GLYCERINE CONCENTRATION The present invention concerns a new modified microorganism for the production of 1 ,3- propanediol. This microorganism is adapted for growth and production of 1 ,3-propanediol from a culture medium with high glycerine content and specifically with a high concentration of industrial glycerine. The invention also concerns culture conditions of said adapted microorganisms and process for the production of 1 ,3-propanediol. The invention concerns, finally, 1 ,3-propanediol produced by the modified microorganism and its applications.

BACKGROUND OF THE INVENTION 1 ,3-propanediol (PDO), also called trimethylene glycol or propylene glycol, is one of the oldest know fermentation products. It was originally identified as early as 1881 by August Freund in a glycerine fermented culture containing Clostridium pasteurianum. PDO is a typical product of glycerine fermentation and has 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. freunddi), Lactobacilli (L brevis and L buchneri) and Clostridia of the C. butyricum and the C. pasteurianum group.

PDO, as a bifunctional organic compound, may potentially be used for many synthesis reactions, in particular as a monomer for polycondensations to produce polyesters, polyethers, polyurethanes, and in particular, polytrimethylene terephtalate (PTT). These structure and reactivity features lead to several applications in cosmetics, textiles (clothing fibers or flooring) or plastics (car industry and packing or coating).

PDO can be produced by different chemical routes but they generate waste stream containing extremely polluting substances and the cost of production is high. Thus, chemically produced PDO can not compete with the petrochemically available diols like 1 ,2-ethanediol, 1 ,2-propanediol and 1 ,4-butanediol. To increase this competitiveness, in 1995, DuPont started a research program for the biological conversion of glucose to PDO. Although this process is environmentally friendly it has the disadvantage to i) use vitamin B12 a very expensive cofactor and ii) be a discontinuous process due to the instability of the producing strain.

Due to the availability of large amounts of glycerine produced by the bio-diesel industry, a continuous, vitamin-B12-free process with a higher carbon yield would on the contrary, be advantageous. It is known in the art that PDO can be produced from glycerine, an unwanted by-product of the biodiesel production that contains roughly 80-85% of glycerine mixed with salts and water.

C. butyricum was previously described as being able to grow and produce PDO from industrial glycerine in batch and two-stage continuous fermentation (Papanikolaou et al., 2000). However, at the highest glycerine concentration, the maximal PDO titer obtained was 48.1 g.L ^"1 at a dilution rate of 0.02 h ^"1 , meaning a productivity of 0.9 g.L ^"1 .h ^"1 . The cultures were conducted with a maximum glycerine concentration in the fed medium of 90g.L ^"1 and in the presence of yeast extract, a costly compound containing organic nitrogen that is known by the man skilled in the art to help increase bacterial biomass production.

Application WO2006/128381 discloses the use of this glycerine for the production of PDO with batch and fed-batch cultures using natural PDO producing organisms such as Klebsiella pneumoniae, C. butyricum or C. pasteuricum. Furthermore, the medium used in WO2006/128381 also contains yeast extract. As described in this patent application, the maximal productivity reached was comprised between 0.8 and 1.1g.l ^"1 .h ^"1 .

The performance of a C. acetobutylicum strain modified to contain the vitamin B12- independent glycerine-dehydratase and the PDO-dehydrogenase from C. butyricum, called "C. acetobutylicum DG1 pSPD5" has been described in Gonzalez-Pajuelo et al., 2005. This strain originally grows and produces PDO in a fed medium containing up to 120 g. ¹ of pure glycerine. In addition, analyses in a fed medium containing a maximum of 60 g. ¹ of pure or industrial glycerine did not point out to any differences. These results have been obtained in presence of yeast extract. Moreover, no test was performed with concentrations of industrial glycerine higher than 60g.l ^"1 . When comparing a wild-type C. butyricum to the modified microorganism "C. acetobutylicum DG1 pSPD5", a globally similar behaviour was observed.

In patent application PCT/EP2010/056078 the inventors have described a process to adapt the strain of C. acetobutylicum DG1 pSPD5 such as described in Gonzalez-Pajuelo et al. (2005) to grow in a medium with a high concentration of industrial glycerine and without yeast extract. The resulting strain is able to produce PDO in medium containing up to 120 g. ¹ of industrial glycerine with a titer up to 53.5 g.L ^"1 of PDO, a yield up to 0.53 g.g ^"1 and a productivity up to 2.86 g.L ^"1 .h ^"1 .

In the present patent application, the inventors highlight the main genetics modifications of the adapted microorganism useful for the production of PDO, such as obtained after adaptation in presence of high concentration of industrial glycerine. BRIEF DESCRIPTION OF THE INVENTION

The present invention concerns a population of Clostridium acetobutylicum useful for the production of 1 ,3-propanediol (PDO), wherein said population comprises at least one strain of a Clostridium acetobutylicum sp. comprising mutations selected among the mutations identified in Table 1 , wherein relative percentages of said mutations are selected among the following gene families:

Particularly, the population of the invention comprises at least one strain of Clostridium acetobutylicum selected among the group consisting of :

- strain DG1 pSPD5 PD0001VE05c01 deposited at CNCM under accession number I-4378;

- strain DG1 pSPD5 PD0001VE05c05 deposited at CNCM under accession number I-4379;

- strain DG1 pSPD5 PD0001VE05c07 deposited at CNCM under accession number I-4380.

