Microbial production of 1,3-propanediol

Transcription

1 Appl Microbiol Biotechnol (1999) 52: 289±297 Ó Springer-Verlag 1999 MINI-REVIEW H. Biebl á K. Menzel á A.-P. Zeng á W.-D. Deckwer Microbial production of 1,3-propanediol Received: 12 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999 Abstract 1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO 2,H 2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH 2, which is mainly a ected by the product distribution (of the oxidative pathway) and depends rst of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a ``green'' polyester is a more cost-e ective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great e orts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the rst bulk chemical produced by a genetically engineered microorganism. Historical outline 1,3-Propanediol (1,3-PD) is one of the oldest known fermentation products. It was reliably identi ed as early H. Biebl á K. Menzel á A.-P. Zeng á W.-D. Deckwer (&) GBF ± Gesellschaft fuè r Biotechnologische Forschung mbh, Biochemical Engineering Division, Mascheroder Weg 1, D Braunschweig, Germany WDD@GBF.de Tel.: Fax: as 1881 by August Freund, in a glycerol-fermenting mixed culture obviously containing Clostridium pasteurianum as the active organism (Freund 1881). Later, in 1914, Voisenet described a wine-spoiling bacillus that produced the substance, but so far no comparable strain has been isolated. Quantitative analysis of the fermentation of di erent enterobacteria producing 1,3-PD (trimethylene glycol, propylene glycol) started at the microbiology school of Delft (Braak 1928) and was successfully continued at Ames, Iowa (Mickelson and Werkman 1940). In the 1960s, interest shifted to the glycerol-attacking enzymes, in particular to the glycerol and diol dehydratases, as these enzymes were peculiar in requiring coenzyme B 12 (see Lin 1976). 1,3-PD-forming clostridia were rst described in 1983 as part of a process to obtain a specialty product from glycerol-excreting algae (Nakas et al. 1983). Previous situation and 1,3-propanediol from an economic perspective 1,3-PD as a bifunctional organic compound could potentially be used for many synthesis reactions, in particular as a monomer for polycondensations to produce polyesters, polyethers and polyurethanes. However, its high price in the past [according to Millet (1993), about 30 U.S. $/kg when chemical synthesis was used] meant that 1,3-PD could not compete with the petrochemically available, and hence low-priced (up to 2 U.S. $/kg), diols like 1,2-ethanediol, 1,2-PD, and 1,4-butanediol. Therefore, in the past 1,3-PD has only found niche applications of negligible market volume [solvent, production of dioxanes, specialty polymers (Sullivan 1993)]. The natural substrate for the microbial production of 1,3-PD is glycerol, and the cost of producing 1,3-PD from the microbial process, including product recovery and puri cation, can be estimated roughly from the relationship reported by Deckwer (1995):

2 290 Price of1;3-pd ˆ a b price of glycerol ˆ 1 2 price of glycerol 1 As glycerol costs at least 1 $/kg, more than 2/3 of the 1,3-PD production costs stem from the raw-material price. Glycerol, being a constituent of natural fats and oils, can be regarded as a regrowing resource and, because of the e orts to make use of agricultural areas for producing non-food materials, such as biodiesel, by transesteri cation of rape seed oil, it was thought that the simultaneous surpluses of glycerol may reduce its market price and, hence, improve the economic feasibility of the microbial glycerol-to-propanediol conversion. However, a signi cant fall in price was not observed as the market assimilated all the surplus glycerol (Murphy 1996). In 1995 the market situation for 1,3-PD changed signi cantly as Shell Chemical Company announced the commercialization of a new polyester, Corterra, based on terephthalic acid and 1,3-PD. This poly(trimethylene terephthalate) is particular appropriate for ber and textile applications and combines excellent properties (good resilience, inherent stain resistance, low static generation) with an environmentally benign manufacturing process (Chuah et al. 1995). Shell developed a new chemical route for 1,3-PD by the reaction of ethylene oxide with carbon monoxide and hydrogen (Sullivan 1993; Stinson 1995). A 4000-t/year plant is already in operation and another t/year plant is expected to go on stream in Also Degussa has announced the design of a new 1,3-PD plant (around t/year) at Wesseling Germany (McCoy 1998), which is based on the conventional route (hydrolysis of acrolein followed by catalytic hydrogenation; Sullivan 1993) with improved catalysts (Arntz et al. 1991). The Degussa process of manufacturing 1,3-PD has been taken over recently by DuPont. However, DuPont's strategic interest is obviously to establish an economically feasible biotechnological route to 1,3-PD, which can be used as a monomer to produce the desired polyester, i.e., poly(trimethylene terephthalate) (McCoy 1998). In a collaborative joint project by DuPont and Genencor International, metabolic engineering is being used to convert glucose to 1,3-PD directly (Potera 1997; Balthuis et al. 1998; Gatenby et al. 1998). By changing the feedstock basis for the fermentative 1,3-PD production from glycerol to