INTRODUCTION

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1,3-Propanediol (1,3-PD) is a three-carbon diol that is currently manufactured by synthetic processes beginning with petroleum derivatives such as acrolein or ethylene oxide (35). An emerging large-volume application of 1,3-PD is as a monomer in the synthesis of polyesters for use in carpet and textile fibers (17, 22, 25, 33). Therefore, there is much interest in developing improved routes to 1,3-PD production. One potential method is via the fermentation of glycerol or, ultimately, of sugars (24).

Klebsiella pneumoniae is one of several organisms that naturally ferment glycerol to 1,3-PD. The conversion is carried out in two enzymatic steps. The first enzyme, glycerol dehydratase (EC 4.2.1.30), which requires adenosylcobalamin (coenzyme B₁₂), removes a water molecule from glycerol to form 3-hydroxypropionaldehyde (3-HPA). The second enzyme, 1,3-PD oxidoreductase (EC 1.1.1.202), transfers a reducing equivalent from NADH to 3-HPA, yielding 1,3-PD. The genes encoding glycerol dehydratase and 1,3-PD oxidoreductase are designated dhaB and dhaT, respectively.

The 1,3-PD pathway was expressed in Escherichia coli in our laboratory (41) by using genes from K. pneumoniae and in the laboratory of Daniel and Gottschalk (10) by using genes from Citrobacter freundii. The main purpose of these endeavors was the characterization of the genes and enzymes responsible for the conversion of glycerol to 1,3-PD. In both cases, the configuration of the 1,3-PD genes and regulatory elements was the same as in the donor organism.

To enhance the utility of the 1,3-PD genes, we investigated their rearrangement into a form that would be adaptable to expression in various hosts under any desired regulation. We sequenced a fragment of K. pneumoniae DNA containing the dhaB and dhaT genes and found that these genes are transcribed in opposite directions. We therefore rearranged the dhaB and dhaT genes into an operon free of the K. pneumoniae regulatory elements. In this report, we describe the construction of the 1,3-PD operon and its performance in E. coli cofermentations of glycerol and glucose.

This novel genetic configuration provides the basis for an improved microbial 1,3-PD process. Several research groups have achieved 1,3-PD concentrations of 60 to 70 g/liter in the fermentation of glycerol, using organisms that can naturally convert glycerol to 1,3-PD (13, 18, 29, 30). Without directed improvement of the host, however, this level of performance is probably a plateau and cannot compete with newly improved synthetic processes. In 1995, Shell Chemical Company announced an improvement to the ethylene oxide hydrocarbonylation process that permits 1,3-PD to be produced at a cost low enough for its use in polypropylene terephthalate carpet fibers (32). Metabolic engineering provides a means to improve the fermentation process. DuPont, for example, recently patented a process to convert sugars to 1,3-PD with various organisms expressing glycerol dehydratase and 1,3-PD oxidoreductase from K. pneumoniae (24). The operon we describe in this report is designed so that any promoter and other desired genetic elements can be readily introduced to enable expression of the 1,3-PD genes in various prokaryotes. As such, it should prove useful in the metabolic engineering of 1,3-PD processes.

	MATERIALS AND METHODS

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DNA sequencing. Sequencing of the K. pneumoniae DNA was performed by the dideoxy chain termination method (31) with a Sequenase 2.0 kit (United States Biochemicals, Cleveland, Ohio) at Lofstrand Labs Limited (Gaithersburg, Md.). Two large pieces of DNA to be sequenced, ApaI-SacI and NheI-ApaI fragments, were separately cloned into the vector pSL301 (Invitrogen, San Diego, Calif.). This vector contains a multiple cloning site flanked by T7 and T3 promoter sequences; therefore, T3 and T7 primers were used to initiate double-stranded sequencing. Primer walking with synthetic 18-mer oligonucleotide primers was used to determine the remainder of the double-stranded sequence, with a new primer synthesized for about every 250 nucleotides sequenced.

