25 °C) and resuspended in 2 ml MM Tet supplemented with 0·5 % xylose and 50 µCi ml–1 (1·85 MBq ml–1) L-[35S]methionine (DuPont-NEN). Cells were incubated for 15 min (37 °C, 200 r.p.m.) and harvested as above. Cell pellets were resuspended in MM Tet supplemented with 0·5 % xylose and 5 mM L-methionine, with 15 mg chloramphenicol l–1, and incubated (37 °C, 200 r.p.m.). Aliquots (0·5 ml) were withdrawn after 5, 10, 15, 30, 60, 90, 120, 150 and 180 min incubation and immediately added to 50 µl stop solution (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 7·5, and 1 mg chloramphenicol ml–1). Cells were incubated on ice (5 min), harvested at 16 000 g (10 min, 25 °C) and stored at –80 °C. Cell pellets were subjected to three cycles of freeze–thaw (–80 °C and 0 °C) to weaken the cell membrane. Pellets were resuspended in lysis solution (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 7·5, and 0·2 mg lysozyme ml–1) and incubated (25 °C, 15 min). Samples (0·02 OD600 units per lane) were boiled (20 min) in SDS-PAGE loading dye (Bio-Rad) and separated by SDS-PAGE. Gels were dried and exposed to X-ray film. A VersaDoc model 1000 with Quantity One Software (Bio-Rad) was used for densitometric readings.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of Gram-positive PDC expression plasmids
Our previous work suggested that codon usage influences the levels of PDC obtained in recombinant E. coli (Talarico et al., 2001). To determine if this was the factor responsible for limiting PDC expression in Gram-positive bacteria, four PDC genes with different G+C content and codon usage were chosen for expression analysis. These included the S. ventriculi pdc gene (Svpdc), which is poorly expressed in E. coli and is the only known PDC from a Gram-positive bacterium. In addition, the Sac. cerevisiae PDC1 (ScPDC1) was analysed based on its close relationship to SvPDC (Talarico et al., 2001; Raj et al., 2001) and its use in corn-to-ethanol production (Dien et al., 2002). The A. pasteurianus pdc (Appdc) (Raj et al., 2001) and Z. mobilis pdc (Zmpdc) were also included (Hoppner & Doelle, 1983; Braü & Sahm, 1986; Bringer-Meyer et al., 1986; Conway et al., 1987; Neale et al., 1987). The latter two genes are from Gram-negative bacteria and have high levels of expression and activity in Gram-negative hosts (Raj et al., 2001, 2002).

To construct the expression plasmids, the pdc genes were initially cloned into pET vectors (Fig. 1, Table 1). DNA fragments containing each pdc gene and the Shine–Dalgarno and T7 terminator from the pET vector were cloned into the B. megaterium expression plasmid pWH1520. This generated a truncation of the xylA gene (xylA'), which encodes xylose isomerase and allowed for induction of pdc expression by xylose in B. megaterium.

Expression of PDC in recombinant B. megaterium
After induction of pdc expression in recombinant B. megaterium, the levels of PDC protein were estimated by Coomassie blue R-250-stained SDS-PAGE gels (Fig. 2). High levels of SvPDC were detected, and were estimated to account for 5 % of protein in clarified cell lysate. In contrast, only low levels of the ZmPDC, ApPDC and ScPDC1 proteins were detected. To determine if the PDC proteins were produced in an active form, the cell lysate of the recombinant B. megaterium strains was assayed for PDC activity (Table 2). Of the strains examined, those cells expressing Svpdc had the highest activity with 5·29 U (mg protein)–1, while those encoding the Zmpdc and ScPDC1 genes had five- to tenfold lower activity. No PDC activity was detected for the strain which encoded the Appdc gene. Thus, most if not all of the SvPDC in the cell lysate of recombinant B. megaterium was active, based on the specific activity of purified SvPDC, which is estimated to be 65–103 U (mg protein)–1 (Lowe & Zeikus, 1992; Talarico et al., 2001). The results demonstrate that SvPDC is not only produced in very high quantity, but is produced in an active form within the B. megaterium host cell.



