US20110217744A1

US20110217744A1 - Use of sucrose as substrate for fermentative production of 1,2-propanediol

Info

Publication number: US20110217744A1
Application number: US13/127,642
Authority: US
Inventors: Francois Voelker; Rainer Figge; Philippe Soucaille
Original assignee: Metabolic Explorer SA
Current assignee: Metabolic Explorer SA
Priority date: 2008-11-07
Filing date: 2008-11-07
Publication date: 2011-09-08
Also published as: CN102272316B; KR101395971B1; BRPI0823256A2; KR20110088546A; AR074092A1; EP2346999A1; BRPI0823256B8; US9617567B2; DK2346999T3; ES2429305T3; MX2011004842A; WO2010051849A1; CA2741427A1; EP2346999B1; JP2012507992A; US20150159181A1; BRPI0823256B1; CN102272316A

Abstract

The present invention is relative to a method for producing 1,2-propanediol by fermentation, comprising: cultivating a microorganism producing 1,2-propanediol in an appropriate medium comprising a source of sucrose, and recovering the 1,2-propanediol being produced, wherein the microorganism is able to utilize sucrose as sole carbon source for the production of 1,2-propanediol. In a preferred aspect of the invention, the source of sucrose is obtained from plant biomass, and is in particular sugar cane juice.

Description

The present invention relates to fermentation processes, to microorganisms and to substrates useful for fermentation. In particular, this invention is related to the production of 1,2-propanediol by fermentation, from a sucrose-containing medium, in particular from plant biomass.
1,2-propanediol or propylene glycol, a C3 dialcohol, is a widely-used chemical. It is a component of unsaturated polyester resins, liquid detergents, coolants, anti-freeze and de-icing fluids for aircraft. Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives.
1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water. Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances. In addition, the hydroperoxide route generates by-products such as tert-butanol and 1-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products. The chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and (S)1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products).