CNCM means "Collection Nationale de Cultures de Microorganismes" at the Pasteur Institute, Paris.

In a particular embodiment of the invention, the population comprises the above strains further mutated with at least one of the following point mutations: C is replaced with T at locus CA_C0175, position 198989 in the Clostridium acetobutylicum genome, coding for a predicted sugar phosphate isomerase, homolog of an eukaryotic glucokinase regulator (carbohydrate metabolism) G is replaced with A at locus CA_C1300, position 1444099 in the Clostridium acetobutylicum genome, coding for an RNA polymerase sigma factor RPOD ( transcription and translation regulation)

- C is replaced with T at locus CA_C2670, position 2787387 in the Clostridium acetobutylicum genome, coding for a Glu-tRNAGIn amidotransferase subunit A (transcription and translation regulation)

- C is replaced with T at locus CA_C3339, position 3512658 in the Clostridium acetobutylicum genome, coding for an ATPase component of an ABC transporter (two ATPase domains)

C is replaced with T at locus CA_C1610, position 1752341 in the Clostridium acetobutylicum genome, coding for a branched-chain amino acid permease (transporter).

The present invention also concerns a method for the production of 1 ,3- propanediol, comprising culturing a population of Clostridium acetobutylicum useful for the production of 1 ,3-propanediol (PDO) according to the invention in a culture medium comprising glycerine as sole source of carbon and recovering the 1 ,3-propanediol produced from the culture medium.

DETAILLED DESCRIPTION OF THE INVENTION

Population of Clostridium acetobutylicum useful for the production of 1,3- propanediol (PDO)

A population of Clostridium acetobutylicum useful for the production of 1 ,3- propanediol means one or more strains of Clostridium acetobutylicum genetically modified for the production of 1 ,3-propanediol from glycerine as sole source of carbon. Such strains are known in the art and disclosed, particularly, in applications WO200104324 and WO2008052595. The population according to the invention may be a combination of several strains, the majority of which comprising the mutations according to the invention, as well as a single strain, and particularly strain DG1 pSPD5 PD0001VE05c01 , DG1 pSPD5 PD0001VE05c05 or DG1 pSPD5 PD0001VE05c07 deposited at CNCM under accession numbers I-4378, I-4379, I-4380 respectively, or strain DG 1 pSPD5 PD0001VE05c08.

Mutations are changes of nucleotides in the strain genome, more particularly SNPs ("Single Nucleotide Polymorphisms"), identified when compared to the mother strain DG1 pSPD5 PD0001VT. Said strain is disclosed in WO200104324 and is derived from strain ATCC824 which genome sequence has been published (Nolling et al., 2001).

Mutations can occur in coding or non-coding sequences. These mutations can be synonymous wherein there is not modification of the corresponding amino acid or non- synonymous wherein the corresponding amino acid is altered. Synonymous mutations do not have any impact on the function of translated proteins, but may have an impact on the regulation of the corresponding genes or even of other genes, if the mutated sequence is located in a binding site for a regulator factor. Non-synonymous mutations may have an impact on the function of the translated protein as well as on regulation depending the nature of the mutated sequence.

The population of Clostridium acetobutylicum useful for the production of 1 ,3- propanediol may preferably comprise one of those deposited strains comprising additional modifications, at least one of the following modifications:

C replaced with T at locus CA_C0175, position 198989 in the Clostridium acetobutylicum genome, coding for a predicted sugar phosphate isomerase, homolog of an eukaryotic glucokinase regulator (carbohydrate metabolism)

- G replaced with A at locus CA_C1300, position 1444099 in the Clostridium acetobutylicum genome, coding for an RNA polymerase sigma factor RPOD ( transcription and translation regulation)

- C replaced with T at locus CA_C2670, position 2787387 in the Clostridium acetobutylicum genome, coding for a Glu-tRNAGIn amidotransferase subunit A (transcription and translation regulation)

- C replaced with T at locus CA_C3339, position 3512658 in the Clostridium acetobutylicum genome, coding for an ATPase component of an ABC transporter (two ATPase domains)

C replaced with T at locus CA_C1610, position 1752341 in the Clostridium acetobutylicum genome, coding for a branched-chain amino acid permease (transporter).

It may preferably comprise any combinations of these mutations, comprising 1 , 2, 3, 4 or 5 of these mutations.

The population of strains of the invention is capable of growing on a medium comprising up to 120 g.L ^"1 of glycerine and more particularly of industrial glycerine.

The strains of the population of the invention may be obtained using standard techniques of mutagenesis and/or gene replacement in Clostridium, such as disclosed in application WO2008040387 which contents are incorporated herein by reference.

