Microorganism and method for improved 1,3-propanediol production by fermentation on a culture medium with high glycerine content

Abstract

The present invention concerns a new method for the production of 1,3-propanediol comprising culturing a recombinant microorganism converting glycerol into 1,3-propanediol and overexpressing the hcpR and/or frdX gene on a medium comprising glycerine. A recombinant microorganism for the production of 1,3 propanediol from glycerol, wherein said microorganism converts glycerol into 1,3-propanediol and 10 overexpresses the hcpR and/or the frdX gene.

Claims

1. A recombinant microorganism for the production of 1,3 propanediol from glycerol, wherein the recombinant microorganism converts glycerol into 1,3-propanediol and overexpresses hcpR (nitric oxide-responsive transcriptional regulator) and frdX (ferredoxin-3 like protein) genes by at least 1.5 fold as compared to an expression level in an unmodified or parental microorganism under the same conditions, and wherein the recombinant microorganism is Clostridium acetobutylicum DG1 pSPD5 and said hcpR and frdX genes are overexpressed by a genetic modification comprising at least one of the following: mutating the promoter regulating the expression of the hcpR and frdX genes, mutating the intergenic region between the hcpR and frdX genes, gene duplication, or overexpressing the hcpR and frdX genes from a plasmid.

2. The recombinant microorganism of claim 1, wherein the recombinant microorganism is adapted to grow on a culture medium having a glycerol concentration in the industrial glycerine which is comprised between 90 g/L and 120 g/L and/or wherein the industrial glycerine comprises at least 5% fatty acids.

3. A method for the fermentative production of 1,3-propanediol, wherein the recombinant microorganism as set forth in claim 1 is cultured on a medium comprising industrial glycerine.

4. The method of claim 3, wherein the hcpR and frdX genes are overexpressed in the recombinant microorganism by intergenic mutation between the hcpR and frdX genes by insertion.

5. The method of claim 4, wherein the insertion occurs in a region of repeating A nucleotides.

6. The method of claim 3, wherein the recombinant microorganism is adapted to grow in the presence of a glycerol concentration in the industrial glycerine which is comprised between 90 g/L and 120 g/L.

7. The method of claim 6, wherein the industrial glycerine comprises at least 5% fatty acids.

8. The method of claim 3, wherein the industrial glycerine is a by-product of biodiesel production.

9. The method of claim 3, wherein the 1,3 propanediol produced in the culture is further purified.

Description

FIGURES

(1) FIG. 1: Obtention of Clostridium acetobutylicum DG1 pSPD5 Type 130P strain by adaptation of the Type 008P strain on raw glycerine. Dynamic of OD.sub.620nm (OD units or ODU, squares), residual glycerol (g/L; hatches), PDO concentration (g/L; triangles) and feed flow rate (mL/h; circles) of the continuous culture are shown as a function of culture duration (days; d).

(2) FIG. 2: Chromosomal organization of the hcpR and frdX genes in C. acetobutylicum. The genes hcpR and frdX are organized as a bi-directional gene pair, with a 123 bp intergenic region located between the two genes. Nucleotide positions within the C. acetobutylicum ATCC 824 genome (NCBI reference sequence: NC_003030.1) are indicated.

(3) FIG. 3: Overexpression of the hcpR and frdX genes by quantitative PCR. Both hcpR and frdX were overexpressed in the 130P strain, as compared to the parent strain Type 008P, as determined by qRT-PCR. Black bars: Type 008P; Grey bars: Type 130P.

EXAMPLES

(4) The present invention is further defined in the following examples. It should be understood that these example, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the person skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modifying the essential means of the invention.