PRIOR ART

The disadvantages of the chemical processes for the production of 1,2-propanediol make biological synthesis an attractive alternative. Two routes have been characterized for the natural production of 1,2-propanediol from sugars by microorganisms. In the first route 6-deoxy sugars (e.g. L-rhamnose or L-fucose) are cleaved into dihydroxyacetone phosphate and (S)-lactaldehyde, which can be further reduced to (S)-1,2-propanediol (Badia et al, 1985). This route is functional in E. coli, but can not yield an economically feasible process due to the elevated cost of the deoxyhexoses. The second route is the metabolism of common sugars (e.g. glucose or xylose) through the glycolysis pathway followed by the methylglyoxal pathway. Dihydroxyacetone phosphate is converted to methylglyoxal that can be reduced either to lactaldehyde or to acetol. These two compounds can then undergo a second reduction reaction yielding 1,2-propanediol. This route is used by natural producers of (R)-1,2-propanediol, such as Clostridium sphenoides and Thermoanaerobacter thermosaccharolyticum. These two organisms have been used to produce 1,2-propanediol from different sugars, namely monosaccharides (D-glucose, D-mannose, D-galactose for the hexoses and D-xylose or L-arabinose for the pentoses) or disaccharides (lactose or cellobiose) or mixtures (Tran Din and Gottschalk, 1985, Cameron and Cooney, 1986, Sanchez-Rivera et al, 1987, Altaras et al, 2001). The best performance obtained was a titer of 9 g/l and a yield from glucose of 0.2 g/g (Sanchez-Rivera et al, 1987). However, the improvement of the performances obtained with these organisms is likely to be limited due to the shortage of available genetic tools. The same synthesis pathway is functional in E. coli or other Enterobacteriaceae and several investigations have been done by the group of Cameron (Cameron et al, 1998, Altaras and Cameron, 1999, Altaras and Cameron, 2000) and the group of Bennett (Huang et al, 1999, Berrios-Rivera et al, 2003) for the production of 1,2-propanediol in this organism with carbon sources limited to D-glucose or D-fructose. The best result obtained in an anaerobic fed-batch fermenter was a production of 4.5 g/l 1,2-propanediol with a yield of 0.19 g/g from glucose (Altaras and Cameron, 2000). Results obtained with the same approach but with lower titers and yields are also described in the patent WO 98/37204, although using different carbon sources, namely galactose, lactose, sucrose and xylose but also glucose. The titers obtained with disaccharides (lactose and sucrose) were very low (6 mg/l and 7 mg/l respectively). Better production results were described with a rationally designed then evolved E. coli strain in patent application WO 2005/073364. A 1,2-propanediol titer of 1.8 g/l was obtained, with a yield of 0.35 g/g of glucose consumed. Production of 1,2-propanediol and hydroxyacetone was also described using recombinant yeast in patent WO 99/28481.
Carbon sources used in fermentation media generally consisted in carbohydrates, mostly derived from plants. Sucrose is obtained from sugar plants such as sugar beet, sugarcane, sweet sorghum, sugar maple, sugar palms or blue agaves. Starch is the most abundant storage carbohydrate in plants. The most important starch sources are cereals (corn, wheat, rice), manioc, sweet potatoes and potatoes. Starch is not metabolized by most microorganisms but can be processed to fermentable feedstocks by the starch industry. Inulin or inulin-like polymers (mainly consisting of fructose units) are the second most abundant storage carbohydrate in plants and are found in chicory, Jerusalem artichoke or dahlia. Lignocellulosic biomass composed of cellulose, hemicellulose and lignin is also a promising source of carbohydrate but still under development (Peters, 2006). As the cost of the biotechnologically produced commodity chemicals are mainly related to the cost of raw material (i.e. the cost of the fermentation substrate), use of refined sugars such as glucose or sucrose is not an economically sustainable choice for industrial scale production. Less expensive substrates are needed that retain a high content of fermentable sugar. In this respect, sucrose containing carbon sources coming from the sugar industry represent a good option.
Sucrose is produced from sugar beet containing 16 to 24% sucrose by sugar beet processing in several steps. The cleaned and washed beets are sliced into long strips called cossettes that are extracted with hot water by diffusion to get a sucrose juice called raw juice and containing 10 to 15% sucrose. The second step is the purification of the raw juice by alkalization and carbonation using lime and then carbon dioxide to remove the impurities and get the thin juice. The evaporation process increases the sucrose concentration in the thin juice by removing water to get the thick juice with a sucrose content of 50 to 65%. This concentrated sucrose juice is then crystallised and the crystals are separated by centrifugation and then washed and dried to get pure sugar. One or more crystallisation steps can be applied to get sucrose of various purity grades. By-products of sugar beet processing include pulp (the exhausted cossettes) and molasses (the remaining mother-liquor from the crystallisation having still a sucrose content of 40 to 60%).