Sequence analysis. The Genetics Computer Group (Madison, Wis.) package was used for analysis of open reading frames (ORFs) and restriction sites and for comparisons to GenBank nucleotide sequences. The BLAST server at the National Center for Biotechnology Information (2) was used for amino acid comparisons with the nonredundant protein databases Swiss-Prot, Protein Information Resource, Brookhaven Protein Data Bank, and GenPept.

Bacterial strains, media, and growth conditions. K. pneumoniae was obtained from the American Type Culture Collection (Rockville, Md.) as strain ATCC 25955. The E. coli strains used in all fermentations were AG1 (Stratagene, La Jolla, Calif.) and TOP10F' (Invitrogen). The temperature for all fermentations and inoculum cultures was 37°C. The volume of the fed-batch fermentation was 4 liters. The volumes of all other fermentations were 300 ml when extracts were prepared or 2 ml when they were not. The fed-batch fermentation was conducted in a Bio-Flo 3000 fermentor (New Brunswick Scientific, Edison, N.J.) with pH controlled at 7.0, agitation at 50 rpm, and nitrogen sparging to minimize dissolved oxygen. Fermentations of the 300-ml total volume were conducted without agitation for at least 10 h in anaerobic flasks. Two-milliliter fermentations were conducted in closed screw-cap tubes with a total liquid volume of 1.8 ml for at least 10 h. Inocula for smaller-scale fermentations were started from stocks frozen in glycerol and grown overnight with shaking in 2 ml of Luria-Bertani medium plus 100 µg of ampicillin per ml (LA medium). The two-milliliter fermentation mixtures were inoculated with 50 µl of the overnight culture, and 300-ml fermentation mixtures were inoculated with 500 µl. The inoculum for the 4-liter fermentation was grown as for the smaller-scale fermentations, and then 100 µl of this culture was added to 50 ml of LA medium and was grown overnight in a closed 50-ml centrifuge tube with no shaking. The entire 50 ml was used to inoculate the 4-liter fermentation.

Preparation of cell extracts. Crude cell extracts were prepared by sonication of cell pastes and subsequent centrifugation. Cell pastes were obtained by centrifugation of fermentation broths at 4,000 rpm for 5 to 10 min at 4°C with a Beckman (Fullerton, Calif.) model J2-21 centrifuge and JA-20 rotor. The pastes were washed in 20 mM Tris buffer (pH 8.0) or 50 mM potassium phosphate buffer (pH 8.0), centrifuged as described above, and resuspended in a small amount of the appropriate assay resuspension buffer. The cells were then disrupted by sonication for 5 min on ice at a duty cycle of 70% with 1-s cycles. Cell debris was removed by centrifugation at maximum speed for 5 to 15 min in a microcentrifuge.

Assays. 1,3-PD oxidoreductase was assayed by the method of Johnson and Lin (21). The initial rate of reduction of NAD⁺ to NADH was measured spectrophotometrically (340 nm) at 25°C for a mixture containing 100 mM 1,3-PD, 35 mM ammonium sulfate, 100 mM potassium bicarbonate buffer (pH 9.0), 0.6 mM NAD⁺, and 10 to 50 µl of crude cell extract in a final volume of 1 ml. A baseline was established prior to the addition of NAD⁺. Glycerol dehydratase activity was measured by a coupled assay with yeast alcohol dehydrogenase (42) or by the MBTH (3-methyl-2-benzothiazolinone hydrazone) method (43).

High-performance liquid chromatography analysis. The metabolites present in fermentation broths were analyzed with a Bio-Rad high-performance liquid chromatography system with a refractive index detector and a Bio-Rad Aminex HPX-87H organic acids column at a flow rate of 0.6 ml/min and a column temperature of 65°C. The mobile phase was 0.01 N sulfuric acid. Samples were filtered through 0.45-µm-pore-size Supor membranes (Gelman Sciences, Ann Arbor, Mich.) prior to analysis.

SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done with a Hoefer (San Francisco, Calif.) SE 600 vertical unit. Each protein sample was diluted with an equal volume of loading buffer (20% [vol/vol] glycerol, 2% [vol/vol] -mercaptoethanol, 120 mM Tris-Cl [pH 6.8], 41 mg of SDS per ml, 0.01 mg of bromophenol blue per ml) and boiled for 5 min prior to being loaded onto a 1.5-mm-thick gel. The stacking gel and 10% separating gel were prepared as described by Ausubel et al. (4). The gel was run at 20 mA until the blue dye entered the separating gel, and subsequently it was run at 30 mA for 3 h.

Construction of plasmids. Standard techniques of recombinant DNA technology as described by Ausubel et al. (4) were used for all DNA manipulations. Restriction and DNA-modifying enzymes were obtained from Promega (Madison, Wis.) or New England Biolabs (Beverly, Mass.) and used according to the instructions of the manufacturer. Plasmids used in experimentation are described in Table 1. Some plasmids that were used only in construction are not listed in Table 1 but are mentioned below.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Construction of pTC48 as outlined in Materials and Methods. Restriction enzyme recognition sites are abbreviated as follows: H, HindIII; K, KpnI; Nd, NdeI; Nh, NheI; No, NotI; P, PstI; Sc, SacI; and Sl, SalI. Brackets indicate the combination of two fragments of DNA by cleavage at the restriction sites shown in the bracket and subsequent ligation to form the product shown beneath the bracket.

Nucleotide sequence accession number. The nucleotide sequence described in this paper was submitted to GenBank under accession no. U30903.

	RESULTS

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General features of the DNA sequence. The sequence of an NheI-SacI fragment of cosmid pTC1 (41), a contiguous sequence of 8,067 nucleotides, was determined as described in Materials and Methods. Several large ORFs (that would encode proteins of 10 kDa or more) were found within the fragment known to contain the dhaB and dhaT genes. The major ORFs were designated as shown in Fig. 2.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Major ORFs within the sequenced K. pneumoniae DNA fragment. The direction of transcription is indicated (arrows). The three rows shown for each transcriptional direction represent the three frames of reference. ORFs incorporated into the 1,3-PD operon are indicated (shaded). Glycerol dehydratase is encoded by ORFs 4, 4a, and 3a. 1,3-PD oxidoreductase is encoded by ORF 2. ORF 3 augments 1,3-PD production by providing an unknown function.

Identification of functional units conferring dhaB and dhaT activities. The main objective in the construction of the 1,3-PD operon was to enable expression of the dhaB and dhaT genes in one transcript under the regulation of a single replaceable promoter/operator. The construction of the 1,3-PD operon therefore required the identification of regions of DNA that confer dhaB and dhaT activities but are independent of their native regulation.

View larger version (90K): [in this window] [in a new window]   FIG. 3.   Coomassie blue-stained SDS-PAGE gel of crude cell extracts of E. coli/pTC42, induced with 0.5 mM IPTG (lane +) and uninduced (lane ). The protein overproduced when the cells are induced is indicated (arrow). Crude cell extracts were prepared as described in Materials and Methods. Lane M, molecular mass marker.

Construction of the 1,3-PD operon. In the native DNA fragment isolated from K. pneumoniae the dhaT and dhaB genes are transcribed in opposite directions from a common region. In Fig. 2, ORF 2 (dhaT) is transcribed from left to right, and ORFs 4, 4a, 3a, and 3 are transcribed from right to left. Therefore, the steps necessary for construction of the 1,3-PD operon were (i) isolation of the functional dhaB and dhaT units without their promoters, (ii) rearrangement of the DNA so that the two units are transcribed in the same direction, and (iii) attachment of a replaceable promoter upstream of the units. A representation of the 1,3-PD operon (plasmid pTC49) is shown in Fig. 4.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Plasmid pTC49, one version of the 1,3-PD operon. The regions sufficient for glycerol dehydratase (dhaB) and 1,3-PD oxidoreductase (dhaT) expression (black segments) and the direction of transcription (arrows) are indicated. The ampR (ampicillin resistance) gene and the pMB1 origin of replication are identical to those in pBR322. Nucleotide positions are also indicated (in parentheses).