glucose, the raw material price could certainly be reduced considerably. However, e orts are also underway to revive the production of glycerol by yeast. Hence, the future economic situation is in uenced by several factors, as outlined by Wilke (1999). Physiology of 1,3-propanediol formation 1,3-PD is a typical product of glycerol fermentation and has not been found in anaerobic conversions of other organic substrates. Only very few organisms, all of them bacteria, are able to form it. They include enterobacteria of the genera Klebsiella (K. pneumoniae), Enterobacter (E. agglomerans) and Citrobacter (C. freundii), lactobacilli (L. brevis and L. buchneri) and clostridia of the C. butyricum and the C. pasteurianum group (Nakas et al. 1983; SchuÈ tz and Radler 1984; Forsberg 1987; Homann et al. 1990; Biebl et al. 1992; Dabrock et al. 1992; Barbirato et al. 1995). Analysis of the fermentation products shows that part of the glycerol is converted to the same products as in sugar fermentation. This conversion provides the necessary energy for growth but, for many of the products, reducing equivalents are released, which are oxidized in a reductive conversion of glycerol leading to the formation of 1,3-PD. Lactobacilli have only this reductive conversion and need an additional fermentation substrate for growth and generation of the reduction equivalents (Veiga da Cunha and Foster 1992). Figure 1 shows the biochemical pathways for glycerol fermentations with 1,3-PD as the end product. The reactions down to the stage of pyruvate are common to all organisms involved. Glycerol is dehydrogenated to dihydroxyacetone which, after phosphorylation, can be converted to pyruvate in the course of the known sequence of glycolytic reactions involving another dehydrogenation and two ATP-forming steps. The reductive glycerol conversion consists of a vitamin B 12 -mediated dehydration to 3-hydroxypropionaldehyde and a reduction of the aldehyde to 1,3-PD. Pyruvate utilization The fate of pyruvate is di erent. In the enterobacteria it is cleaved to acetyl-coa and formate in a reaction catalyzed by the enzyme pyruvate formate-lyase. From acetyl-coa, acetic acid is formed via acetyl-phosphate, yielding extra ATP as well as ethanol, involving two NADH-oxidizing steps with acetaldehyde as the intermediate. Formate is usually cleaved to hydrogen and carbon dioxide by a formate lyase. As in sugar fermentation, pyruvate can also be condensed to a-acetolactate to give acetoin nally and 2,3-butanediol. Lactic acid, a reduction product of pyruvate, and succinic acid, which originates from phosphoenolpyruvate, also appear among the end-products of the enterobacterial fermentation. In C. butyricum and related strains, virtually two products are formed in addition to 1,3-PD: acetic and butyric acids. Butyric acid is formed after condensation of two molecules of acetyl-coa in a reaction chain that involves two NADH-oxidizing steps and generation of ATP. Small amounts of ethanol are also found. C. pasteurianum forms butanol in addition, which sometimes becomes the predominating product (Dabrock et al. 1992; Biebl, unpublished results). Yields of product and ATP Assuming an average cell mass composition corresponding with C 4 H 7 O 2 N, the substrate glycerol is more

3 291 Fig. 1 Biochemical pathways of glycerol fermentation. Pyruvate utilization is indicated for di erent organisms. Butyrate and n-butanol are produced by clostridia, while 2,3- butanediol is only formed by enterobacteria. Acetate and ethanol are produced by both bacterial groups reduced than cell material. Hence, biomass growth also yields reducing equivalents, which can be used for the production of PD. Yields of 1,3-PD coupled with any single by-product shown in Fig. 1 are compiled in Table 1. Also given are the ATP yields. Of course, the data calculation took biomass formation into account (for details see Zeng et al and Zeng 1996) and Table 1 Yields of 1,3-propanediol (1,3-PD) and biomass (ATP) for di erent by-products of glycerol metabolism Products Yield (mol/100 mol glycerol) 1 mol H 2 /mol CO 2 formed H 2 not formed 1,3-PD ATP 1,3-PD ATP Acetic acid ,3-Butanediol Butyric acid Lactic acid Ethanol Butanol Succinic acid should be regarded as approximate theoretical values. It is obvious that cultures that produce, in addition to CO 2 and H 2, only acetic acid as the by-product (of pyruvate formation and utilization) give the highest 1,3-PD yield, namely 64% of the glycerol consumed. In contrast, if the two molecules of NADH liberated during the generation of one molecule of pyruvate are completely reused for further pyruvate processing, i.e. the formation of ethanol or butanol, the 1,3-PD yield is the lowest but the biomass yield is the highest. As will be shown below, the 1,3-PD yield can be still improved by reduced evolution of gaseous hydrogen. In reality, the various pathways schematically outlined in Fig. 1 are used simultaneously by the microorganisms. Therefore, the product distributions are complicated functions of all the factors in- uencing cell physiology, gene regulation, gene product activities etc. In uences of product formation As in other fermentations, product formation in the 1,3- PD fermentation depends mainly on the availability of

