QLK5-1999-01364 PROBIO: 1,3-Propanediol - a versatile bulk chemical from renewable resources by novel biocatalysts and process strategies

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Contract No:	QLK5-1999-01364
Source:	Progress Report - June 2001 - Abstract

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Progress Report - June 2001 - Abstract

Objectives

The overall aim of this project is to develop improved methods for the production of propanediol by fermentation. The objectives for the reporting period (and corresponding work packages) were to:

• improve glycerol formation in yeast (WPl)
• develop a strain of Escherichia coli that converts glucose to glycerol (WP2)
• express a glycerol pathway in Clostridium acetobutylicum (WP3)
• improvement of 1,3-propanediol (1,3-PD) production in Klebsiella pneumoniae (WP4)
• analyse chemical flux and control mechanisms in wild type strains (WP5)
• develop a two-stage process for 1,3-propanediol production (WP6)

Results and Milestones

For an improved production of glycerol in yeasts both genetic and physiological approaches have been applied. The industrial yeast strain CENPK2 with appropriate auxotrophies (his3, leu2, trpl and ura3) was first used as a model strain for the over-expression of glycerol phosphate dehydrogenase (GPD1), a key enzyme for the formation of glycerol from glucose. The successful overexpression of GPD1 led to a ten times increase of glycerol formation in this strain. Work is going on to genetically modify several other steps in glycerol synthesis. In relation to the development of a two-stage process for 1,3-PD production glucose fermentation to glycerol was also studied with an osmotolerant strain Pichia farinosa. This strain can produce more than 70 g/l glycerol with a yield of 0.35 g glycerol/g glucose.

Significant progress has been achieved in genetically constructing E coli strains that convert glucose into glycerol. A synthetic operon containing the GPD1 and GPP2 (encoding glycerol-3-phosphate phosphatase) genes from S. cerevisiae was constructed and successfully expressed in E coli K12 with a pS3 shuttle vector. To increase the glycerol yield, the triose phosphate isomerase of E. coli K12 was deleted from the chromosome, leading to a very efficient deltatpiA E. coli K12 strain (MG 1655). Using a fed-batch culture with this strain, glycerol was produced from glucose at both high concentration (160 g/1) and yield (0.5 g glycerol/g glucose).

Progress has also been made with respect to genetically engineering Clostridium acetobutyricum to convert glucose (starch) to glycerol. The glycerol yield was however lower than that of the deltatpiA E. coli K12 strain, mainly due to formation of butyrate. To avoid butyrate formation, a new shuttle vector pTLS3 was constructed to express the GPD1 and GPP2 genes in a butyrate minus strain of C. acetobutyricum ATCC 824. The functionality of the genes in this vector was confirmed in E. coli deltaAtpia. Work is continuing in order to transform the butyrate minus strain of C. acetobutyricum ATCC 824 with pTLS3 and to express a synthetic 1,3-PD operon.

To improve 1,3-PD production in K. pneumoniae a synthetic operon over-expressing the genes dhaB (for glycerol dehydratase) and dhaT (for 1,3-PD oxidoreductase) has been constructed and successfully expressed in this strain, leading to a 46 to 68-folds increase in the activities of these enzymes compared to the wild-type strain in shake flask cultures. The productivity of 1,3-PD and regulation of gene expression in the recombinant strain are being investigated under real fermentation conditions. In an effective metabolic engineering step the dha regulon of K. pneumoniae has been reconstructed and expanded the from genomic data and compared it with the dha regulon from other organisms. Several new regulatory proteins and domains have been identified.

The two-stage process was first studied with glycerol production from glucose by the yeast P. farinosa and subsequent conversion of the glycerol to 1,3-PD by K. pneumoniae. It turned out that the culture broth of P. farinosa contained metabolites that strongly inhibit the growth of K. pneumoniae and alter the product spectrum. Acetate was identified as a major metabolite that affected the conversion of glycerol to 1,3-PD. Work has been done to remove acetate by using the acetate consuming methanogenic bacterium Methanosarcina barkeri. Preliminary results showed that the growth of M. barkeri on acetate is too slow for this process.

The metabolic fluxes and activities of key enzymes of several Clostridium strains with or without genetic modifications have been quantitatively studied. The recombinant C. acetobutyricum DG1 pSPD5 was shown to produce 1,3-PD at a higher yield (0.64 mol/mol) than the wild-type strain C. butyricum (0.58 mol/mol) under similar conditions. The metabolic flux analysis gives useful hints for further genetic improvement of clostridia to produce glycerol and 1,3-PD. As suggested from the flux analysis, a butyrate minus Clostridium strain with a simultaneously suppressed butanol pathway is under development and should be optimised for 1,3-PD production.

Benefits and Beneficiaries

The recombinant deltatpiA E coli K12 strain (MG 1655) with a high glycerol concentration (160 g/1) and yield represents a significant achievement for the development of the envisaged one and two-stage processes for the conversion of glucose to 1,3-propanediol. The results with E. coli are also beneficial for metabolic engineering of K. pneumoniae, clostridia and yeasts.