The person skilled in the art may start from one of the strains disclosed in applications WO200104324 and WO2008052595 as well as use one of the strains c01 , c05 or c07 deposited at CNCM under accession numbers 1-4378, 1-4379, 1-4380 respectively, and introduce additional mutations.

In a preferred embodiment, the population of the invention comprises strain DG1 pSPD5 PD0001VE05c08, which mutations are identified in Table 1. The person skilled in the art knows how to introduce the mutations into a Clostridium strain to generate a strain similar to strain DG1 pSPD5 PD0001VE05c08, starting from one of strains DG1 pSPD5 PD0001VE05c01 , DG1 pSPD5 PD0001VE05c05 or DG1 pSPD5 PD0001VE05c07, deposited at CNCM under accession numbers I-4378, I-4379, I-4380 respectively and using standard gene replacement and recombination techniques.

Culture medium comprising glycerine

An "appropriate culture medium" or a "culture medium" refers to a culture medium optimized for the growth and the diol-production of the Clostridium strains or population. The fermentation process is generally conducted in reactors with a synthetic, particularly inorganic, culture medium of known defined composition adapted to the Clostridium species used and containing glycerine.

The term "synthetic medium" means a culture medium comprising a chemically defined composition on which organisms are grown. In the culture medium of the present invention, glycerine is advantageously the single source of carbon.

The terms "glycerine" and 'glycerol" are synonymous and used interchangeably in this invention to refer to the same molecule.

In a particular embodiment, glycerine is added to the medium in the form of glycerine composition comprising at least 50% of glycerine, preferably at least 85% of glycerine.

Advantageously, the glycerine used in the culture medium of the invention is industrial glycerine. "Industrial glycerine" means a glycerine product obtained from an industrial process without substantial purification. I ndustrial glycerine can also be designated as "raw glycerine". Industrial glycerine comprises more than 70% of glycerine, preferably more than 80%, water and impurities such as mineral salts and fatty acids. The maximum content of glycerine in industrial glycerine is generally 90%, more generally about 85%.

Industrial processes form which industrial glycerine is obtained are, inter alia, manufacturing methods where fats and oils, particularly fats and oils of plant origin, are processed into industrial products such as detergent or lubricants. In such manufacturing methods, industrial glycerine is considered as a by-product.

In a particular embodiment, the industrial glycerine is a by-product from biodiesel production and comprises known impurities of glycerine obtained from biodiesel production, comprising about 80 to 85% of glycerine with salts, water and some other organic compounds such as fatty acids. Industrial glycerine obtained from biodiesel production has not been subjected to further purification steps.

Preferably, the culture medium comprises high concentrations of glycerine.

The terms "high glycerine content" or "high concentration of glycerine" means more than 90 g.L ^"1 of glycerine in the culture medium . I n preferred embodiments, the concentration is comprised between 90 and 200 g.L ^"1 of glycerine, more particularly between 90 and 140 g/L of glycerine, preferably about 120 g.L ^"1 of glycerine.

Preferably, the culture medium is a synthetic medium without addition of organic nitrogen.

Such culture media are disclosed in the art, particularly in PCT/EP2010/056078 filed on 05/05/2010 and PCT/EP2010/064825 filed on 5/10/2010, which contents are incorporated herein by reference.

Culturing the microorganisms

In the method of the invention, the production is advantageously done in a batch, fed-batch or continuous process. Culturing microorganisms at industrial scale for the production of 1 ,3-propanediol i s kn own i n th e a rt, pa rti cu l arl y d i s cl osed i n PCT/EP2010/056078 filed on 05/05/2010 and PCT/EP2010/064825 filed on 5/10/2010, which content are incorporated herein by reference.

1,3-propanediol recovery

Methods for recovering and eventually purifying 1 ,3-propanediol from a fermentation medium are known to the skilled person. 1 ,3-propanediol may be isolated by distillation. In most embodiments, 1 ,3-propanediol is distilled from the fermentation medium with a by-product, such as acetate, and then further purified by known methods.

A particular purification method is disclosed in applications WO2009/0681 10 and WO 2010/037843, which content is incorporated herein by reference.

FIGURES

Figure 1 describes the evolution of 1 ,3-propanediol production and glycerine consumption of the population and clone c08 during the chemostat cultures from inoculation up to D = 0.06h ^"1 . EXAMPLES

EXAMPLE 1 Isolation of clones from the evolved population

Clone isolation was performed on agar plates starting from a growing flask culture of the population strain Clostridium acetobutylicum DG1 pSPD5 PD0001 VE05. The synthetic media used for flask culture contained per liter of deionized water : glycerine, 30g; KH ₂ P0 ₄ , 0.5g; K ₂ HPO ₄ , 0.5g; MgS0 ₄ , 7H ₂ 0, 0.2g; CoCI ₂ 6H ₂ 0, 0.01g; acetic acid, 99.8%, 2.2ml; NH ₄ CI, 1.65g; MOPS, 23.03g, biotin, 0.16mg; p-amino benzoic acid, 32mg; FeS0 ₄ , 7H ₂ 0, 0.028g; resazurin, 1 mg and cysteine, 0.5g. The pH of the medium was adjusted to 6.5 with NH ₄ OH 6N.