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Fig. 2. PDC proteins synthesized in recombinant B. megaterium. After 3 h induction with 0·5 % xylose, cell lysate (6 µg) was separated by reducing SDS-PAGE and stained with Coomassie blue R-250. Lane 1, molecular mass standards (5 µg). Lanes 2–6, cell lysate of B. megaterium WH320 transformed with: lane 2, plasmid pWH1520 (vector alone); lane 3, pJAM420 (carries Svpdc); lane 4, pJAM430 (carries Appdc); lane 5, pJAM432 (carries Zmpdc); lane 6, pJAM435 (carries ScPDC1). SvPDC (lane 3), ZmPDC (lane 5) and ScPDC1 (lane 6) proteins are indicated by arrowheads.

 

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Table 2. PDC activity of B. megaterium strains transformed with pdc expression plasmids

 
It was previously reported that production of SvPDC in recombinant E. coli was limited to specific activities in cell lysate of 0·16 U (mg protein)–1, even when using a BL21-CodonPlus-RIL strain augmented with accessory tRNAs for AUA and AGA codons (Talarico et al., 2001). No tRNA augmentation was necessary for SvPDC production in recombinant B. megaterium, and yet there was a 33-fold increase in the specific activity in cell lysate compared to recombinant E. coli. This indicates that B. megaterium is a better host for the production of the SvPDC while it is suboptimal for the production of the Gram-negative PDCs ZmPDC and ApPDC, which are expressed more efficiently in E. coli. Previous studies have determined the specific activity of ZmPDC to be 6·2–8 U (mg protein)–1 in cell lysate (Neale et al., 1987; Raj et al., 2002) when produced in recombinant E. coli, in contrast to the 1·1 U (mg protein)–1 in B. megaterium for this study.

Analysis of PDC transcript levels
The factors responsible for low-level production of PDC protein in recombinant Gram-positive bacteria are unknown (Gold et al., 1992, 1996; Barbosa et al., 1994; Nichols et al., 2003). In order to determine if transcript levels were limiting the production of PDC in Gram-positive hosts, we analysed the transcript levels specific for the various pdc genes when expressed in recombinant B. megaterium. Total RNA was isolated and quantitative reverse transcriptase PCR was performed to determine if transcript levels correlated with PDC production. The transcript levels were similar for all four pdc genes, ranging from 12 to 24 % of total RNA, with the transcript for Zmpdc the lowest (12·2±1·2 %) and ScPDC1 the highest (24·5±1·7 %). The levels of Appdc- and Svpdc-specific transcript were intermediate at 14·6±2·1 and 21·6±9·5 % of total RNA, respectively.

In contrast to SvPDC protein and its activity levels, which were at least fivefold higher than those of the other PDC proteins, there was not an abundance of Svpdc transcript compared to the other pdc transcripts. Thus, the pdc-specific mRNA levels did not correlate with the levels of PDC protein in the recombinant B. megaterium strains. These results indicate that the level of transcript is not the factor influencing protein levels of PDC in the cell. This is not unexpected, due to the use of the same inducible promoter, transcription terminator and vector for the construction of all four pdc gene expression plasmids. The reason the levels of pdc-specific transcript are so high for the four constructs may be due to the highly inducible xylA promoter used in this study. Alternatively, the method used to prepare total RNA may not completely release the rRNA from the thick cell wall of this Gram-positive bacterium.

PDC protein stability in recombinant B. megaterium
Gram-positive bacteria, particularly Bacillus species, are well known for an abundance of proteases (Wong, 1995). This is often a problem when producing heterologous proteins in these hosts (Wong et al., 1994; Wong, 1995). To determine if protein degradation was responsible for limiting PDC production in B. megaterium, pulse–chase analysis was performed. The SvPDC and ZmPDC were chosen for analysis. After induction of pdc transcription (15 min), protein was labelled with L-[35s]methionine (15 min) and chased with excess unlabelled L-methionine in the presence of the protein synthesis inhibitor chloramphenicol. This enabled the rate of protein degradation after induction of pdc gene transcription to be monitored over a period of several hours. No significant degradation of either PDC protein was detected during the entire chase (data not shown). Thus, both of the recombinant PDCs examined (SvPDC and ZmPDC) were relatively stable, yet the amounts of the SvPDC protein and activity detected after 3 h induction were dramatically higher than those of the other PDCs (Fig. 1, Table 2). Protein degradation is, therefore, not a factor influencing the levels of active PDC protein in recombinant B. megaterium.