4 292 the carbon and energy substrate and on the hydrogen ion concentration. The importance of glycerol availability can be assessed easily by continuous cultures of K. pneumoniae grown at xed glycerol inlet concentration and variable dilution rate. When glycerol is limiting, cell mass formation is optimized and large amounts of ethanol are produced, but as soon as glycerol appears in the medium, because of increasing inhibition by the products, ethanol formation ceases and the 1,3-PD yield approaches its maximum value. With increasing glycerol excess, lactic acid can be found in yields representing more than 10% of the glycerol, and 2,3-butanediol in slightly lower yields; succinic acid always remains below 2%. 2,3-Butanediol is the characteristic product of acidic media; in ph-uncontrolled batch cultures a product spectrum consisting of 1,3-PD, 2,3-butanediol and ethanol can be obtained. The acetic acid originally formed is reused in such cultures (Biebl et al. 1998). Butyrate formation, which lowers the 1,3-PD yield (Table 1) in clostridia, is somewhat comparable to ethanol formation in Klebsiella, but it seems to be more dependent on growth rate. Butyrate decreases rapidly with the dilution rate, even in the absence of substrate excess. In any case, changes in the acetate/butyrate ratio do not have such a great impact on the 1,3-PD yield. The controlling factors for butanol formation in C. pasteurianum could not be gured out, as the product equilibrium was extremely labile in continuous cultures of this organism (Biebl, unpublished results). Zeng et al. (1994) have formulated a mathematical model describing selective product formation, which describes growth as a function of ph, glycerol and product concentration using the K S value for glycerol and the critical product concentrations as constants. The critical product concentrations c P, i.e. the concentrations at which growth is entirely inhibited by the respective product, were determined both experimentally by using a ph-auxostat and added products (Biebl 1991) and by parameter estimation, and were found to be equal for K. pneumoniae and C. butyricum. The growth rates calculated from steady-state data of di erent types of continuous cultures, di erent ph values and di erent organisms corresponded well to the dilution rate actually measured. In batch fermentation with Enterobacteriaceae, Barbirato et al. (1996) also observed the accumulation of 3-hydroxypropionaldehyde in the culture after a certain amount of glycerol had been consumed. 3-Hydroxypropionaldehyde is a toxic metabolite and acts as an inhibitor for both growth and 1,3-PD formation. For E. agglomerans cessation of growth and product formation were found, while only transient accumulation of 3-hydroxypropionaldehyde and its complete exhaustion at the end of the batch cultivation were observed for K. pneumoniae and C. freundii. The reasons for the appearance of the toxic metabolite are not yet elucidated. It is, however, understood that this behavior may have a signi cant e ect on the performance and optimization of 1,3-PD production processes. Role of hydrogen release During acetyl-coa formation from pyruvate, reducing equivalents are released as formate or hydrogen, which are lost for the formation of 1,3-PD. Nevertheless, in both Clostridium and Klebsiella cultures, a higher yield of 1,3- PD was observed than expected if 1 mol formate or hydrogen was formed/mol acetyl-coa, and indeed a lower production of hydrogen gas was demonstrated (Biebl et al. 1992; Zeng et al. 1993). For clostridia this is not surprising, as electrons can be transferred from reduced ferredoxin to NAD by an NAD:ferredoxin oxidoreductase rst described for Clostridium acetobutylicum (Petitdemange et al. 1976) and later also for glycerolfermenting C. butyricum (Saint-Amans and Soucaille 1995; Abbad-Andaloussi et al. 1996a,b). In Klebsiella, however, the reducing equivalents released as formate by the pyruvate formate lyase cannot be recycled. Therefore, Zeng et al. (1993) postulated a second pyruvate-cleaving enzyme system, and recently Menzel et al. (1997a) presented evidence for this hypothesis: Klebsiella is able to use the pyruvate dehydrogenase normally active in aerobic catabolism, thus making additional NADH available for 1,3-PD formation (Table 1). The authors showed that up to 30% of pyruvate can be cleaved by pyruvate dehydrogenase; the highest activity occurred under conditions of glycerol excess. Process development and optimization Both enterobacteria and clostridia appear to be suitable for a 1,3-PD production process. K. pneumoniae and C. freundii, as facultative anaerobes, might be easier to handle, but since all strains of these species are classi ed as opportunistic pathogens, special safety precautions are required to grow them. As shown in Table 2, the 1,3- PD concentrations achieved in batch and fed-batch cultures are similar for both bacterial types, con rming the validity of the common growth model proposed by Zeng et al. (1994). A product content of 5.5%±7.3% appears to be low in comparison to the alcoholic fermentation (up to 15%) but high if compared to the acetone/butanol process (2.5%). The fermentation time, as expressed by the average volumetric productivity, Q, is in the same range in both groups, but the yield per substrate is distinctly lower in Klebsiella owing to the formation of by-products that provide little or no reducing equivalents for 1,3-PD formation, such as lactate and ethanol. Among the Klebsiella strains, the type strain ATCC exhibits the highest fermentation rates (see also Homann et al. 1990), among the clostridia the DSM 5431 strain grows fastest. The high 1,3-PD concentrations reached by some other Clostridium strains have obviously been attained at the expense of a markedly longer fermentation time (Saint-Amans et al. 1994; Petitdemange et al. 1995). GuÈ nzel et al. (1991) studied 1,3-PD production by C. butyricum (DSM 5431) in stirred-tank and airlift re-