Example 1

Continuous Culture of Clostridium acetobutylicum DG1 (pSPD5) on Raw Industrial Glycerol and Obtention of the Microorganism Type 130P

(5) Bacterial Strains:

(6) Type 008P: C. acetobutylicum strain DG1 pSPD5 adapted on high concentrations of raw glycerine as described in patent application WO 2010/128070 Type 130P: C. acetobutylicum strain DG1 pSPD5 Type issued from a continuous culture form C. acetobutylicum strain DG1 pSPD5 Type 008P on high concentrations of raw industrial glycerine, and overexpressing the hcpR and frdX genes, as described herein
The synthetic media used for clostridia batch cultivations contained, per liter of tap water: glycerol, 30 g; KH.sub.2PO.sub.4, 0.5 g; K.sub.2HPO.sub.4, 0.5 g; MgSO.sub.4, 7H.sub.2O, 0.2 g; CoCl.sub.2 6H.sub.2O, 0.01 g; H.sub.2SO.sub.4, 0.1 ml; NH.sub.4Cl, 1.5 g; biotin, 0.16 mg; p-amino benzoic acid, 32 mg; FeSO.sub.4, 7H.sub.2O, 0.028 g. The pH of the medium was adjusted to 6.3 with NH.sub.4OH 3N. Commercial glycerol purchased from SDS Carlo_Erba (purity 99%) was used for batch cultivation. The feed medium for continuous cultures contained, per liter of tap water: glycerol from raw glycerine, 105 g; KH.sub.2PO.sub.4, 0.50 g; K.sub.2HPO.sub.4, 0.50 g; MgSO.sub.4, 7H.sub.2O, 0.2 g; NH.sub.4Cl, 1.5 g; CoCl.sub.2 6H.sub.2O, 0.026 g; biotin, 0.16 mg; p-amino benzoic acid, 32 mg; FeSO.sub.4, 7H.sub.2O, 0.04 g; anti-foam, 0.05 ml; ZnSO.sub.4, 7H.sub.2O, 8 mg; CuCl.sub.2, 2H.sub.2O, 4 mg; MnSO.sub.4, H.sub.2O, 0.04 g; H.sub.3BO.sub.3, 2 mg; Na.sub.2MoO.sub.4, 2H.sub.2O, 0.8 mg. Medium pH was adjusted between 3.5 and 4 with H.sub.2SO.sub.4 96%.

(7) Raw glycerine, from the transesterification process for biodiesel, was provided by two different providers and had the following composition: from ADM (Rolle, Switzerland) (using vegetable oil; purity 80.9%; Moisture 12.6%; MONG 0.39%; Ash 6.2%), from Greenergy (London, UK) (using cooking oil; purity 76.5%; Moisture 10.2%; MONG 7.1%; Ash 6.3%).

(8) Optionally, these glycerines were pretreated by acidification.

(9) The purity and MONG composition has an incidence on the toxicity of the glycerine on the microorganism. The Greenergy (London, UK) glycerine is both less pure and more dirty, therefore more toxic for the microorganism, than the ADM (Rolle, Switzerland) glycerine, because of the high concentration of MONG. Indeed, MONG concentration in the Greenergy (London, UK) glycerine is above 5%.

(10) The following example shows the adaptation of the strain Type 008P on coarser glycerine (from ADM to Greenergy) to get a new strain named Type 130P, that is able to grow and produce PDO on less refined industrial glycerine. This adaptation is highly advantageous, as less refined industrial glycerine is a cheaper raw material for the fermentation process.

(11) Experimental Set-Up:

(12) Continuous cultures were performed in a 5 l Tryton bioreactor (Pierre Guerin, France) with a working volume of 2000 ml. The culture volume was kept constant at 2000 ml by automatic regulation of the culture level. Cultures were stirred at 200 RPM, the temperature was set to 35° C. and pH was maintained constant at 6.5 by automatic addition of NH.sub.4OH 5.5N. To create anaerobic conditions, the sterilized medium in the vessel was flushed with sterile O.sub.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 sterilization, the feed medium was also flushed with sterile O.sub.2-free nitrogen until room temperature was reached and kept under nitrogen to avoid O.sub.2 entry.

(13) Analytical Procedures:

(14) Cell concentration was measured turbidimetrically at 620 nm (OD.sub.620nm) and correlated with cell dry weight, which was determined directly. Glycerol, PDO, ethanol, lactate, acetic and butyric acid 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 0.5 mM; flow rate 0.5 ml/min, temperature, 25° C.