Construction of a portable ethanol production operon for Gram-positive bacteria
B. megaterium strain WH320 (the host used in this study) is capable of growth on xylose minimal medium. The strain also grows at temperatures up to 42 °C and at the relatively low pH of 5·0. Based on these characteristics, this strain appears to be a suitable candidate to perform preliminary tests on ethanol production with a portable pyruvate to ethanol (PET) operon, and may prove useful in large-scale ethanol production under acidic conditions. To construct a Gram-positive (G+) PET operon, the adh gene from G. stearothermophilus (Gsadh) was cloned behind the Svpdc gene in the B. megaterium pWH1520 expression vector. This vector was chosen based on successful use in the high-level synthesis of SvPDC (Fig. 2). The resulting plasmid pJAM423, which carried the G+ PET operon, was transformed into B. megaterium. After induction of the G+ PET operon with xylose, a considerable portion of the lysate of this strain was composed of the SvPDC and GsADH proteins (Fig. 3). Similar to SvPDC, the GsADH synthesized in this strain was active, with ADH activity levels of 1·8 U (mg protein)–1 in cell lysate. In contrast, no ADH activity was detected for the B. megaterium strain which carried the vector alone (pWH1520).



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Fig. 3. Induction of S. ventriculi PDC and G. stearothermophilus ADH in B. megaterium. Proteins were separated by reducing SDS-PAGE using 12 % polyacrylamide gels and stained with Coomassie blue R-250. Lanes: 1, molecular mass standard (5 µg); 2, cell lysate (20 µg) of B. megaterium transformed with pWH1520 (vector alone) induced with xylose; 3, 4, cell lysate (20 µg) of B. megaterium WH320 transformed with pJAM423 (plasmid with Gram-positive PET operon) uninduced (lane 3) and xylose induced (lane 4).

 
Based on these results, the B. megaterium strain which harboured the G+ PET operon (pJAM423) was further examined for its ability to produce ethanol during growth at low levels of xylose (0·5 %). Approximately 20 mmol ethanol was produced per litre, 36 % of the maximum theoretical yield.

The basis for such low yields was investigated by examining cell-free lysates of the B. megaterium strains with and without the G+ PET operon (i.e. pJAM423 versus pWH1520) in the presence of excess NADH. Although only trace levels of ethanol were observed with glucose and xylose, ethanol (72 mM) was produced from 150 mM pyruvate, a yield 11-fold that observed with the plasmid lacking ethanol genes (pWH1520).

These results demonstrate that the G+ PET operon of pJAM423 facilitates the production of high levels of SvPDC and GsADH in B. megaterium cells, and that these recombinant enzymes are fully functional in the conversion of pyruvate to ethanol. One explanation for the low levels of ethanol produced by whole cells of B. megaterium (pJAM423) is that the cytosolic pools of pyruvate may be limiting. Other metabolic pathways may compete for the available pyruvate and limit the ability of SvPDC to channel pyruvate ultimately to ethanol. This would be consistent with the differences in kinetic constants of SvPDC compared to other enzymes that utilize pyruvate. For example, SvPDC has a KM for pyruvate 100-fold higher than those of the lactate dehydrogenases of Bacillus species (i.e. 5·7 mM versus 50 µM) (Jackson et al., 1992; Raj et al., 2002), which are likely to compete for pyruvate pools.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
For the production of ethanol in Gram-positive bacteria to become a viable alternative for the production of fuels, it will be necessary to find a PDC that can be expressed at high enough levels to rapidly channel pyruvate to acetaldehyde. Until now, there has not been a PDC that has been expressed well in a recombinant Gram-positive bacterium (Gold et al., 1992, 1996; Barbosa et al., 1994; Nichols et al., 2003).