Sucrose is also produced from sugar cane (7 to 20% sucrose content) by the sugar industry. The harvested sugar cane is cleaned before the milling process for extraction of the juice. The structure of the cane is broken and then grinded and at the same time the sucrose is extracted with water to get the raw juice. The crushed cane exhausted from sugar is called bagasse. This residue is primarily used as fuel source to generate process steam. The raw juice is then clarified by adding lime and heating and the clarified juice is separated from the precipitate. The lasts steps of the process, evaporation to get a concentrated syrup and crystallisation are essentially the same as for the sugar beet processing. The by-products of sugarcane processing include bagasse, filter cake from clarification of raw juice and different kind of molasses, still containing significant amount of sucrose.
The different sucrose containing intermediates, products or by-products from the sugar processes (raw juice, thin or clarified juice, thick juice, sucrose syrup, pure sucrose, molasses) may serve as fermentation feedstock. For example, the sugar industry in Brazil is using the clarified sugarcane juice for ethanol production in order to use it as a substitute to gasoline. Recent examples in literature using crude sucrose containing products include ethanol production from sugar beet diffusion juice by Zymomonas mobilis (Beckers et al., 1999), production of D-lactate from molasses by E. coli (Shukla et al., 2004) and production of D-lactate from sugarcane molasses, sugarcane juice or sugar beet juice by Lactobacillus delbrueckii (Calabia et al., 2007).
Two different systems have been characterized for the uptake and utilization of sucrose in sucrose-positive bacteria (i.e. bacteria able to utilize sucrose)
The first one is based on a phosphoenol pyruvate (PEP)-dependent sucrose phosphotransferase system (sucrose PTS) where sucrose is taken up and phosphorylated using phosphoenol pyruvate (PEP) as a donor to yield intracellular sucrose-6-phosphate. Sucrose-6-phosphate is then hydrolysed to D-glucose-6-phosphate and D-fructose by an invertase. D-fructose is further phosphorylated to D-fructose-6-phosphate by an ATP-dependent fructokinase and can then enter the central metabolism. Such a system has been described in several bacterial species, gram-positive as well as gram-negative. Among Enterobacteriaceae, more than 90% of wild-type Klebsiella but less than 50% of Escherichia and less than 10% of Salmonella strains are sucrose positive.
A conjugative plasmid pUR400 bearing the genes scrKYABR coding for the sucrose PTS has been isolated from Salmonella (Schmid et al., 1982, Schmid et al., 1988).
A second non-PTS system was discovered more recently in E. coli EC3132 (Bockmann et al., 1992). This system involve the genes cscBKAR coding for a sucrose:proton symport transport system (CscB), a fructokinase (CscK), an invertase (CscA) and a sucrose-specific repressor (CscR).
Escherichia coli K12 and its derivatives (including MG1655) cannot utilize sucrose. However, this ability can be conferred by the transfer of the genes coding for the two previously described systems. This has been demonstrated by transferring the plasmid pUR400 in E. coli K12 (Schmid et al, 1982) or different plasmids (including pKJL101-1) bearing the cscBKAR genes in a sucrose negative strain of E. coli (Jahreis et al., 2002). As for industrial application, tryptophan production from sucrose has been documented in E. coli K12 (Tsunekawa et al., 1992), hydrogen production was shown in E. coli carrying the pUR400 plasmid (Penfold and Macaskie, 2004) and production of different amino-acids by transferring both systems, PTS and non-PTS was reported in patent application EP1149911.
Production of 1,2-propanediol from sucrose is mentioned in Clostridium thermosaccharolyticum (later renamed T. thermosaccharolyticum) by Cameron and Cooney (1986) but only traces were recorded whereas amount higher than 3 g/l with yields higher than 0.1 g/g substrate were obtained with other carbon sources.
Production of 1,2-propanediol from sucrose is also mentioned in patent application WO98/37204. However, the strain E. coli AA200 transformed with the plasmid pSEARX produces only 7 mg/l of 1,2-propanediol from 10 g/l of sucrose, whereas the same microorganism produces from 49 to 71 mg/l of 1,2-propanediol from monosaccharides. These very low figures of production cast doubt about the true ability of these organisms to produce 1,2-propanediol from sucrose. In our opinion, the strain AA200, which is derived from E. coli K12, should not have the ability to import and then metabolize sucrose.
These previous reports clearly indicated to the man skilled in the art that the use of sucrose to produce 1,2-propanediol was not a good option.
Surprisingly, by introducing different systems for sucrose utilization in E. coli strains unable to utilize sucrose, the inventors of the present invention were able to obtain improved yield for 1,2-propanediol production from sucrose.
Furthermore, the inventors demonstrated that any sucrose-containing medium, such as a juice or molasses from a plant feedstock, could be used as a substrate for the fermentative production of 1,2-propanediol.