(15) Batch and Continuous Cultures Process and Results:

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

(17) Cultures were first grown in batch mode. At the early exponential growth phase, we performed a pulse of glycerol with the feed medium (the same as described for feed culture). Glycerol from raw glycerine was added at a static flow rate during 3 hours (i.e. an addition of 18 g/L of glycerol). Then, the growth continued in batch mode and before the end of the exponential growth phase the continuous feeding started with a dilution rate of 0.035 h.sup.−1 of feed medium containing 105 g/L of glycerol from raw glycerine provided by ADM (Rolle, Switzerland) only. As can be seen in FIG. 1, after 3 days with a dilution rate of 0.035 h.sup.−1, glycerol accumulation started and reached 46.6 g/L at 6.5 residence times (RT, calculated according to the formula shown below), corresponding to the first peak of residual glycerine. This accumulation was coupled with a decrease of PDO production (up to 31 g/L instead of 52 g/L) and biomass production (1.8 ODU instead of 5.6 ODU). This accumulation was followed by a quick re-consumption, after 9 RT at a dilution rate of D=0.035 h.sup.−1 residual glycerol was drop down at 2.9 g/L. At this time (12 days after the inoculation), the dilution rate was increased from 0.035 h.sup.−1 to 0.070 h.sup.−1 in five days. After 9 RT at a dilution rate of D=0.07 h.sup.−1, performances stabilized at 5.5±1.1 g/L of glycerol and 51.6±0.7 g/L of PDO.

(18) After this stabilization, (28 days after the inoculation (see FIG. 1), raw glycerine of the feed was changed to a blend of raw glycerine provided by ADM (50%; Rolle, Switzerland) and by Greenergy (50%; London, UK), thereby increasing the level of MONG, and glycerine toxicity for microorganisms.

(19) This modification of the feed composition induced cycles of glycerol accumulation (max at 35.1 g/L) and drops of PDO production (min at 37.4 g/L) during 13 days. The culture was monitored for stabilization via the key factors (OD, residual glycerol and PDO concentration), and the new adapted strain 130P was identified for storage at day 45 (see FIG. 1).

(20) At this step, the new strain was sequenced and compared to the sequence of 008P. We identified the intergenic mutation described in Example 3 below.

(21) Performances of the resulting strain Type 130P are presented below in Table 2.

(22) Formula for the Calculation of Residence Time from Dilution Rate

(23) $\mathrm{RT}=\frac{1}{\mathrm{DR}}$$\mathrm{RT}\text{:}\mathrm{residence}\mathrm{time}\left(h\right)$$\mathrm{DR}\text{:}\mathrm{dilution}\mathrm{rate}\left(h^{-1}\right)$

(24) TABLE-US-00002 TABLE 2 Performances of the C. acetobutylicum DG1 pSPD5 strain type 130P. The feed medium contained 105 g/L of glycerol from raw glycerine provided by ADM (Rolle, Switzerland) and Greenergy (London, UK) at a dilution rate of 0.070 h.sup.−1. PDO Production performances Type 130P strain Feed glycerol 109 (ADM/Greenergy) (g.l.sup.−1) PDO (g.l.sup.−1) 52.0 YPDO (g.g.sup.−1) 0.48 QPDO (g.l.sup.−1.h.sup.−1) 3.62 Dilution rate (h.sup.−1) 0.070 Residual glycerol (g.l.sup.−1) 7.50 Biomass (g.l.sup.−1) 1.4 Acetic acid (g.l.sup.−1) 5.1 YPDO: PDO yield (g/g of glycerol engaged) QPDO: PDO volumetric productivity

Example 2

PDO Production Performances of C. acetobutylicum DG1 pSPD5 Strains Type 008P and 130P in a Chemostat by Continuous Culture with High Concentration of Raw Glycerine