Different media were used for isolation on agar plates : synthetic agar medium (the same as described above) with either commercial glycerine or raw glycerine and CGM (Clostridial Growth Medium) agar medium which contains per liter of deionized water : commercial or raw glycerine, 30g; yeast extract, 5g; KH ₂ P0 ₄ , 0.75; K ₂ HP0 ₄ , 0.75g; MgS0 ₄ , 7H ₂ 0, 0.4g; asparagine, 2g; (NH ₄ ) ₂ S0 ₄ , 2g; NaCI, 1 g; MnS0 ₄ , H20, 10mg; FeS0 ₄ , 7H ₂ 0, 10mg; MOPS, 23.03g; resasurin, 1 mg and cysteine, 15g. The pH of the medium was adjusted to 6.6 with NH ₄ OH 3N.

Cells were plated from a flask culture (Table 2) in four different ways:

- on agar plates of synthetic medium with commercial glycerine ;

on agar plates of synthetic medium with raw glycerine ;

on agar plates of rich medium with commercial glycerine ;

on agar plates of rich medium with raw glycerine.

Isolated clones were considered pure after three subsequent subcultures on agar plates. Pure clones were then transferred into liquid rich medium which contained either commercial or raw glycerine (Table 2). Subsequently, growing liquid cultures were conserved on glycerine 20% at -80°C until further characterization.

Clones were then characterized in the following way:

Measurement of viability after conservation : evaluation of growth rate of cells on synthetic medium ;

Evaluation of growth and metabolism : measurement of OD ₆₂ o _n m during culture and PDO/glycerine yield on synthetic medium ;

Genetic evaluation : PCR analysis to confirm the genotype of the strain;

Chemostat culture to compare the performances of isolated clones with those of the population (example 2) ;

gDNA extraction for sequence analysis of the clones (example 3). Table 2: Synthetic agar media and liquid media used for the isolation of 4 clones from the population.

Clone Agar media for isolation Liquid media for clone culture number before conservation

c01 Synthetic medium with Rich medium with commercial commercial glycerine glycerine

c05 Rich medium with raw glycerine Rich medium with commercial glycerine

c07 Synthetic medium with Rich medium with raw glycerine

commercial glycerine

c08 Rich medium with commercial Rich medium with raw glycerine

glycerine EXAMPLE 2 Performances of clone c08 in a chemostat culture with high concentrations of raw glycerine

Bacterial strain:

Isolated clone of C. acetobutylicum strain DG1 pSPD5 PD0001 VE05 (strain was 1/ cured from pSOL1 21 transformed with plasmid pSPD5 harbouring dhaB1, dhaB2 and dhaT genes, ie 1,3-propanediol operon, and 3/ evolved on high concentrations of raw glycerine). The isolation protocol was described in example 1.

Culture media:

The synthetic media used for Clostridia batch cultivations contained per liter of deionized water: glycerine, 30g; KH ₂ P0 ₄ , 0.5g ; K ₂ HP0 ₄ , 0.5g ; MgS0 ₄ , 7H ₂ 0, 0.2g ; CoCI ₂ 6H ₂ 0, 0.01g ; H ₂ S0 ₄ , 0.1ml ; NH ₄ CI, 1.5g ; biotin, 0.16mg ; p-amino benzoic acid, 32mg and FeS0 ₄ , 7H ₂ 0, 0.028g. The pH of the medium was adjusted to 6.3 with NH ₄ OH 3N. Commercial glycerine purchased from Sigma (purity 99.5%) was used for batch cultivation. The feed medium for continuous cultures contained per liter of tap water : raw glycerine, 105g ; KH ₂ P0 ₄ , 0.5g ; K ₂ HP0 ₄ , 0.5g ; MgS0 ₄ , 7H ₂ 0, 0.2g ; CoCI ₂ 6H ₂ 0, 0.026g ; NH ₄ CI, 1.5g ; biotin, 0.16mg ; p-amino benzoic acid, 32mg ; FeS0 ₄ , 7H ₂ 0, 0.04g ; anti- foam, 0,05ml ; ZnS0 ₄ , 7H ₂ 0, 8mg ; CuCI ₂ , 2H ₂ 0, 4mg ; MnS0 ₄ , H ₂ 0, 40mg ; H ₃ B0 ₃ , 2mg ; Na ₂ Mo0 ₄ , 2H ₂ 0, 0.8mg. Medium pH was not adjusted in this case. Raw glycerine, from the transestenfication process for biodiesel, was supplied by Novance (Venette, France) and had the following purity : glycerine 84.8% (w/w).