DESCRIPTION OF THE INVENTION

The present invention is relative to a method for producing 1,2-propanediol by fermentation, comprising:

- cultivating a microorganism producing 1,2-propanediol in an appropriate medium comprising a source of sucrose, and
- recovering the 1,2-propanediol being produced,
  wherein the microorganism is able to utilize sucrose as sole carbon source for the production of 1,2-propanediol.

In particular, the invention describes the use of a modified microorganism able to use sucrose as a sole source of carbon, said sucrose being obtained from biomass, in particular from plant biomass.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms may be used for interpretation of the claims and specification.
According to the invention the terms ‘cultivating’, ‘culture’, ‘growth’ and ‘fermentation’ are used interchangeably to denote the growth of bacteria in an appropriate growth medium containing a simple carbon source. Fermentation is a classical process that can be performed under aerobic, microaerobic or anaerobic conditions.
The term ‘appropriate medium’ according to the invention denotes a medium of known molecular composition adapted to the growth of the micro-organism. In particular, said medium contains at least a source of phosphorus and a source of nitrogen. Said appropriate medium is for example a mineral culture medium of known set composition adapted to the bacteria used, containing at least one carbon source. Said appropriate medium may also designate any liquid comprising a source of nitrogen and/or a source of phosphorus, said liquid being added and/or mixed to the source of sucrose. In particular, the mineral growth medium for Enterobacteriaceae can thus be of identical or similar composition to M9 medium (Anderson, 1946), M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al. (1999), and in particular the minimum culture medium named MML11PG1_—100 described below:

TABLE 1

composition of minimal medium MML11PG1_100
with glucose as carbon source.

	EDTA	0.0084
	CoCl₂6H₂O	0.0025
	MnCl₂4H₂O	0.0150
	CuCl₂2H₂O	0.0015
	H₃BO₃	0.0030
	Na₂MoO₄2H₂O	0.0025
	Zn(CH₃COO)₂2H₂O	0.0130
	Fe(III) citrate H₂O	0.1064
	Citric acid	1.70
	KH₂PO₄	1.65
	K₂HPO₄3H₂O	0.92
	(NH₄)₂HPO₄	0.40
	(NH₄)₂SO₄	4.88
	MgSO₄7H₂O	1.00
	CaCl₂2H₂O	0.08
	Thiamine	0.01
	Glucose	20.00
	MOPS buffer	40.00