5 Table 2 1,3-Propanediol (1,3-PD) nal concentrations, yields (Y PD ) and volumetric productivities (Q PD ) of di erent strains and mutants in batch (B) and fed-batch (Fb) culture 293 Species Strain Batch/ fed-batch Yeast extract (g l )1 ) 1,3-PD (g l )1 ) Y PD (mol/mol) Q PD (g l )1 h )1 ) Reference K. pneumoniae ATCC B Tag 1990 ATCC Fb Held 1996 ATCC Fb Held 1996 C. butyricum DSM 5431 B Biebl et al DSM 5431 Fb GuÈ nzel et al DSM 5431 Fb Reimann and Biebl 1996 DSM 5431 Fb Abbad-Andaloussi et al mutant 2/2 Fb Reimann and Biebl 1996 VPI 3266 B Saint-Amans et al VPI 3266 Fb Saint-Amans et al E5 Fb Petitdemange et al actors of various scales (up to 2 m 3 and 1.2 m 3 respectively). Gassing with an inert gas (N 2 for quick desorption of fermentative gases such as CO 2 and H 2 ) and changing the stirrer speed had no signi cant e ect, nor did the reactor type and scale. Inhibition by the initial substrate concentration meant that fed-batch operation led to optimal results for this strain, as shown in Table 2. On the basis of the results of GuÈ nzel et al. (1991), one can conclude that the scaling-up of the microbial 1,3-PD production to industrial reactor sizes will not cause serious problems. As the airlift reactor is less expensive (lower investment and operational costs), its use appears more attractive. The recovery of 1,3-PD from the fermentation broth and its puri cation will be based on mechanical and thermal operations. The entire downstream processing costs are most signi cantly affected by the 1,3-PD concentration achievable in the fermentation. Rough cost estimates are reported by Deckwer (1995). Some progress in fed-batch cultivation was made by controlling the nutrient supply. Saint-Amans et al. (1994) used CO 2 as the control parameter, Reimann and Biebl (1996) combined ph correction by KOH with nutrient addition. By careful calculation of the ratio of the control parameter to nutrient addition, a slight but constant excess of glycerol was maintained, which is necessary to keep the amount of butyrate low. Control via glycerol concentration, which would mean safer process management, has not yet been achieved. As shown above, continuous cultures have contributed considerably to the knowledge of product formation in the 1,3-PD fermentation. If considered for a production process, continuous cultures are usually considered for their high productivity, but the product concentrations achieved are often too low for e cient downstream processing. In fact, in some continuously run processes, product concentrations were obtained that were very close to those of batch fermentations. With K. pneumoniae and a dilution rate of 0.1 h )1, Menzel et al. (1997b) arrived at a 1,3-PD concentration of 48 g/l and a molar yield of 63%, but this required a glycerol excess of more than 50 g/l. At low glycerol excess the corresponding gures were only 33 g/l and 46%. Boenigk et al. (1993) used a two-stage continuous culture of C. freundii at a dilution rate of 0.05 h )1 in the second stage. These conditions resulted in a 1,3-PD concentration of 42 g/l and a yield of 62% with a high glycerol excess of 17 g/l, but the productivity was only 2.1 g l )1 h )1 ) in comparison to 4.8 g l )1 h )1 or 3.3 g l )1 h )1 for K. pneumoniae. For clostridia, no continuous-culture data for a low dilution rate and high glycerol feed concentration are available, but they seem to be in the same range, if the data from Reimann (1997) are used for estimation; she came up with 23 g/l 1,3-PD and a yield of 68% at a dilution rate of 0.27 h )1, corresponding to a productivity of 6.2 g l )1 h )1. Devices to increase productivity by cell retention have been also applied to glycerol fermentation. P ugmacher and Gottschalk (1994) worked with Citrobacter cells immobilized on polyurethane foam in a xed-bed reactor. In comparison to the corresponding stirred-tank reactor culture (Boenigk et al. 1993) the propanediol productivity was doubled, but the concentration could not be increased beyond 19 g/l. Similar results were obtained with C. butyricum when a cross ow ltration technique was used (Reimann et al. 1998). The productivity was increased to four times that of the conventional continuous culture, while the maximum product concentration (26 g/l) could not be increased signi cantly by cell recycling. Product-tolerant mutants of C. butyricum Genetic approaches to strain improvement in C. butyricum have been undertaken only very recently (Abbad- Andaloussi et al. 1995). Chemically generated mutants of strain DSM 5431 were selected in the presence of high propanediol concentrations and on bromide/bromate/ glucose medium to obtain mutants that sustained substantially higher product concentrations and were strongly reduced in hydrogen evolution (Reimann et al.