Experimental set-up:

Continuous cultures were performed in a 5I bioreactor Tryton (Pierre Guerin, France) with a working volume of 2000ml. The culture volume was kept constant at 2000ml by automatic regulation of the culture level. Cultures were stirred at 200 RPM, the temperature was set to 35°C and pH maintained constant at 6.5 by automatic addition of NH4OH 5.5N. The POR measurement (mV) was controlled during the entire culture. To create anaerobic conditions, the sterilized medium in the vessel was flushed with sterile 0 ₂ -free nitrogen for one hour at 60°C and flushed again until 35°C was attained (flushing during 2 hours). The bioreactor gas outlet was protected from oxygen by a pyrogallol arrangement (Vasconcelos et al, 1994). After sterilisation the feed medium was also flushed with sterile 0 ₂ -free nitrogen until room temperature was attained and maintained under nitrogen at 200 mbar to avoid 0 ₂ entry. Batch and continuous cultures process:

The process used to evaluate has been described in patent application PCT/EP2010/056078 (example 2).

A culture growing in a 100ml flask on synthetic medium (the same as described above for batch culture but with addition of acetic acid, 2.2 g.L ^"1 and MOPS, 23.03g.L ^"1 ) taken at the end of exponential growth phase was used as inoculum (5% v/v).

Cultures were first grown batchwise. At the early exponential growth phase we performed a pulse of commercial glycerine: For the pulse synthetic medium (the same as described for batch culture) with 105 g.L ^"1 raw glycerine was added at a static flow rate during 3 hours (i.e. an addition of 18 g.L ^"1 of glycerine). Then the growth continued batchwise and before the end of the exponential growth phase the continuous feeding started with a dilution rate of 0.025 h ^"1 : The feed medium contains 105 g.L ^"1 of raw glycerine. 8-10 days after inoculation of the bioreactor and after 3 residence times the dilution rate was increased from 0.025 h ^"1 to 0.060h ^"1 by different stages: Increase of 0.01 h ^"1 units in 48 hours - no change for 24-hours - increase of 0.01 h ^"1 units in 48 hours - no change for 24hours - increase of 0.015 h ^"1 unit in 48 hours. After that, stabilisation of the culture was followed by 1 ,3-propanediol production and glycerine consumption (Figure 1 ) using the HPLC protocol described below. Particularly we waited until the concentration of residual glycerine was as low as possible.

The overall performances of c08 clone are presented in Table 3 and compared with performances of the population under the same conditions and with performances of the strain C. acetobutylicum DG1 pSPD5 PD0001VT such as described in Gonzalez-Pajuelo et al. (2005).

Analytical procedures:

Cell concentration was measured turbidimetrically at 620nm and correlated with cell dry weight determined directly. Glycerine, 1 ,3-propanediol, ethanol, butanol, acetic and butyric acids concentrations were determined by HPLC analysis. Separation was performed on a Biorad Aminex HPX-87H column and detection was achieved by refractive index. Operating conditions were as follows: mobile phase sulphuric acid 0.5mM; flow rate 0.5ml/min, temperature, 25°C.

Table 3: performances of the C. acetobutylicum DG1 pSPD5 population PD0001VE05 (mean data from 4 chemostats), of clone c08 PD0001VE05c08. The feed medium contained 105g.L ^"1 of raw glycerine, dilution rate was 0.060h ^"1 and 0.025h ^"1 . Values correspond to the average of samples analyzed after at least 3 residences times at dilution rate of 0.060h ^"1 .

Y _I ,3- _PDO : PDO yield (g/g of glycerol consumed)

Q _I ,3 _PD O : PDO volumetric productivity

Nl: no information. The PD0001VT strain can not grow in a medium lacking yeast extract.

These results show that the adapted population of C. acetobutylicum DG1 pSPD5 is able to grow on higher concentrations of industrial glycerine and thus exhibits a better titer and productivity of PDO on industrial glycerine, than the non adapted strain C. acetobutylicum DG1 pSPD5 PD0001VT from Gonzalez-Pajuelo et al. (2005) which can not grow in a medium lacking yeast extract. Example 3

Genomic DNA extraction

Genomic DNA from strains PD0001VT, PD0001VE05, PD0001 VE05c01 , PD0001VE05c05, PD0001 VE05c07 and PD0001VE05c08 was extracted using Qiagen Genomic kit 500G (Qiagen, Inc., Valencia, CA). Briefly, cells were grown anaerobically respectively in rich or synthetic glycerine medium (as described in example 1 and 2) in penicillin vials (70 ml_) to late exponential phase (A ₆ 2o 1 .5 to 2.0). Strictly anaerobic conditions were maintained throughout cell lysis. Cells were collected and washed twice in SET buffer (25% sucrose, 0.05 M Tris-HCI, 0.05 M EDTA). Cell pellets were suspended in 1 1 ml_ B1 kit buffer with 44 μΙ_ RNase, 30 mg/mL lysozyme and 100 μg/mL proteinase K. The mixtures were incubated at 37°C for 45 min, centrifuged and supernatants were used for DNA extraction according to the Qiagen DNA purification kit instructions. The DNAs were then suspended in 50 μΙ_ of 10 mM Tris-HCI (pH8.0). Sequencing analysis

Genomes of the native DG1 pSPD5 PD0001VT strain and the evolved population DG1 pSPD5 PD0001 VE05 were sequenced using the Roche GS FLX technology. The sequencing project was performed by Eurofins Genomics MWG/ Operon (ZA de Courtabeauf-9 Avenue de la Laponie, 91978 Les Ulis Cedex) with for each strain 1 Long- Tag paired end libraries (8 Kb), generation of sequence and scaffolding of the contigs with GS FLX Titanium series chemistry using a half run (max. 600 000 reads, max 180 000- 300 000 true paired end reads).