The pH of the medium is adjusted to 6.8 with sodium hydroxide.
The carbon source ‘glucose’ can be replaced in this medium by any other carbon source, in particular by sucrose or any sucrose-containing carbon source such as sugarcane juice or sugar beet juice.
The growth medium for Clostridiaceae can be of identical or similar composition to Clostridial Growth Medium (CGM, Wiesenborn et al., 1987) or a mineral growth medium as given by Monot et al. (1982) or Vasconcelos et al. (1994).
The term ‘sucrose’ designates a disaccharide of glucose and fructose linked by a α(1,2) glycosidic bond, with the molecular formula C₁₂H₂₂O₁₁. Its systematic name is α-D-glucopyranosyl-(1
2)-β-D-fructofuranoside.
The term ‘sucrose source’ or ‘source of sucrose’ designates any medium, liquid or solid, containing sucrose in different concentrations, in particular from 1 to 100% of sucrose.
The term ‘producing 1,2-propanediol’ means that the production of the microorganism in the culture broth can be recorded unambiguously by standard analytical means known by those skilled in the art. The limit of quantification of HPLC for 1,2-propanediol, which is the preferred technique used to quantify this compound, is 25 mg/l. Therefore, the term “producing 1,2-propanediol” means according to the invention that the production levels have to be above 25 mg/l.
The term ‘able to utilize sucrose as sole carbon source’ indicates that the microorganism can grow in a medium containing sucrose as unique carbon source. Therefore, the definition of a “microorganism able to utilize sucrose as sole carbon source for the production of 1,2-propanediol” means that the microorganism, when grown in a medium containing sucrose as sole carbon source, can produce at least 25 mg/l of 1,2-propanediol. It is however understood that in the method for producing 1,2-propanediol according to the invention, the sucrose source in the culture medium can comprise additional carbon sources in addition to sucrose such as hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, disaccharides (such as sucrose, cellobiose or maltose)), oligosaccharides, starch or its derivatives, hemicelluloses, glycerol and combinations thereof.
According to a preferred aspect of the invention, the microorganism has been genetically modified to be able to utilize sucrose as sole carbon source, for the production of 1,2-propanediol.
In a specific embodiment of the invention, the microorganism comprises functional genes coding for a PTS sucrose utilization system. A PTS sucrose utilization system is a system for sucrose utilization based on the transport of sucrose by a phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (Sucrose-PTS). A phosphotransferase system couples the transport of a sugar (e.g. sucrose or glucose) with the phosphorylation of the sugar using PEP as phosphate donor. After transport into the cell, the sucrose-phosphate is cleaved into glucose-6-phosphate and fructose by an invertase. Fructose is then phosphorylated into fructose-6-phosphate by a fructokinase. The genes coding for this PTS sucrose utilization system can be controlled by a regulatory protein.
In a preferred aspect of the invention, the microorganism expresses naturally or has been modified with the introduction of the genes: scrKYABR (scrK coding for a fructokinase, scrY coding for a porin, scrA coding for the Protein IIBC, scrB coding for a sucrose-6-P invertase, scrR coding for a repressor) from Salmonella. A conjugative plasmid pUR400 bearing said genes scrKYABR might be used to transform the microorganism. These genes can be used all together in combination, or in any combination comprising at least one of these genes. In particular, the gene scrR can be omitted.
The designation of these genes has a more general meaning according to the invention, and covers the corresponding genes in other micro-organisms. Using the GenBank references of the genes from Salmonella, those skilled in the art can determine equivalent genes in other organisms than Salmonella.
The means of identification of the homologous sequences and their percentage homologies are well-known to those skilled in the art, and include in particular the BLAST programmes that can be used on the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website. The sequences obtained can be exploited (aligned) using for example the programmes CLUSTALW (http://www.ebi.ac.uk/clustalw/), with the default parameters indicated on these websites.
The PFAM database (protein families database of alignments and hidden Markov models http://www.sanger.ac.uk/Software/Pfam/) is a large collection of alignments of protein sequences. Each PFAM makes it possible to visualise multiple alignments, view protein domains, evaluate distributions among organisms, gain access to other databases and visualize known protein structures.
COGs (clusters of orthologous groups of proteins http://www.ncbi.nlm.nih.gov/COG/) are obtained by comparing protein sequences derived from 66 fully sequenced unicellular genomes representing 14 major phylogenetic lines. Each COG is defined from at least three lines, making it possible to identify ancient conserved domains.
Several techniques are currently used by the man skilled in the art for introducing DNA into a bacterial strain. A preferred technique is electroporation, which is well known to those skilled in the art.