In this study, B. megaterium expression vectors were designed in such a way as to transcribe all four pdc genes at similar rates by using the same xylA promoter, Shine–Dalgarno sequence and T7 terminator. Using this approach, the PDC protein of S. ventriculi was synthesized at high levels in the recombinant Gram-positive host. The SvPDC protein levels and activity were at least fivefold higher than those of the PDCs of Z. mobilis, A. pasteurianus, or Sac. cerevisiae. To assess the biological reason for these differences, quantitative reverse transcriptase PCR and pulse–chase experiments were performed. The recombinant Gram-positive host synthesized similar levels of pdc-specific transcript, and PDC protein degradation was minimal. Thus, in the Gram-positive host examined in this study, protein synthesis limited the production of PDC proteins from yeast and Gram-negative bacterial genes.

It was previously demonstrated that the addition of accessory tRNAs is necessary for the enhancement of SvPDC protein levels in E. coli by tenfold (Talarico et al., 2001). This is not the case when ApPDC and ZmPDC are expressed in E. coli. Both PDCs are produced at very high levels in this Gram-negative host without the addition of accessory tRNA. In B. megaterium, however, SvPDC is expressed at very high levels, while expression of ApPDC and ZmPDC is poor. The results of the expression of the PDC proteins in E. coli and B. megaterium indicate that codon usage of the pdc genes is one of the primary factors influencing synthesis of these proteins in Gram-positive hosts (Talarico et al., 2001; Raj et al., 2002). The contrasting codon usage of the pdc genes used in this study becomes evident when analysing the percentage G+C in the wobble position. B. megaterium has a wobble position percentage G+C of 30·8 %. The Svpdc gene has the lowest percentage G+C in the wobble position at 12·3 %, the Appdc gene has the highest at 74·2 %, and the Zmpdc and ScPDC1 genes have similar percentages of 54·6 and 51·5 %, respectively. These values vary quite dramatically, and correspond with the general trend of efficiency of expression in B. megaterium demonstrated by this study. Other studies have shown that changing rare codons to codons optimal for the recombinant host can increase protein levels. For example, expression of cyt2Aa1 of Bacillus thuringiensis in Pichia pastoris is improved (Gurkan & Ellar, 2003) and production of antigen 85A from Mycobacterium tuberculosis in E. coli is increased 54-fold (Lakey et al., 2000).

Thus, future research is aimed at engineering Gram-positive hosts for ethanol production using the only known PDC that is expressed well in a Gram-positive host, SvPDC. Alternatively, a pdc gene with optimized codon usage could be synthesized for high-level production of alternative PDCs. SvPDC has qualities that make it unique among bacterial PDCs, including its substrate activation and elevated pH optimum (Lowe & Zeikus, 1992; Talarico et al., 2001). However, in comparison to other PDCs, the KM of SvPDC for pyruvate is over tenfold higher (Raj et al., 2002).

The results of this study also demonstrate the generation of a Gram-positive operon that is functional in the synthesis of PDC and ADH proteins that actively convert pyruvate to ethanol. Our construction and expression of this G+ PET operon using the SvPDC and G. stearothermophilus ADH have demonstrated that high-level synthesis of active PDC and ADH no longer limits ethanol production in Gram-positive biocatalysts. Optimization of the conversion of biomass to pyruvate and minimization of the pathways that compete for available pyruvate are now needed to ensure high yields of ethanol in recombinant Gram-positive bacteria.


   ACKNOWLEDGEMENTS
 
We thank Francis Davis and Jack Shelton for DNA sequencing, and Sean York for gas chromatographic analyses of ethanol. Thanks also to Frank Jordan for generously supplying pScPDC1. This work was supported in part by the US Department of Energy (DOE) (DE-FG36-04GO-14019).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 26 July 2005; revised 9 September 2005; accepted 14 September 2005.



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