The significance of ORF 3. Tobimatsu et al. (38) reported that ORF 3 does not encode a subunit of glycerol dehydratase. We observed a loss of dehydratase activity when the SfiI site within ORF 3 of pTC9 was disrupted by digestion and blunt-end ligation (44). The relevance of ORF 3 to the 1,3-PD operon was tested by conducting fermentations with strain TOP10F' carrying either pTC49 or pTC63 (pTC49 with an NruI-NruI deletion within ORF 3). TOP10F'/pTC63 produced about 40% as much 1,3-PD as TOP10F'/pTC49 (Table 2), indicating that ORF 3 is not necessary for glycerol dehydratase activity, but it may serve some other function that permits 1,3-PD synthesis to be more effective.

Requirement for coenzyme B₁₂ addition. We determined to what extent coenzyme B₁₂ addition was necessary to effect 1,3-PD production in the transgenic E. coli cells. Figure 5 shows the final 1,3-PD concentrations of fermentations in which TOP10F'/pTC49 was grown in the standard 1,3-PD production medium except with various low concentrations of coenzyme B₁₂. 1,3-PD was not formed by TOP10F'/pTC49 when coenzyme B₁₂ was not added, and TOP10F' cells did not synthesize 1,3-PD without the K. pneumoniae genes, confirming that the cloned K. pneumoniae genes are responsible for 1,3-PD synthesis. Coenzyme B₁₂ was no longer limiting to 1,3-PD synthesis when provided at concentrations above 10 nM, far below the concentration in our standard production medium (1 µM).

View larger version (16K): [in this window] [in a new window]   FIG. 5.   1,3-PD production in E. coli TOP10F' carrying pTC49. The 2-ml fermentations were carried out as described in Materials and Methods in standard 1,3-PD production medium but with various low concentrations of coenzyme B12. Symbols: , final 1,3-PD concentration (in grams per liter); , molar yield of 1,3-PD from glycerol (moles of 1,3-PD produced per mole of glycerol consumed).

Fed-batch fermentation. We conducted a 4-liter fed-batch cofermentation of glycerol and glucose with E. coli AG1/pTC53 without induction to determine what concentration of 1,3-PD could be achieved in the final broth. pTC53 is essentially the same as pTC49 except that it contains the constitutive lac repressor gene (lacI^q). pTC49, although it can be somewhat more effective in 1,3-PD synthesis, was not used because of its tendency to rearrange. pTC49 and pTC53 are stable plasmids, but pTC49 often did not retain its original configuration over many generations. It is possible that the addition of the lacI^q gene to pTC49 prevents excessive transcription and that rearrangement in pTC53 does not provide a significant growth advantage. Figure 6 shows the time course of the AG1/pTC53 fermentation, in which glucose and glycerol were adjusted to 2 g/liter each time the concentrations of both components were nearing zero. The results of the fermentation are summarized in Table 3. The final concentration of 1,3-PD was 6.3 g/liter. The fermentation had two distinct phases, one in which the cells grew and produced predominantly 1,3-PD and acetate, and another after the cells ceased to grow in which pyruvate became the dominant acid product.

View larger version (16K): [in this window] [in a new window]   FIG. 6.   Time course of the 4-liter fed-batch fermentation of E. coli AG1 carrying pTC53. (A) Substrate consumption, growth, and 1,3-PD production. Concentrations in the fermentor are shown for glycerol (), glucose (), and 1,3-PD (), as well as the optical density of cells at 660 nm (). (B) By-product formation. Concentrations in the fermentor are shown for acetate (), formate (), lactate (), pyruvate (), and succinate ().