In another embodiment of the invention, the microorganism comprises functional genes coding for a non-PTS sucrose utilization system. A non-PTS sucrose utilization system is a system for sucrose utilization based on transport of sucrose by a system independent of phosphoenolpyruvate. After transport into the cell, the sucrose is cleaved into glucose and fructose by an invertase. Fructose is then phosphorylated into fructose-6-phosphate by a fructokinase and glucose is phosphorylated into glucose-6-phosphate by a glucokinase. The genes coding for this non-PTS sucrose utilization system can be controlled by a regulatory protein. In a preferred aspect of the invention, the microorganism expresses naturally or has been modified with the introduction of the genes from E. coli EC3132 i.e. the genes cscBKAR coding for a sucrose:proton symport transport system (cscB), a fructokinase (cscK), an invertase (cscA) and a sucrose-specific repressor (cscR). These genes can be used all together in combination or in any combination comprising at least one of these genes. In particular, the gene cscR can be omitted. Homologous genes from other organisms can also be used.
The designation of these genes has a more general meaning according to the invention, and covers the corresponding genes in other micro-organisms. Using the GenBank references of the genes from E. coli, those skilled in the art can determine equivalent genes in other organisms than E. coli (see above).
In a specific aspect of the invention, the microorganism is characterized by an improved activity of the biosynthesis pathway of 1,2-propanediol. Microorganisms optimized for the production of 1,2-propanediol have been extensively disclosed in patent applications WO 2005/073364, WO 2008/116853, WO 2008/116852 and WO 2008/116848, that are here incorporated as references.
The microorganisms according to the invention are bacteria, yeast or fungi.
Preferentially, the microorganism is selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae and Corynebacteriaceae.
More preferentially, the microorganism is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides or Saccharomyces cerevisiae.
The culture conditions for the fermentation process can be readily defined by those skilled in the art. In particular, bacteria are fermented at temperatures between 20° C. and 55° C., preferably between 25° C. and 40° C., and preferably at about 35° C. for Clostridiaceae and at about 37° C. for Enterobacteriaceae.
This process can be carried out either in a batch process, in a fed-batch process or in a continuous process. Fermentation is a classical process that can be performed under aerobic, microaerobic or anaerobic conditions.
‘Under aerobic conditions’ means that oxygen is provided to the culture by dissolving the gas into the liquid phase. This could be obtained by (1) sparging oxygen containing gas (e.g. air) into the liquid phase or (2) shaking the vessel containing the culture medium in order to transfer the oxygen contained in the head space into the liquid phase. Advantages of the fermentation under aerobic conditions instead of anaerobic conditions is that the presence of oxygen as an electron acceptor improves the capacity of the strain to produce more energy in form of ATP for cellular processes. Therefore the strain has its general metabolism improved.
Micro-aerobic conditions are defined as culture conditions wherein low percentages of oxygen (e.g. using a mixture of gas containing between 0.1 and 10% of oxygen, completed to 100% with nitrogen), is dissolved into the liquid phase.
Anaerobic conditions are defined as culture conditions wherein no oxygen is provided to the culture medium. Strictly anaerobic conditions are obtained by sparging an inert gas like nitrogen into the culture medium to remove traces of other gas. Nitrate can be used as an electron acceptor to improve ATP production by the strain and improve its metabolism.
In a specific aspect of the invention, the sucrose source is obtained from biomass, in particular from plant biomass. The whole plant or any specific part of a plant can be used to prepare the raw material used as sucrose source. The preparation can be based on any treatment known by those skilled in the art to extract sucrose from a sucrose-containing plant biomass.
In a preferred aspect of the invention, the sucrose source is obtained from a plant chosen among the group consisting of: sugarcane, sugar beet, sweet sorghum, sugar maple, sugar palm and blue agave.
The source of sucrose may in particular be obtained from sugarcane or sugar beet.
Different forms of sucrose source can be used according to the invention, such as a juice, a concentrated juice, a syrup, a clarified juice, molasses or crystallized sucrose.
A preferred form is the raw juice from sugar cane, directly extracted from the plant without any treatment. Briefly, the harvested sugar cane is cleaned before the milling process for extraction of the juice. The structure of the cane is broken and then grinded, and at the same time the sucrose is extracted with water to get the raw juice.
The raw juice may then be clarified by adding lime and heating and the clarified juice is separated from the precipitate. Concentrated syrup is obtained by evaporation.
In another embodiment of the invention, the sucrose source may be a final product, an intermediate product or a by-product of the sugarcane or sugar beet industry.
As some crude sucrose sources, particularly those obtained from biomass as mentioned above, contain other nutrients that can be used for growth of microorganisms in addition to the carbon source, an appropriate medium for the growth of microorganisms can be designed either by using the sucrose source alone, i.e. the appropriate medium consists of the source of sucrose, or by complementing the sucrose source with a source of phosphorus and/or a source of nitrogen.
Preferentially, the sucrose source comprises at least 7% of sucrose.