Isolated clones from the evolved population were sequenced using the comparative genomic sequencing (CGS) method developed by NimbleGen (Roche NimbleGen Inc. 500 S. Rosa Rd. Madison Wl 53719). The CGS analysis was performed in two phases: in phase 1 , regions of genomic difference were identified by a comparative hybridization of DNA of the native strain and the evolved clones. In phase 2, only the identified regions of genomic differences were sequenced so as to produce a set of fully characterized single nucleotide polymorphisms (SNPs).

SNP analysis

Bioinformatics and SNP analysis of the evolved population were performed by Eurofins Genomics MWG / Operon. For this analysis, the read sets of both strains were separately mapped to the Genbank reference sequence (Clostridium acetobutylicum ATCC 824 http://www.ncbi.nlm.nih.gov/nuccore/AE001437) using the software gsMapper (Roche 454, V2.3) . Three SN Ps files were delivered comparing DG 1 pSPD5 PD0001VT to ATCC824, DG1 pSPD5 PD0001VE05 to ATCC824 and DG1 pSPD5 PD0001VT to DG1 pSPD5 PD0001VE05. Unique SNPs between the native and the evolved strains are presented below. Low coverage (<25) and low variant frequency (<85%) were removed resulting in 160 unique SNPs distributed in 17 families according to the KEGG database used for the family group annotations.

SNP analysis of the isolated clones was performed by NimbleGen (Roche). The SNP files we re d e l i ve red co m pa ri n g n ati ve D G 1 pS P D 5 P D0001 VT to DG 1 pS P D5 PD0001VE05C01 , DG1 pSPD5 PD0001VE05c05, DG1 pSPD5 PD0001VE05c07 or DG1 pSPD5 PD0001VE05c08 using Genbank reference sequence (Clostridium acetobutylicum ATCC 824 http://www.ncbi.nlm.nih.gov/nuccore/AE001437).

The sequence results are presented in Table 1 which contains the following information:

RefStart the start position within the reference sequence, where the difference occurs

RefNuc the reference nucleotide sequence at the difference location

VarNuc the differing nucleotide sequence at the difference location

VarFreq the percentage of different reads versus total reads that fully span the difference location

Type Lists whether or not an SNP is found within an annotated gene, or between annotated genes. SNPs in genes are designated as coding. SNPs between genes are designated as intergenic

AA change categorizes coding SNPs base on whether or not they change the amino acid sequence of a protein. S indicates synonymous SNPs (no amino acid change). N indicates nonsynonymous SNPs (altered amino acid). FC (Frame-Change) indicates a modification in protein translation because of insertion or deletion of a nucleotide

ORIG AA the am i no acid associ ated with the reference seq uence for the corresponding SNP position

SNP AA the amino acid associated with the test sequence, for the corresponding

SNP position

Locus Tag locus tag of the corresponding gene from Genbank

Function the function of the gene as described in Genbank

Family the family of the gene from KEGG

Table 1 : Mutations between native and evolved strains. Mutations were first identified in the adapted population and then presence of each mutat was verified in isolated clones (four last columns: Y for presence and N for absence of mutation).

2114483 A G >99% C N V A CA_C2003 Predicted permease Transporters Y Y Y

2123888 T C >99% C s L L CA_C2010 Predicted Fe-S Energy metabolism Y Y Y oxidoreductase

2171503 c T >99% c N D N CA_C2068 Sporulation factor spollM, Sporulation Y Y Y uncharacterized membrane

protein

2231570 c - >99% c FC CA_C2137 Cation transport P-type Transporters N N N

ATPase

2294764 G A >99% c N T I CA_C2201 Hypothetical protein Hypothetical proteins Y Y Y

2299326 C G >99% c N s T CA_C2205 Flagellar hook-associated Cell motility Y Y Y protein FliD

2307214 C T >99% c N G R CA_C2215 Flagellar switch protein FliY, Cell motility Y N Y contains CheC-like domain

2342826 G c >99% c N P A CA_C2247 Site-specific recombinase, Transcription translation Y Y Y

DNA invertase Pin homolog regulation

2392178 C T >99% c N V CA_C2288 Acyl-protein synthetase, luxE Lipid metabolism Y Y Y

2450006 C T >99% c S P P CA_C2340 DNA mismatch repair protein Transcription translation Y Y Y mutS, YSHD B.subtilis regulation