	DISCUSSION

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We have constructed an operon containing the glycerol dehydratase and 1,3-PD oxidoreductase genes of K. pneumoniae. The operon enables the production of 1,3-PD in E. coli and provides the basis for future expression of the 1,3-PD pathway under novel regulation in other organisms. It also provides a basis for extending the 1,3-PD pathway to include fermentable sugars. Laffend et al. (24) have already demonstrated that the sugars-to-1,3-PD pathway is realizable. Expression of the 1,3-PD genes in an organism that can naturally produce glycerol from sugars would complete a microbial sugars-to-1,3-PD pathway, averting the possibly toxic effects of high glycerol concentrations and providing a microbial route to 1,3-PD from fermentable sugars, which are more abundant, less expensive, and utilizable by a wider range of organisms than glycerol.

The 1,3-PD operon is very flexible in that it contains unique restriction sites in several strategic locations (Fig. 4). The promoter region can be replaced with any BglII-BglII fragment; PCR can be used to isolate any promoter region of interest bounded by BglII sites, and the promoter region can be ligated into this location. We observed that the trc promoter fragment could be replaced by the E. coli lac or phoA promoter with the retention of the ability to produce 1,3-PD, but removal of the promoter caused a dramatic decrease in 1,3-PD production (data not shown). The BglII sites flanking the promoter are also significant because DNA cleaved with BglII can be ligated to DNA cleaved with Sau3AI without the creation of blunt ends with DNA polymerase. Therefore, Sau3AI digests of genomic DNA of any organism can be randomly ligated into this location to screen for promoters effective in the production of 1,3-PD under any conditions desired, an implementation not possible with unmanipulated K. pneumoniae DNA. The upstream regions of ORF 2 or 4 can be replaced with any KpnI-MluI or SalI-NotI fragment, respectively. This allows the introduction of different ribosome binding sites or leader sequences at either of these locations. Additional genes, promoters, and terminator structures can be ligated into the operon before or after the promoter, dehydratase region, or oxidoreductase region.

During the construction of the 1,3-PD operon, we determined that ORFs 4, 4a, 3a, and 3 are sufficient for glycerol dehydratase activity. Two important points concerning this determination should be addressed here: the demonstration of the activity and the significance of ORF 3. We observed that cells containing pTrcB1 (ORFs 4, 4a, 3a, and 3 under control of the trc promoter) possessed glycerol dehydratase activity. These cells converted 1,2-PD to 1-propanol when the medium was supplemented with coenzyme B₁₂. Glycerol dehydratase converts glycerol to 3-HPA, which is a potent antimicrobial agent (15, 36), but it converts 1,2-PD to propionaldehyde, which is less toxic to E. coli than is 3-HPA (15, 23). We therefore used 1,2-PD as a substrate instead of glycerol to demonstrate the activity in whole cells.

Tobimatsu et al. (38) reported that the K. pneumoniae ORF corresponding to our ORF 3 is not necessary for glycerol dehydratase activity. We found this to be true also, but we found that ORF 3 seems to augment 1,3-PD synthesis by providing an unknown function. ORF 3 has some identity to coenzyme B₁₂-dependent enzymes. A generally recognized conserved sequence in such enzymes is DxHxxG (7). This motif is not encoded in ORF 3; however, the translation of ORF 3 shares a VGxSSL motif with the coenzyme B₁₂-dependent enzymes methylmalonyl-coenzyme A mutase (8, 28), methyleneglutarate mutase (7), glutamate mutase (27), and methionine synthase (6). This motif, however, does not occur in all coenzyme B₁₂-dependent enzymes, as it is not contained in ethanolamine ammonia-lyase (14, 16). The similarity of ORF 3 to several genes encoding coenzyme B₁₂-dependent enzymes suggests that the gene product of ORF 3 may interact with coenzyme B₁₂ or perhaps with the glycerol dehydratase holoenzyme.