EXAMPLES

Example 1

Construction of Two Strains of E. coli Producing 1,2-Propanediol and Able to Utilize Sucrose as Sole Carbon Source

The E. coli strain MG1655 lpd*, ΔtpiA, ΔpflAB, ΔadhE, ΔldhA::Cm, ΔgloA, ΔaldA, ΔaldB, Δedd evolved in chemostat culture under anaerobic conditions and adapted for growth in minimal medium was obtained as described in WO 2008/116852. This strain was named evolved strain E. coli MG1655 lpd*, ΔtpiA, ΔpflAB, ΔadhE, ΔldhA::Cm, ΔgloA, ΔaldA, ΔaldB, Δedd.
The chloramphenicol resistance cassette was eliminated in said evolved strain and the presence of the modifications previously built in the strain was checked as previously described in WO 2008/116852.
Two plasmids were used to confer the ability to utilize sucrose to said E. coli strain:

- pUR400, bearing the genes coding for the sucrose-PTS system such as described in Schmid et al. (1982)
- pKJL101.1, bearing the genes coding for the sucrose permease and kinase system such as described in Jahreis et al. (2002).

These plasmids were introduced separately into the evolved strain E. coli MG1655 lpd*, ΔtpiA, 66 pflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA, ΔaldB, Δedd by electroporation.
The two strains obtained were named respectively:

- evolved E. coli MG1655 lpd*, ΔtpiA, ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA, ΔaldB, Δedd (pUR400), and
- evolved E. coli MG1655 lpd*, ΔtpiA, ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA, ΔaldB, Δedd (pKJL101.1).

Example 2

Production of 1,2-Propanediol with Sucrose as Sole Carbon Source

The strains obtained as described in Example 1 and the strain without plasmid used as control were cultivated in an Erlenmeyer flask assay under aerobic conditions in minimal medium MML11PG1_—100 (see composition above) with 20 g/l glucose or sucrose as sole carbon source. Glucose as carbon source was used as control.
The culture was carried out at 34° C. and the pH was maintained by buffering the culture medium with MOPS.
At the end of the culture, 1,2-propanediol, and residual glucose or sucrose in the fermentation broth were analysed by HPLC and the yields of 1,2-propanediol over glucose or the yields of 1,2-propanediol over sucrose were calculated.

TABLE 2

production of 1,2-propanediol in minimal medium with
glucose or sucrose as carbon source.

	1,2-propanediol	1,2-propanediol
	titer	yield (g/g
Strain/Carbon source	(g/l)	carbon source)

Evolved E. coli MG1655 lpd*, ΔtpiA,	ND	—
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 1)	(n = 1)
ΔaldB, Δedd/sucrose
Evolved E. coli MG1655 lpd*, ΔtpiA,	3.89 +/− 0.12	0.20 +/− 0.01
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 3)	(n = 3)
ΔaldB, Δedd (pUR400)/glucose
Evolved E. coli MG1655 lpd*, ΔtpiA,	2.26 +/− 0.27	0.12 +/− 0.01
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 6)	(n = 6)
ΔaldB, Δedd (pUR400)/sucrose
Evolved E. coli MG1655 lpd*, ΔtpiA,	3.55 +/− 0.21	0.19 +/− 0.01
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 3)	(n = 3)
ΔaldB, Δedd (pKJL101.1)/glucose
Evolved E. coli MG1655 lpd*, ΔtpiA,	4.86 +/− 0.77	0.26 +/− 0.03
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 6)	(n = 6)
ΔaldB, Δedd (pKJL101.1)/sucrose

ND means ‘not detected’.
n is the number of repetitions of the same experiment.
The figures given are the mean and standard deviation of the figures obtained for n repetitions.

Example 3

Production of 1,2-Propanediol with Sugarcane Juice as Carbon Source

The strains obtained as described in Example 1 were cultivated in an Erlenmeyer flask assay under aerobic conditions in minimal medium MML11PG1_—100 with sugarcane juice (20 g/l sucrose equivalent) as carbon source.
The sugarcane juice used in this experiment was obtained from a sugar mill in the south-east asia area and was collected right after the clarification with lime of the raw juice
The culture was carried out at 34° C. and the pH was maintained by buffering the culture medium with MOPS. At the end of the culture, 1,2-propanediol, and residual sucrose, glucose and fructose in the fermentation broth were analysed by HPLC and the yield of 1,2-propanediol over the sum of carbon sources was calculated.

TABLE 3

production of 1,2-propanediol in minimal medium with sugarcane
juice as sucrose source.

	1,2-propanediol	1,2-propanediol
	titer	yield (g/g
Strain/Carbon source	(g/l)	carbon source)

Evolved E. coli MG1655 lpd*, ΔtpiA,	3.43 +/− 0.22	0.15 +/− 0.01
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 2)	(n = 2)
ΔaldB, Δedd (pUR400)/sugarcane juice
Evolved E. coli MG1655 lpd*, ΔtpiA,	3.94 +/− 0.94	0.26 +/− 0.01
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 3)	(n = 3)
ΔaldB, Δedd (pKJL101.1)/sugarcane
juice

n is the number of repetitions of the same experiment.
The figures given are the mean and standard deviation of the figures obtained for n repetitions.

