Elsevier

Metabolic Engineering

Volume 7, Issues 5–6, September–November 2005, Pages 329-336
Metabolic Engineering

Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol

https://doi.org/10.1016/j.ymben.2005.06.001 Get rights and content

Abstract

Clostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better “vitamin B12-free” biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3-propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104 mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L−1 h−1) and titer (788 mM).

Introduction

For a long time, 1,3-propanediol (1,3-PD) has been considered a specialty chemical. However, the recent development of a new polyester called poly(propylene terephtalate), with unique properties for the fiber industry (Miller, 2000; Rudie, 2000), necessitates a drastic increase in the production of this chemical. There are currently two processes for the chemical synthesis of 1,3-propanediol. Both of these processes produce toxic intermediates and require a reduction step under high hydrogen pressure (Sullivan, 1993). The biological production of 1,3-propanediol from glycerol was demonstrated for several bacterial strains, e.g., Lactobacillus brevis and buchnerii (Schütz and Radler, 1984; Sobolov and Smiley, 1959), Bacillus welchii (Humphreys, 1924), Citrobacter freundii, Klebsiella pneumoniae (Lin and Magasanik, 1960; Ruch et al., 1957; Streekstra et al., 1987), Clostridium pasteurianum (Luers et al., 1997) and Clostridium butyricum (Biebl et al., 1992; Heyndrickx et al., 1991; Saint-Amans et al., 2001). Among those microorganisms, C. butyricum is to our knowledge the best “natural producer” both in terms of yield and titer of 1,3-propanediol produced (Saint-Amans et al., 1994). However, to develop an economical process of 1,3-propanediol production, it is necessary to further improve the process by a metabolic engineering approach on the strain. No genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. On the other hand, we recently characterized, from a biochemical (Saint-Amans et al., 2001) and a molecular point of view (Raynaud et al., 2003), the B12-independent pathway converting glycerol to 1,3-propanediol in C. butyricum. This work opens the possibility to convert other clostridia to 1,3-propanediol producers by the heterologous expression of the genes encoding the B12-independent 1,3-propanediol pathway. Among the clostridia, Clostridium acetobutylicum is a microorganism of choice as (i) it has already been used for the industrial production of solvent (Cornillot and Soucaille, 1996) and (ii) the genetic tools for gene knockout or gene over-expression are currently available (Mermelstein and Papoutsakis, 1993; Green et al., 1996). The objective of the present work is to develop a recombinant strain of C. acetobutylicum for the conversion of glycerol to 1,3-propanediol at higher titer and productivity and if possible higher yield than those obtained in C. butyricum. We succeeded for the first two objectives but we failed in the yield improvement due to the metabolic flexibility of C. acetobutylicum.

Section snippets

Bacterial strains and plasmids

All bacterial strains and plasmids used or derived from this study are listed in Table 1.

DNA isolation and manipulation

Plasmid DNA was extracted from Escherichia coli with the Qiaprep Kit (Qiagen, Courtaboeuf, France). DNA restriction enzymes, CIP enzyme and T4 DNA ligase were obtained from New England Biolabs (Beverly, Mass) or GIBCO/BRL (Life Technologies, Cergy Pontoise, France) and used according to the manufacturer's instructions. DNA fragments were purified from agarose gels with the QIAquick gel purification kit

Engineering of C. acetobutylicum for the production of 1,3-propanediol

The conversion of glycerol to 1,3-propanediol in C. butyricum occurs in two steps catalyzed by the B12-independent glycerol dehydratase and the 1,3-propanediol dehydrogenase, consuming 1 mol of NADH. The pSPD5 plasmid (Raynaud et al., 2003) carrying the 1,3-propanediol operon from C. butyricum and the pIMP1 as a control plasmid were introduced in C. acetobutylicum ATCC 824 and the DG1 mutant, which is cured from the pSOL1 megaplasmid and which is then unable to produce solvents and sporulate (

Discussion

C. acetobutylicum cannot grow on glycerol as it cannot re-oxidize the excess of NADH generated in glycerol catabolism (Vasconcelos et al., 1994; Girbal et al., 1995). On the other hand, when the NADH consuming 1,3-propanediol pathway from C. butyricum was introduced in C. acetobutylicum growth on glycerol was achieved and 1,3-propanediol was the main fermentation product. C. acetobutylicum DG1 (pSPD5) was the most efficient recombinant strain for the conversion of glycerol to 1,3-propanediol.

Acknowledgments

This work was financially supported by the European Committee Fourth Project (contract no. QLK5-CT-1999-01364) and the Agence de l’Environnement et de la Maitrise de l’Energie (contract no. 00 01 027). M. González-Pajuelo was supported by PRAXIS XXI with a Ph.D. grant (BD/16036/98).

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    María González-Pajuelo and Isabelle Meynial-Salles contributed equally to this work.

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