ortholog

2477825 C T >99% c S S S CA_C2367 Uncharacterized protein Cell adhesion Y Y Y containing predicted cell

adhesion domain and ChW- repeats

2493211 T c >99% c S H H CA_C2385 Hypothetical protein Hypothetical proteins Y Y Y

2595349 G A >99% c N A V CA_C2486 Transcriptional regulator, Transcription translation Y Y Y

MarR/EmrR family regulation

2693354 C T >99% c N E K CA_C2588 Glycosyltransferase Carbohydrate metabolism Y Y Y

2787387 C T >99% c N M I CA_C2670 Glu-tRNAGIn Transcription translation Y Y Y amidotransferase subunit A regulation

2833384 T c >99% c N I V CA_C2709 Electron transfer flavoprotein Energy metabolism Y Y Y alpha-subunit

2836979 G A >99% c N A V CA_C2713 AT-rich DNA-binding protein Transcription translation Y Y Y regulation

2901642 C T >99% c N V CA_C2770 Amino acid transporter Transporters Y Y Y

2969858 G A >99% c N M I CA_C2838 Predicted nucleotide-binding Transcription translation Y Y Y protein, YjeE family regulation

3001642 G A >99% c S L L CA_C2867 FoF1 -type ATP synthase Energy metabolism Y Y Y alpha subunit

3032956 T C >99% c N H R CA_C2898 Stage II sporulation protein R Sporulation Y Y Y

3140918 T C >99% I I I Y Y Y

3174743 G A >99% C s D D CA_C3032 Galactose mutarotase Carbohydrate metabolism Y N Y related enzyme

3251276 G C >99% C N T S CA_C3099 Pseudouridylate synthase, Nucleic acid metabolism Y Y Y

TRUA

3337937 G - >99% I I I N N N

3392124 G A >99% C N G R CA_C3242 Uncharacterized Fe-S Energy metabolism Y Y Y protein, PfIX (pyruvate

formate lyase activating

protein) homolog

3462380 C T >99% C S N N CA_C3297 D-alanyl-D-alanine Hypothetical proteins Y Y Y carboxypeptidase family

hydrolase, YODJ B.subtilis

ortholog

3509372 C T >99% C S E E CA_C3335 Short-chain alcohol Energy metabolism Y Y Y dehydrogenase family

enzyme

3512658 C T >99% C S Y Y CA_C3339 ATPase component of ABC Transporters Y Y Y transporter (two ATPase

domains)

3518240 T c >99% C S Y Y CA_C3345 Transcriptional regulator, Transcription translation Y Y Y

AcrR family regulation

3541557 T c >99% C N I V CA_C3363 Hypothetical protein Hypothetical proteins Y Y Y

3565291 c T >99% c N T I CA_C3387 Pectate lyase Cellulase Y Y Y

3576865 T c >99% c N H R CA_C3392 NADH-dependent butanol Energy metabolism Y Y Y dehydrogenase

3583724 c T >99% I I I Y Y Y

3608511 c T >99% c S S S CA_C3422 Suganproton symporter Transporters Y Y Y

(possible xylulose)

3614985 c T >99% c S K K CA_C3428 6Fe-6S prismane cluster- Energy metabolism Y Y Y containing protein

3674358 T c >99% I I I Y Y Y

3707038 T c >99% c S L L CA_C3510 Membrane associated Membrane proteins Y Y Y methyl-accepting chemotaxis

protein (with HAMP domain)

3747653 G A >99% c N A V CA_C3551 Na+ ABC transporter (ATP- Transporters Y Y Y binding protein), NATA

3821135 C T >99% c S N N CA_C3617 Uncharacterized membrane Hypothetical proteins Y Y Y protein, YHAG B.subtilis

diphosphate-sugar

epimerase and GAF domain

1717948 G A 97% C N V I CA_C1572 Fructose-1 ,6-bisphosphatase Carbohydrate metabolism Y Y Y

(YYDE B.subtils ortholog)

2004797 C T 97% C N S N CA_C1852 Magnesium and cobalt Transporters Y Y Y transport protein

2134058 G A 97% c S A A CA_C2020 Molybdopterin bioSthesis Energy metabolism Y Y Y enzyme, MoeA, fused to

molibdopterin-binding

domain

2331746 G A 97% c N G R CA_C2237 ADP-glucose Lipid metabolism Y Y Y pyrophosphorylase

2391588 G A 97% c N P L CA_C2288 Acyl-protein Sthetase, luxE Lipid metabolism Y Y Y

2452705 C T 97% c N C Y CA_C2341 Collagenase family protease Proteases/Peptidases Y Y Y

2739459 T c 97% c N I V CA_C2630 Uncharaterized conserved Hypothetical proteins Y Y Y protein, YOME B.subtilis

ortholog

2775979 c T 97% c N A T CA_C2660 Pyruvate carboxylase, PYKA Carbohydrate metabolism Y Y Y

2813985 G - 97% I I I N N N

3082247 C T 97% c N L F CA_C2948 ATPase components of ABC Transporters Y Y Y transporter with duplicated

ATPase domains (second

domain is inactivated)

3242900 G c 97% c N V L CA_C3088 NtrC family transcriptional Transcription translation Y N N regulator, ATPase domain regulation

fused to two PAS domains

3442855 T c 97% c N M V CA_C3282 ABC-type Transporters Y Y Y multidrug/protein/lipid

transport system, ATPase

component

3498584 c T 97% c N L F CA_C3327 Amino acid ABC-type Transporters Y Y Y transporter, ATPase

component

3643224 G A 97% c S L L CA_C3447 Protein-disulfide isomerases Sporulation Y Y Y

DsbC/DsbG

3663477 - T 97% c FC CA_C3464 Uncharacterized conserved Hypothetical proteins N N N protein (fragment)