Example 4

Production of 1,2-Propanediol with Sugarcane Juice Alone or Supplemented with Nutrients

The strains obtained as described in Example 1 were cultivated in an Erlenmeyer flask assay under aerobic conditions in a medium containing diluted sugarcane juice (20 g/l sucrose equivalent) either without supplementation or supplemented with phosphate and ammonium ((NH₄)₂HPO₄2.5 g/l), iron (Fe Citrate, H₂O 0.1 g/l) and thiamine (0.02 g/l).
The culture was carried out at 34° C. and the pH was maintained by buffering the culture medium with MOPS (40 g/l). At the end of the culture, 1,2-propanediol, and residual sucrose, glucose and fructose in the fermentation broth were analysed by HPLC and the yield of 1,2-propanediol over the sum of carbon sources was calculated.

TABLE 4

production of 1,2-propanediolin sugarcane juice without supplementation
or in supplemented sugarcane juice.

	1,2-propanediol	1,2-propanediol
	titer	yield (g/g
Strain/Carbon source	(g/l)	carbon source)

Evolved E. coli MG1655 lpd*, ΔtpiA,	0.42	0.05
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 1)	(n = 1)
ΔaldB, Δedd (pUR400)/sugarcane juice
Evolved E. coli MG1655 lpd*, ΔtpiA,	1.07	0.12
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 1)	(n = 1)
ΔaldB, Δedd (pKJL101.1)/sugarcane
juice
Evolved E. coli MG1655 lpd*, ΔtpiA,	2.29 +/− 0.02	0.14 +/− 0.00
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 2)	(n = 2)
ΔaldB, Δedd (pUR400)/supplemented
sugarcane juice
Evolved E. coli MG1655 lpd*, ΔtpiA,	4.08 +/− 0.02	0.26 +/− 0.00
ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA,	(n = 2)	(n = 2)
ΔaldB, Δedd (pKJL101.1)/supplemented
sugarcane juice

n is the number of repetitions of the same experiment.
The figures given are the mean and standard deviation of the figures obtained for n repetitions

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Claims

1. A Method for producing 1,2-propanediol by fermentation, comprising:

cultivating a microorganism producing 1,2-propanediol in an appropriate medium comprising a source of sucrose, and

recovering the 1,2-propanediol being produced,

wherein the microorganism is able to utilize sucrose as sole carbon source for the production of 1,2-propanediol.

2. A Method according to claim 1, wherein the microorganism has been genetically modified to be able to utilize sucrose as said sole carbon source.

3. A Method according to claim 1, wherein the microorganism has functional genes coding for a PTS sucrose utilization system.

4. A Method according to claim 3 wherein the microorganism has been modified by introduction of genes scrKYABR.

5. A Method according to claim 1, wherein the microorganism has functional genes coding for a non-PTS sucrose utilization system.

6. A Method according to claim 5 wherein the microorganism has been modified with the introduction of genes cscBKAR.

7. A Method according to claim 1, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.

8. A Method according to claim 7 wherein the microorganism is selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae and Corynebacteriaceae.

9. A Method according to claim 8 wherein the microorganism is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides and Saccharomyces cerevisiae.

10. A Method according to claim 1, wherein said source of sucrose is obtained from biomass.

11. A Method according to claim 10, wherein said source of sucrose is obtained from a plant selected from the group consisting of: sugarcane, sugar beet, sweet sorghum, sugar maple, sugar palm and blue agave.

12. A Method according to claim 10, wherein said source of sucrose is under form of a juice, a concentrated juice or syrup, a clarified juice, molasses and/or crystallized sucrose.

13. A Method according to claim 10 wherein said sucrose source is a final product, an intermediate product and/or a by-product of sugarcane and/or sugar beet industry.

14. A Method according to claim 1, wherein the appropriate medium consists of the source of sucrose.

15. A Method according to claim 1, wherein the appropriate medium comprises at least a source of phosphorus and/or a source of nitrogen in addition to the source of sucrose.

16. A Method according to claim 1, wherein said sucrose source comprises at least 7% sucrose.

17. A method of claim 10, wherein said biomass comprises plant biomass.