(25) The synthetic media used for clostridia batch cultivations contained per liter of tap water: glycerol, 30 g; KH.sub.2PO.sub.4, 0.5 g; K.sub.2HPO.sub.4, 0.5 g; MgSO.sub.4, 7H.sub.2O, 0.2 g; CoCl.sub.2 6H.sub.2O, 0.01 g; H.sub.2SO.sub.4, 0.1 ml; NH.sub.4Cl, 1.5 g; biotin, 0.16 mg; p-amino benzoic acid, 32 mg and FeSO.sub.4, 7H.sub.2O, 0.028 g. The pH of the medium was adjusted to 6.3 with NH.sub.4OH 3N. Commercial glycerol purchased from SDS Carlo_Erba (purity 99%) was used for batch cultivation. The feed medium for continuous cultures contained per liter of tap water: glycerol from raw glycerine, 105 g; KH.sub.2PO.sub.4, 0.50 g; K.sub.2HPO.sub.4, 0.50 g; MgSO.sub.4, 7H.sub.2O, 0.2 g; NH.sub.4Cl, between 0 to 1.5 g; CoCl.sub.2 6H.sub.2O, between 0.013 to 0.026 g; biotin, between 0.08 to 0.16 mg; p-amino benzoic acid, between 16 to 32 mg; FeSO.sub.4, 7H.sub.2O, 0.04 g; anti-foam, 0.05 ml; ZnSO.sub.4, 7H.sub.2O, 8 mg; CuCl.sub.2, 2H.sub.2O, 4 mg; MnSO.sub.4, H.sub.2O, 0.02 g to 0.04 g; H.sub.3BO.sub.3 between 0 to 2 mg; Na.sub.2MoO.sub.4, 2H.sub.2O, between 0 to 0.8 mg. Medium pH was adjusted between 3.5 and 4 with H.sub.2SO.sub.4 96%.

(26) Raw glycerine, from the transesterification process for biodiesel, was obtained from several different sources and had the following composition: Novance (Compiegne, France) (using vegetable oil; purity between 82 to 85%; Moisture between 8 to 13%; MONG between 0.1 to 0.3%; Ash 1.4%) ADM (Rolle, Switzerland) (using vegetable oil; purity 80.9%; Moisture 12.6%; MONG 0.39%; Ash 6.2%), used in a blend with Greenergy glycerine Greenergy (London, UK) (using cooking oil; purity 76.5%; Moisture 10.2%; MONG 7.1%; Ash 6.3%), used in a blend with ADM glycerine

(27) Optionally, these glycerine were pretreated by acidification.

(28) As explained above in Example 1, the purity and MONG composition has an incidence on the toxicity of the glycerine on the microorganism.

(29) Experimental set-up is as described in Example 1, above.

(30) Batch and Continuous Cultures Process:

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

(32) Cultures were first grown in batch mode. At the early exponential growth phase we performed a pulse of glycerol with the feed medium (the same as described for feed culture). Glycerol from raw glycerine was added at a static flow rate during 3 hours (i.e. an addition of 18 g/L of glycerol). Then, the growth continued in batch mode and before the end of the exponential growth phase the continuous feeding started with a dilution rate of 0.035 h.sup.−1. Five to eight days after inoculation of the bioreactor, the dilution rate was increased from 0.035 h.sup.−1 to 0.070 h.sup.−1 in five days. After that, stabilization of the culture was followed by PDO production and glycerol consumption using the HPLC protocol described in example 1 in Analytical procedures.

(33) TABLE-US-00003 TABLE 3 Performances of the C. acetobutylium Type 008P and of the Type 130P strains in continous culture. The feed medium contained 105 g/L of glycerol from raw glycerine at dilution of 0.070 h.sup.−1. Mean data from respectively 8 and 17 chemostats. Providers of glycerine used in the cultures are indicated for each strain. Novance (Compiègne, France) corresponds to a relatively clean and pure glycerine while ADM (Rolle, Switzerland) and Greenergy (London, UK) provide glycerine that is less pure with more contaminants, and therefore more toxic to the microorganism. Type 008P strain Type 008P strain Type 130P strain Raw Raw glycerine Raw glycerine glycerine used: ADM/ used: Novance or used: Greenergy ADM/ Novance blend Greenergy blend* Feed glycerol (g.l.sup.−1) 105 104 106 1,3-propanediol (g.l.sup.−1) 49.8 41.7 52.3 YPDO (g.g.sup.−1) 0.47 0.40 0.49 QPDO (g.l.sup.−1.h.sup.−1) 3.55 2.90 3.70 Dilution rate (h.sup.−1) 0.072 0.070 0.071 Residual 5.7 18.6 3.6 glycerol (g.l.sup.−1) Biomass (g.l.sup.−1) 2.3 1.7 2.3 Acetic acid (g.l.sup.−1) 2.5 2.8 3.1 Butyric acid (g.l.sup.−1) 10.8 8.2 10.6 YPDO: PDO yield (g/g of glycerol engaged) QPDO: PDO volumetric productivity *Performances for strain Type 130P did not change significantly when different glycerine types were used (i.e. Novance or ADM/Greenergy blend)