6 ). In fed-batch culture, the best system was able to convert up to 130 g glycerol to about 70 g 1,3-PD. Thus it tolerated even more product than the new isolates obtained by the same group (Petitdemange et al. 1995) and than the collection strain used by Saint-Amans et al. (1994). If these strains are subjected to genetic improvement, further increases in the amount of product can be expected. Attempts to generate a strain from the producttolerant mutant that generated less H 2 and was defective in or had an altered butyrate production failed though several methods were used. In contrast, it was possible to obtain butyrate-reduced mutants from the isolate E5 by selection on allyl alcohol (Abbad-Andaloussi et al. 1996c). These mutants were not much changed in propanediol production but exhibited a hydrogen production that was near the physiological maximum. This result, in addition to fermentation data and experiments that showed stimulation of 1,3-propanediol production in the presence of an aldehyde, prompted the authors to assume that glycerol dehydration, the product of which is the toxic 3-hydroxypropionaldehyde, is the rate-limiting step (Abbad-Andaloussi et al. 1996a). Consequently a certain amount of reducing equivalents will always have to be disposed of via butyrate and/or hydrogen, as the 1,3-propanediol pathway can be varied only within a narrow range. Genetic improvement of pathways As already pointed out, the microbial route to 1,3-PD could be an attractive alternative to chemical synthesis (Tran-Dinh and Hill 1987; Gottschalk and Averho 1990; Deckwer 1995; La end et al. 1996; McCoy 1998; Balthuis et al. 1998; Gatenby 1998). However, before the microbial 1,3-PD plays a signi cant commercial role, the cost must be considerably reduced. As indicated by the cost function (Eq. 1), costs can be reduced by decreasing 1. The term a in Eq. 1, which characterizes the fermentation and downstream processing costs 2. The raw-material costs, by improving the product selectivity (reduction of b) and by use of cheap glycerol. Of course, even more promising is to use a cheaper alternative substrate instead of glycerol, glucose for example. Several strategies are therefore being pursued to reduce the costs of the biotechnological process: 1. Improvement of already existing 1,3-PD fermentations by increasing the gene dosage for limiting steps and/or by knocking-out genes responsible for undesired by-product formation. Abbad-Andaloussi et al. (1996a) and Ahrens et al. (1998) identi ed glycerol dehydratase to be the limiting enzyme for 1,3-PD production in C. butyricum and K. pneumoniae respectively. The overexpression of this enyzme is an interesting starting point for higher 1,3-PD productivity and is currently under investigation. Thus, the quantity a in the cost function (Eq. 1) can presumably be decreased. 2. Use of glucose, which is considerably cheaper than glycerol. However, there is no microbial wild-type strain capable of converting glucose directly into 1,3-PD. Microorganisms can either produce glycerol from glucose, like yeast, as shown in Fig. 2A, or convert glycerol to 1,3-PD. The latter are the 1,3-PD-producers, as indicated in Fig. 2B. A glucose-to-1,3-pd conversion process can therefore be achieved by a mixed culture or a two-stage process with yeast and Enterobacteriaceae in two consecutive stages (Haynie and Wagner 1996). However, because of the repression of microbial 1,3-PD formation by glucose (Sprenger et al. 1989), using a mixed culture appears not to be very favorable. A more timely, but highly promising technique is certainly to combine the two pathways of glycerol formation and its conversion into 1,3-PD in one microorganism. Several approaches are under discussion for metabolically engineering the 1,3-PD pathway, starting from glucose in a single organism. One way is to express the genes of the 1,3-PD pathway heterologously. The genes coding for the enzymes of glycerol metabolism, i.e. glycerol dehydratase (dhab,c,e), 1,3-PD oxidoreductase (dhat), glycerol dehydrogenase (dhad), dihydroxyacetone kinase (dhak) and a putative regulatory gene (dhar) belong to one and the same regulon, named dha (Forage and Lin 1982). The genes from K. pneumoniae and C. freundii have been cloned and sequenced (Sprenger et al. 1989; Tong et al. 1991; Tobimatsu et al. 1991; Daniel and Gottschalk 1992; Daniel et al. 1995a,b; Seyfried et al. 1996). Strictly speaking, the dehydratase is encoded by three structural genes glda, gldb and gldc in the case of K. pneumoniae (Tobimatsu et al. 1996) or dhab, dhac and dhae in the case of C. freundii (Seyfried et al. 1996). The enzyme activities expressed in E. coli were comparable to those reported for the natural 1,3-PD-producing microorganisms and 1,3-PD concentrations of 6.5 g/l could be achieved (Sprenger et al. 1989; Tong and Cameron 1992). Skraly et al. (1998) recently constructed a variety of synthetic operons with dhab and dhat under the control of a single trc promoter, which are adaptable for expression in di erent hosts. Glycerol/glucose fed-batch cofermentations of E. coli AG1 harbouring these synthetic operons resulted in a nal concentration of 6.33 g/ l 1,3-PD (Skraly et al. 1998). Another approach is to express dhab,c,e and dhat in a glycerol producer like Saccharomyces cerevisiae (see Fig. 3D) (Nevoigt and Stahl 1997). The di erent expression systems in procaryotes and eucaryotes, however, require a completely new structure of dhab,c,e and dhat. While the dhat gene has only one open reading frame (ORF), dhab,c,e consists of four ORF labelled dhab3, dhab3a, dhab4 and dhab4a (Skraly et al. 1998; Tobimatsu et al. 1996). The dhab3 (ORF4 of K. pneumoniae, ORFZ of C. freundii and C. pas-