204202 G A 96% c N G E CA_C0180 Oligopeptide ABC Transporters Y Y Y transporter, ATP-binding

protein

803682 C T 96% C N T I CA_C0695 Altronate oxidoreductase Carbohydrate metabolism Y Y Y

892875 G A 96% C N M I CA_C0770 Glycerine uptake facilitator Glycerine metabolism Y Y Y protein, permease

1009389 C T 96% c N P s CA_C0879 ABC-type polar amino acid Transporters Y Y Y transport system, ATPase

component

1690355 C T 96% c S G G CA_C1546 Pyrimidine-nucleoside Nucleic acid metabolism Y Y Y phosphorylase

1752341 C T 96% c N G R CA_C1610 Branched-chain amino acid Transporters Y Y Y permease

3217481 A c 96% c S L L CA_C3067 Predicted membrane protein Membrane proteins Y Y Y

3238489 T c 96% c S S S CA_C3086 Protein containing cell Cell adhesion Y Y Y adhesion domain

447460 A - 95% I I I N N N

670931 G A 95% c S N N CA_C0578 Cobalamine-dependent Amino acid metabolism N Y Y methionine synthase I

(methyltransferase and

cobalamine-binding domain)

994575 G A 95% c N A T CA_C0864 Histidine kinase-like ATPase Transcription translation Y Y Y regulation

3657101 A - 95% c FC CA_C3459 Homolog of cell division Cell division N N N

GTPase FtsZ, diverged

1 142263 T - 94% c FC CA_C0995 Predicted membrane protein Membrane proteins N N N

1823156 G A 94% c S E E CA_C1674 Small subunit of NADPH- Amino acid metabolism Y Y Y dependent glutamate

synthase

19891 17 C T 94% c N R K CA_C1837 Mismatch repair protein Transcription translation Y Y Y

MutS, ATPase regulation

3481651 G A 94% c S S s CA_C331 1 TPR-repeat domain fused to Carbohydrate metabolism Y Y Y glycosyltransferase

126942 G A 93% c N E K CA_C01 16 Carbone-monoxide Energy metabolism Y Y Y dehydrogenase, beta chain

302716 - T 93% c FC CA_C0270 Hypothetical protein Hypothetical proteins N N N

2551 103 G A 93% c S S s CA_C2434 Membrane associate Transcription translation Y N Y histidine kinase with HAMP regulation

domain

1834077 C T 92% c N S L CA_C1684 TYPA/BIPA type GTPase Energy metabolism Y Y Y

3927304 G A 92% I I I Y N Y

786649 - T 91 % c FC CA_C0680 Predicted membrane protein Membrane proteins N N N

2640439 C T 91 % C N E K CA_C2532 Protein containing ChW- Cell adhesion Y Y Y repeats

3601904 A 91 % C FC CA_C3415 ABC-type Transporters N N N multidrug/protein/lipid

transport system, ATPase

component

838350 A - 89% c FC CA_C0723 Transcriptional regulator, Transcription translation N N N

AcrR family regulation

3721023 G A 89% c S S S CA_C3523 Hypothetical protein, CF-7 Hypothetical proteins Y Y Y family

803924 G A 88% c N A T CA_C0695 Altronate oxidoreductase Carbohydrate metabolism Y Y Y

3478420 C T 87% c N G E CA_C3309 Predicted membrane protein Membrane proteins N Y N

3853836 T c 87% c N N D CA_C3652 Acetolactate synthase Amino acid metabolism Y Y Y

244464 c T 86% c N S L CA_C0220 Hypothetical protein Hypothetical proteins Y Y Y

899104 G A 86% c N M I CA_C0776 NCAIR mutase (PurE)- Nucleic acid metabolism Y N Y related protein

658665 T - 85% c FC CA_C0569 SACPA operon Transcription translation N N N antiterminator (sacT) regulation

REFERENCES

Gonzalez-Pajuelo M, Meynial-Salles I, Mendes F, Andrade JC, Vasconcelos I, and Soucaille P. 2005. Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1 ,3-propanediol from glycerol. Metabolic Engineering 7: 329-336.

Gonzalez-Pajuelo M, Meynial-Salles I, Mendes F, Soucaille P. and Vasconcelos I. 2006. Microbial conversion of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG (pSPD5). Applied and Environmental Microbiology, 72: 96-101.

Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf Yl, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR. 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. Journal of bacteriology 183(16):4823 - 4838

Papanikolaou S, Ruiz-Sanchez P, Pariset B, Blanchard F and Fick M. 2000. High production of 1 ,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. Journal of Biotechnology. 77: 191-208.

Vasconcelos I, Girbal L, Soucaille P. 1994. Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. Journal of bacteriology. 176(5): 1443-1450.

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