(34) These results show that the Type 130P strain bearing an intergenic mutation (in this case between the nucleotides at positions 1014234 to 1014240 on the chromosome according to C. acetobutylicum ATCC 824) that induces overexpression of hcpR and frdX genes surprisingly exhibits a better PDO production with higher titer and yield and a lesser residual glycerine than its parental strain, 008P.

(35) These results also demonstrate the great advantage of the strain Type 130P which grew and produced much more PDO than the mother strain Type 008P which does not carry the genetic modification (Table 3). Indeed, all key industrial parameters (higher titer and yield of PDO and less residual glycerol) were improved for the 130P when compared to the 008P in culture conditions with industrial glycerine more toxic than usually used with Type 008P strain.

(36) Thus, upon overexpression of the hcpR and frdX genes, the C. acetobutylicum DG1 pSPD5 strain produces more PDO and is more robust and therefore more suitable for an industrial process.

Example 3

Intergenic Mutation Description

(37) Unexpectedly, a single nucleotide insertion in the intergenic region between CA_C0884 and CA_C0885 genes (illustrated in FIG. 2, SEQ ID NO: 15) has the effect of improving the production of PDO and the resistance to impurities MONG present in glycerin.

(38) The nucleotide insertion was detected by nucleic acid sequencing of PCR fragment amplified on DNA of strain Clostridium acetobutylicum DG1 pSPD5 Type 008P compared to DNA of Clostridium acetobutylicum DG1 pSPD5 Type 130P using oligonucleotides Intergenic region forward primer (SEQ ID NO: 18) and Intergenic region reverse primer (SEQ ID NO: 19). The ‘A’ insertion mutation was identified in the intergenic region between CA_C0884 and CA_C0885 genes in a region of repeating ‘A’ nucleotide as mentioned in SEQ ID NO: 17 compared to the parental type sequence (SEQ ID NO: 16). The gene CA_C0884 hcpR (SEQ ID NO: 1) codes for a nitric oxide-responsive transcriptional regulator (SEQ ID NO: 2) and the gene CA_C0885 frdX (SEQ ID NO: 3) codes for a ferredoxin 3-like protein (SEQ ID NO: 4).

Example 4

CA_C0884 and CA_C0885 Gene Expression with or without the Intergenic Nucleotide Insertion

(39) RNA Isolation

(40) RNA was extracted from 2 ml of flask culture, transferred into a 6 ml mixture of phenol (5%)/ethanol (95%) and centrifuged at 3000 g at 4° C. for 5 minutes. Pellet was homogenized in 100 μL lysozyme 100 mg/mL, incubated 30 minutes at 37° C. and RNA was extracted using the Maxwell RSC Simply RNA Tissue kit (Promega) in a Maxwell RSC instrument (Promega).

(41) Quantification of a Specific Ribonucleic Acid Sequence by Quantitative Reverse Transcription PCR (qRT-PCR)

(42) As RNA cannot serve as a PCR template, the first step in gene expression profiling by qRT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.

(43) Reverse transcription was performed with 0.2 μg of total RNA and reverse transcribed into cDNA using SuperScript ViloIV (Invitrogen) in the presence of random primers and oligo dT primers.

(44) The reverse transcriptase reaction was done in a total volume of 20 μl. After completion of the reaction, the mixture was held at 85° C.