7 295 Fig. 2A±F Simpli ed pathways of naturally occurring 1,3-propanediol producers, glycerol producers and other glucose-metabolizing microorganisms (A±C). Also shown is the possible metabolic engineering that would render microorganisms able to convert glucose into 1,3-PD (D±E) or to coferment glucose and glycerol (F). DHAP dihydroxyacetone phosphate, G-3-P glycerol 3-phosphate, GA-3-P glyceraldehyde 3-phosphate, 3-HPA 3-hydroxypropionaldehyde, 1,3- PD 1,3-propanediol, GPPI glycerol-3-phosphatase gene, dhab (also includes dhac and dhae, i.e., the structural genes of the glycerol dehydratase), dhat 1,3-propanediol oxidoreductase gene, dhad glycerol dehydrogenase gene, dhak dihydroxyacetone kinase gene teurianum) is not a structural subunit of the dehydratase (Tobimatsu et al. 1996; Seyfried et al. 1996) but is involved in reactivation of the suicide-inactivated enzyme. For expression in yeasts, each single ORF from dhab and dhat has to be put under the control of an indigenous eucaryotic promoter. This strategy has been used by La end et al. (1996) and Cameron et al. (1998). Both groups measured only very low enzyme activities, and 1,3-PD was either detected in very low concentrations (less than 0.1 g/l) (La end et al. 1996) or was even hardly detectable by HPLC (Cameron et al. 1998). A completely new and, until now, untested strategy is the expression of the glycerol-3-phosphatase in 1,3-PD producers or in organisms already transformed with dhab and dhat (Fig. 2E) (Norbeck et al. 1996). The genes, named GPPI and GPP2, which encode two isoenzymes of the glycerol-3-phosphatase, have recently been cloned from S. cerevisiae. The glycerol-3-phosphatase catalyzes the conversion of glycerol 3-phosphate in glycerol by glycerol producers. Overexpression of this enzyme in the other organisms would link glucose metabolism and 1,3-PD formation because the substrate glycerol 3-phosphate is an intermediate in lipid synthesis in all microorganisms. The yields and productivities of 1,3-PD achievable with metabolically engineered microorganisms have to be carefully evaluated and require detailed analysis. Important aspects in this regard include (1) the balance of reducing equivalents, (2) the toxicity of 1,3-PD and other by-products, (3) the regulation of gene expression, and (4) protein stability in the recombinant microorganisms. Until now the 1,3-PD productivities and, in particular, the product concentrations obtainable with engineered organisms harboring the 1,3-PD pathway

8 296 have been low (less than 0.1 g/l with recombinant S. cerevisiae and 6.5 g/l with recombinant E. coli AG1) compared to those of natural 1,3-PD producers (for example 60±70 g/l 1,3-PD in batch and 50±60 g/l in continuous cultivations with K. pneumoniae) (Tong and Cameron 1992; Held 1996; Menzel et al. 1997b; Skraly et al. 1998). Further investigations and improvement of metabolically engineered organisms are therefore essential to make them competitive to naturally occurring 1,3- PD producers. Outlook Today there is considerable industrial interest in microbial 1,3-PD as it could compete with 1,3-PD made by petrochemistry. The combination of the appropriate genes from yeast and bacteria o ers the possibility to produce the diol directly from a cheap substrate, as claimed by several patents. However, the yields and productivities for such metabolically tailored pathways are still insu cient. It is therefore a challenge for both biochemical and metabolic engineering to develop improved biotechnological processes. These processes could be based on either 1. Two genetically and physiologically optimized organisms in one or two-stage fermentation, or 2. A single-stage fermentation with one organism having the combined pathways together with improved gene regulation and the desired cellular functions. It should be pointed out that the ``green'' polyesters (Potera 1997) are not necessarily biodegradable. With regard to biodecomposition, polyesters made from 1,3- PD and terephthalic acid do not di er from those made from other diols such as 1,4-butanediol (Witt et al. 1994). However, biodegradability can be attained by partially substituting the aromatic diacid by an aliphatic diacid such as adipic acid. Indeed, by varying the ratio of the aromatic to aliphatic acid, co-polyesters can be prepared from 1,3-PD having a wide spectrum of useful properties, i.e., mechanical strength, thermal stability (melting) and biodegradability (Witt et al. 1995, 1996). References