A 4-liter fed-batch fermentation culture with E. coli AG1 carrying plasmid pTC53 accumulated 6.3 g of 1,3-PD per liter. This is about the same level as in a fermentation with AG1/pTC9, in which 6.5 g of 1,3-PD accumulated per liter (39). pTC9 contains the K. pneumoniae 1,3-PD genes in an unaltered form. Therefore, the 1,3-PD operon has retained the ability to effectively direct 1,3-PD synthesis while having the advantage that it can be expressed in any prokaryotic organism, provided that an appropriate promoter is inserted and, if necessary, that the ribosome binding sites are adjusted. As dhaT is an octamer of a single subunit (ORF 2), it may be advantageous in the future to optimize the ratio of dhaB to dhaT expression in the operon, perhaps by providing ribosome binding sites of various strengths or even a second promoter upstream of dhaT. The flexible construction of the 1,3-PD operon allows such changes to be made readily.

Theoretically, 1 mol of 1,3-PD can be produced for every mol of glycerol consumed, given that the cells are provided with a cosubstrate such as glucose from which to derive reducing power (40). We observed a yield of 82% in the AG1/pTC53 fermentation, which means that 18% of the glycerol consumed was not converted to 1,3-PD. In the absence of respiration, E. coli cannot use glycerol as the sole source of carbon and energy because neither of the two known sn-glycerol-3-phosphate dehydrogenases of E. coli can use NAD⁺ as an electron acceptor (26). One of these dehydrogenases can donate electrons to the fumarate reductase complex, which produces succinate from fumarate, but the quantity of succinate produced in the AG1/pTC53 fermentation could account for the metabolism of only about 4% of the glycerol in this manner. Biomass cannot account for the disparity, because in the second phase of the fermentation (Table 3), very little biomass was produced while most of the glycerol was consumed, and the molar yield of 1,3-PD was 83%. Therefore, it may be that background activities resembling glycerol dehydrogenase (3, 20) and dihydroxyacetone kinase (19) are responsible for the conversion of a small portion of the glycerol to dihydroxyacetone phosphate, a glycolytic intermediate.

E. coli with the 1,3-PD operon gives a product distribution not previously observed in 1,3-PD fermentations. Pyruvate accumulated during 1,3-PD synthesis after the AG1/pTC53 cells in the 4-liter fermentation culture had ceased to grow. Presumably, this is because the enzymes of anaerobic pyruvate dissimilation are repressed somewhat when E. coli enters stationary phase. Pyruvate accumulation has been observed in E. coli fermentations where glucose consumption exceeds the growth requirement (37). 1,3-PD fermentations must yield some side product, usually predominantly acetate, in addition to 1,3-PD so that NADH can be regenerated. The pathways from glucose to either acetate or pyruvate have the same NADH yield, but the pyruvate pathway yields half as much ATP as the acetate pathway. Acetate is a main by-product of the AG1/pTC53 fermentation while the cells are growing and require more ATP, but pyruvate becomes the dominant acid product when growth ceases and the ATP requirement decreases.

Overexpression of the 1,3-PD genes in organisms other than E. coli may enable improved 1,3-PD production. For example, this could be accomplished by expressing the 1,3-PD operon in natural producers of 1,3-PD or in organisms highly tolerant to 1,3-PD. It has been shown elsewhere (1) that glycerol dehydratase activity is the limiting factor in 1,3-PD production in Clostridium butyricum. Therefore, increasing the dehydratase activity in such an organism could increase the productivity of the 1,3-PD fermentation. K. pneumoniae, a natural 1,3-PD producer, can no longer grow in the presence of about 70 g of 1,3-PD per liter (39). This is also approximately the highest 1,3-PD concentration reported for a K. pneumoniae fermentation (18). The mechanism of the inhibition is not well understood. If the inhibition is not specific to the 1,3-PD pathway expression of the operon in K. pneumoniae could increase the rate of production, but not the final concentration, of 1,3-PD. Expression of the operon in organisms with greater tolerance of 1,3-PD could lead to higher concentrations of 1,3-PD than are currently possible.