(45) Relative quantification in samples was determined by quantitative PCR using the SsoAdvanced Universal SYBR Green Supermix (Bio-rad Mitry Mory, France). Quantitative PCR was performed on a Bio-Rad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System (Bio-Rad).

(46) PCR reactions mixtures consisted of 1×Sso Advanced Universal SYBR Green Supermix (Bio-Rad), 6 μL of a mix of forward (F) and reverse (R) primers (1 μM), 2 μL of diluted sample and nuclease free water to reach a final volume of 20 μL. Amplification was achieved according to the following thermal cycling program: initial melting at 98° C. for 2 min (1 cycle) followed by 40 cycles of melting at 98° C. for 10 sec, annealing of primers and elongation at 60° C. for 30 sec. (Melt Curve 65 to 95° C., increment 0.5° C. every 5 sec). For each experiment, threshold levels (Ct) were set during the exponential phase of the qPCR reaction using CFX Manager™ 3.1 software (Bio-rad).

(47) The expression level of each gene was determined by quantitative reverse transcription PCR (qRT-PCR). The CA_C0884 gene based primers used were CA_C0884 gene based forward primer (SEQ ID NO:20) and CA_C0884 gene based reverse primer (SEQ ID NO:21) and the CA_C0885 gene based primers used were CA_C0885 gene based forward primer (SEQ ID NO:22) and CA_C0885 gene based reverse primer (SEQ ID NO:23). The amount of each target gene relative to the housekeeping gene DNA gyrase subunit A (gyrA: CA_C1628, primers used CA_C1628 gene based forward primer (SEQ ID NO:24) and CA_C1628 gene based reverse primer (SEQ ID NO:25) was determined for each sample using the comparative threshold cycle (Ct) method, with serial dilutions of ATCC824 genomic DNA at known concentrations used as the calibrator for each experiment. Approximately equal efficiencies of the primers were confirmed using serial dilutions of ATCC824 genomic DNA templates in order to use the comparative Ct method.

(48) The relative expression level of both genes CA_C0884 and CA_C0885 was significantly higher in strain Type 130P carrying the intergenic mutation compared to the strain Type 008P with the parental type intergenic region (FIG. 3).

(49) These data demonstrate that the nucleotide insertion occurring in the intergenic region between CA_C0884 and CA_C0885 of recombinant Clostridium acetobutylicum DG1 psPD5 strain producing PDO, allows the overexpression of the two said genes hcpR and frdX. In the presence of this mutation, PDO production performance features are improved and the strain is much more resistant to dirty, high content MONG compounds present in industrial glycerine.

REFERENCES

(50) Altschul S, Gish W, Miller W, Myers E, Lipman D J (1990). J. Mol. Biol; 215 (3): 403-410. Chambers et al. (1988). Gene; 68(1): 139-49. Davis J J & Olsen G J. (2011). Mol. Biol. Evol.; 28(1):211-221. Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, Wolf H, Wagner R. (2011). González-Pajuelo M, Meynial-Salles I, Mendes F, Andrade J C, Vasconcelos I, and Soucaille P. 2005. Metabolic Engineering, 7: 329-336. González-Pajuelo M, Meynial-Salles I, Mendes F, Soucaille P. and Vasconcelos I. (2006). Applied and Environmental Microbiology, 72: 96-101. Graf M, Bojak A, Deml L, Bieler K, Wolf H, Wagner R. (2000). J. Virol.; 74(22): 10/22-10826. Lee S, Bennett G, Papoutsakis E, (1992). Biotechnology Letters, 14(5): 427-432. Papanikolaou S, Ruiz-Sanchez P, Pariset B, Blanchard F and Fick M. (2000), Journal of Biotechnology, 77: 191-2008. Tummala et al. (1999). Appl. Environ. Microbiol., 65(9): 3793-3799. Sambrook and Russell, (2001), Molecular Cloning: 3.sup.rd edition, Cold Spring Harbor Vasconcelos I, Girbal L, Soucaille P., (1994), Journal of Bacteriology, 176(5): 1443-1450.