Abbad-Andaloussi S, Maginot-DuÈ rr, Amine J, Petitdemange E, Petitdemange H (1995) Isolation and characterization of Clostridium butyricum DSM 5431 mutants with increased resistance to 1,3-propanediol and altered production of acids. Appl Environ Microbiol 61: 4413±4417 Abbad-Andaloussi S, Guedon E, Spiesser E, Petitdemange H (1996a) Glycerol dehydratase activity: the limiting step for 1,3- propanediol production by Clostridium butyricum. Lett Appl Microbiol 22: 311±314 Abbad-Andaloussi S, DuÈ rr C, Raval G, Petitdemange H (1996b) Carbon and electron ow in Clostridium butyricum in chemostat culture on glycerol and glucose. Microbiology 142: 1149±1158 Abbad-Andaloussi S, Amine J, Gerard P, Petitdemange E, Petitdemange H (1996c) Properties of allylalcohol resistent mutants of Clostridium butyricum grown on glycerol. Appl Environ Microbiol 62: 3499±3501 Ahrens K, Menzel K, Zeng A-P, Deckwer W-D (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture. III. Enzymes and uxes of glycerol dissimilation and 1,3-propanediol formation. Biotechnol Bioeng 59: 544±552 Arnzt D, Haas T, MuÈ ller A, Wiegand N (1991) Kinetische Untersuchung zur Hydratisierung von Acrolein. Chem Ing Tech 63: 733±735 Balthuis BA, Gatenby AA, Hynie SL, Hsu AK-H, Lareau RD (1998) Method for the production of glycerol by recombinant organisms. WO (E I DuPont de Nemours and Genencor International) Barbirato F, Camarasca-Claret C, Bories A, Grivet J-P (1995) Description of the glycerol fermentation by a new 1,3-propanediol producing microorganism: Enterobacter agglomerans. Appl Microbiol Biotechnol 43: 786±793 Barbirato F, Grivel JP, Soucaille P, Bories A (1996) 3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 62: 1448±1451 Biebl H (1991) Glycerol fermentation to 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a ph-auxostat. Appl Microbiol Biotechnol 35: 701±705 Biebl H, Marten S, Hippe H, Deckwer W-D (1992) Glycerol conversion to 1,3-propanediol by newly isolated Clostridia. Appl Microbiol Biotechnol 36: 592±597 Biebl H, Zeng A-P, Menzel K, Deckwer W-D (1998) Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae. Appl Microbiol Biotechnol 50: 24±29 Boenigk R, Bowien S, Gottschalk G (1993) Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii. Appl Microbiol Biotechnol 38: 453±457 Braak HR (1928) Onderzoeking over vergisting von glycerine. Dissertation, Delft Cameron DC, Altaras NE, Ho man ML, Shaw AJ (1998) Metabolic engineering of propanediol pathways. Biotechnol Progress 14: 116±125 Chuah HH, Brown HS, Dalton PA (1995) Corterra poly (trimethylene terephthalate). A new performance carpet ber. Int Fiber J, Oct 1995 Dabrock B, Bahl H, Gottschalk G (1992) Parameters a ecting solvent production by Clostridium pasteurianum. Appl Environ Microbiol 58: 1233±1239 Daniel R, Gottschalk G (1992) Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii regulon. FEMS Microbiol Lett 100: 281±286 Daniel R, Boenigk R, Gottschalk G (1995a) Puri cation of 1,3- propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli. J Bacteriol 177: 2151±2156 Daniel R, Stuertz K, Gottschalk G (1995b) Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. J Bacteriol 177: 4392±4401 Deckwer W-D (1995) Microbial conversion of glycerol to 1,3- propanediol. FEMS Microbial Rev 16: 143±149 Forage RG, Lin ECC (1982) dha systems mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 151: 591±599 Forsberg C (1987) Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clostridium species. Appl Environ Microbiol 53: 639±643 Freund A (1881) UÈ ber die Bildung und Darstellung von Trimethylenalkohol aus Glycerin. Monatsh Chem 2: 636±641 Gatenby AA, Haynie SL, Nagarajan (1998) Method for the production of 1,3-propanediol by recombinant organisms. WO (E I DuPont de Nemours and Genencor International) Gottschalk G, Averho B (1990) Process for the microbial preparation of 1,3-propanediol from glycerol. EP A1

9 297 GuÈ nzel B, Yonsel S, Deckwer W-D (1991) Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2 m 3. Appl Microbiol Biotechnol 36: 289±295 Haynie SL, Wagner LW (1996) Process for making 1,3-propanediol from carbohydrates mixed microbial culture. WO (E I DuPont de Nemours) Held AM (1996) The fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae. Master's thesis, University of Wisconsin-Madison, USA Homann T, Tag C, Biebl H, Deckwer W-D, Schink B (1990) Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl Microbioil Biotechnol 33: 121±126 La end LA, Nagarajan V, Nakamura CE (1996) Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. WO96/53796 (E I DuPont de Nemours and Genencor International) Lin ECC (1976) Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol 30: 535±578 McCoy M (1998) Chemical makers try biotech paths. Chem Eng News 22 (June): 13±19 Menzel K, Zeng A-P, Deckwer W-D (1997a) Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol 56: 135±142 Menzel K, Zeng A-P, Deckwer W-D (1997b) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microbiol Technol 20: 82±86 Mickelson MN, Werkman CH (1940) Formation of trimethylenglycol from glycerol by Aerobacter. Enzymologia 8: 252±256 Millet P (1993) Retournement de la situation de la glyceâ rine. Informations Chimie 345: 102±104 Murphy DJ (1996) Engineering oil production in rape seed and other oil crops. Trends Biotechnol 14: 206±213 Nakas JP, Schaedle M, Parkinson CM, Coonley CE, Tanenbaum SW (1983) System development for linked-fermentation products of solvents from algal biomass. Appl Environ Microbiol 46: 1017±1023 Nevoigt E, Stahl U (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Revi 21: 231±241 Norbeck J, Pahlman A-K, Akhtr N, Blomberg A, Adler L (1996) Puri cation and characterization of two isoenzymes of DLglycerol-3-phosphatase form Saccharomyces cerevisiae. J Biol Chem 271: 13875±13881 Petitdemange H, Cherrier C, Raval G, Gay R (1976) Regulation of the NADH-ferredoxin oxidoreductase in Clostridia of the butyric group. Biochim Biophys Acta 421: 334±347 Petitdemange E, DuÈ rr C, Abbad-Andaloussi S, Raval G (1995) Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. J Ind Microbiol 15: 498±501 P ugmacher U, Gottschalk G (1994) Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii. Appl Microbiol Biotechnol 41: 313±316 Potera C (1997) Genencor & DuPont create ``green'' polyester. Gen Eng News 17: 17 Reimann A (1997) Produktion von 1,3-Propandiol aus Glycerin durch Clostridium butyricum DSM 5431 und produkttolerante Mutanten. Dissertation, University of Braunschweig Germany Reimann A, Biebl H (1996) Production of 1,3-propanediol by Clostridium butyricum DSM 5431 and product tolerant mutants in fedbatch culture. Feeding strategy for glycerol and ammonium. Biotechnol Lett 18: 827±832 Reimann A, Biebl H, Deckwer W-D (1998) Production of 1,3- propanediol by Clostridium butyricum in continuous culture with cell recycling. Appl Microbiol Biotechnol 49: 359±363 Saint-Amans S, Soucaille P (1995) Carbon and electron ow in Clostridium butyricum grown in chemostat culture on glucoseglycerol mixtures. Biotechnol Lett 17: 211±216 Saint-Amans S, Perlot P, Goma G, Soucaille P (1994) High production of 1,3-propanediol from glycerol by Clostridium butyricum VPI 3266 in a simply controlled fed-batch system. Biotechnol Lett 16: 832±836 SchuÈ tz H, Radler F (1984) anaerobic reduction of glycerol to propanediol-1,3 by Lactobacillus brevis and Lactobacillus buchneri. Syst Appl Microbiol 5: 169±178 Seyfried M, Daniel R, Gottschalk G (1996) Cloning, sequencing and overexpression of the genes encoding Coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii. J Bacteriol 178: 5793±5796 Skraly FA, Lytle BL, Cameron DC (1998) Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol 64: 98±105 Sprenger GA, Hammer GA, Johnson EA, Lin ECC (1989) Anaerobic growth of Escherichia coli on glycerol by importing genes of the dha regulon from Klebsiella pneumoniae. J Gen Microbiol 135: 1255±1262 Stinson SC (1995) Fine and intermediate chemical makers emphasize new projects and processes. Chem Eng News 17: 10±14 Sullivan CJ (1993) Propanediols, in Ullmann's Encyklopedia of Industrial Chemistry, vol A 22. VCH, Weinheim Tag CG (1990) Mikrobielle Herstellung von 1,3-Propandiol. Dissertation, University of Oldenburg, Germany Tobimatsu T, Azuma M, Matsubara H, Tskatori H, Niida T, Nishimoto K, Satoh H, Hayashi R, Toraya T (1996) Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamin-dependent glycerol-dehydratase of Klebsiella pneumoniae. J Biol Chem 271: 22352±22357 Tong I-T, Cameron DC (1992) Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing Klebsiella pneumoniae dha regulon. Appl Biochem Biotechnol 34/35: 149±159 Tong I-T, Liao HH, Cameron DC (1991) 1,3-Propanediol production by Escherichia coli expresing genes from the Klebsiella pneumoniae dha regulon. Appl Environ Microbiol 57: 3541±3546 Tran-dinh K, Hill FF (1987) Verfahren zur Herstellung von 1,3- Propandiol. DE A1 Veiga da Cunha M, Foster MA (1992) Sugar-glycerol cofermentations in lactobacilli: the fate of lactate. J Bacteriol 174: 1013± 1019 Voisenet E (1914) Sur un ferment contenu dans les eaux agent de deâ shydratation de la glyceâ rine. Ann Inst Pasteur 28: 807±818 Wilke D (1999) Chemicals from biotechnology: molecular plant genetics will challenge the chemical and the fermentation industry. Appl Microbiol Biotechnol (in press) Witt U, MuÈ ller R-J, Widdecke H, Deckwer W-D (1994) Syntheses, properties and biodegradability of polyesters based on 1,3- propanediol. Makromol Chem Phys 195: 793±802 Witt U, MuÈ ller R-J, Deckwer W-D (1995) New biodegradable polyester-copolymers from commodity chemicals with favorable use properties. J Environ Polym Degrad 3: 215±223 Witt U, MuÈ ller R-J, Deckwer W-D (1996) Biologisch abbaubare Polyester-Copolymere mit aromatischen Anteilen. DE (GBF) Zeng A-P (1996) Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioproc Eng 14: 169±175 Zeng A-P, Biebl H, Schlieker H, Deckwer W-D (1993) Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product inhibition. Enzyme Microb Technol 15: 771±779 Zeng A-P, Ross A, Biebl H, Tag C, GuÈ nzel B, Deckwer W-D (1994) Multiple product inhibition and growth modeling of Clostridium butyricum and Klebsiella pneumoniae in glycerol fermentation. Biotechnol Bioeng 44: 902±911