Patent Application
20150368678
The present invention relates to methods of making solvents, such as butanol, acetone or ethanol. In particular, the invention relates to a process for producing a solvent, comprising the step of culturing a solventogenic Clostridium sp. in a culture medium in a culture vessel in the presence of a polysaccharide, wherein the Clostridium sp. is one which is capable of producing a CGTase, and wherein the polysaccharide is one which is a substrate for the CGTase, and harvesting solvent from the culture medium. Preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum N1-4(HMT) or N1-504. The invention also relates to butanol, acetone and ethanol made by such a process.
Solvent-producing Clostridia were first used during the 1920's and 1930's for the industrial production of acetone, butanol and ethanol. During the 1950's the establishment of more efficient petrochemical techniques to synthesise these solvents lead to the abandonment of such large-scale bacterial fermentations. However, in the present environment, with rising oil prices and increasing pressure for the development of biofuels, the interest in Clostridial fermentations for the production of solvents is being renewed. This has also been helped by advancements in the biological understanding of these solventogenic Clostridia and the development of microarray assays. These areas of research have opened up the possibility of engineering new strains capable of over-producing butanol, further improving the economic use of solventogenic fermentations.
It has long been known that growth by solventogenic Clostridia is biphasic, with exponential growth being characterised by profuse acid production and solvent production occurring at the onset of the stationary phase (review by Jones and Woods, 1986, Microbiological Reviews, 50:484-524). There remains a need, however, for enhanced methods of producing ABE.
Thang et al. (Appl. Biochem. Biotechnol. (2010) May; 161(1-8):157-70) describes the production of acetone-butanol-ethanol (ABE) in direct fermentation of cassava by Clostridium saccharoperbutylacetonicum N1-4. This paper refers to the hydrolysis of cassava starch into maltose and glucose by the bacteria's own α-amylase and glucoamylase enzymes. These enzymes are said to continue hydrolysing starch throughout the fermentation. Batch fermentations were performed in stirred-tank fermenters wherein the initial pH was 6.2 and the temperature was maintained at 30° C. Anaerobic conditions were ensured by sparging the fermentation broth with oxygen-free nitrogen and by flushing the fermentation broth at the beginning of the fermentation with nitrogen gas.
It has now been found, however, that certain other Clostridium saccharoperbutylacetonicum strains do not have obvious gene homologues which encode α-amylase or glucoamylase. In these other strains—which include Clostridium saccharoperbutylacetonicum N1-4(HMT) and N1-504—the hydrolysis of starch-based materials such as cassava actually occurs by means of a novel cyclodextrin glucanotransferase (CGTase) enzyme.
This new knowledge has allowed the conditions for solvent-producing processes involving certain Clostridium saccharoperbutylacetonicum strains to be optimised in order to enhance the activity of the CGTase and hence to increase the production of acetone, butanol and/or ethanol. In particular, the CGTase has been found to perform optimally at a temperature which is significantly different from that used in Thang et al. (supra). Additionally, it has been found that the CGTase is not affected by the presence of oxygen and that growing cultures of some Clostridium saccharoperbutylacetonicum strains may in fact be inoculated aerobically.
It should be noted that wherein Clostridium thermohydrosulfuricus was previously classified as a Clostridial species, it has now been reclassified as Thermoanaerobacter thermohydrosulfuricus (Collins, et al (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Bacteriol., 44(4), 812-26). The genus Thermoanaerobacter has now clearly established by sequence analysis and shown that it forms a separate and distinct genus from Clostridium sensu stricto (Cluster I) (Stackebrandt et al. (1999) Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol. Med. Microbiol., 24(3), 253-8).
In one embodiment, therefore, the invention provides a process for culturing a micro-organism, the process comprising the step:
The invention also provides a process for producing a solvent, the process comprising the step:
In a further embodiment, the invention provides a process for producing butyric acid or an acid derivative thereof, the process comprising the steps:
Preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum, more preferably a N1-strain. Even more preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum N1-4(HMT) or N1-504. In some embodiments, the Clostridium sp. is cultured at a temperature of 32-47° C.
The Clostridium sp. is one which produces or is capable of producing a cyclodextrin glucanotransferase (CGTase). CGTases are also known as cyclodextrin glycosyl transferases and cyclodextrin glucosyltransferases. Whilst CGTases are generally capable of catalysing more than one reaction, the most important activity is the production of cyclic dextrins from substrates such as starch, amylose and other polysaccharides. In this process, the polysaccharide chain is cleaved and the ends are joined by the CGTase in order to produce a cyclic dextrin, i.e. a cyclodextrin. The size of the cyclodextrin (i.e. the number of sugar residues it incorporates) is dependent on the distance apart of the ends.
The CGTases fall within the general EC classification 2.4.1 (hexosyltransferases). In some embodiments, the CGTase falls within classification EC 2.4.1.248 (cycloisomaltooligosaccharide glucanotransferase). In other embodiments of the invention, the CGTase falls within classification EC 2.4.1.19 (cyclomaltodextrin glucanotransferase).
In one embodiment, the amino acid sequence of the CGTase:
Variants or derivatives of the polypeptide of SEQ ID NO: 1 or 3 may also be used. The CGTase may be altered in various ways including substitutions, deletions, truncations, and/or insertions of one or more (e.g. 2-5, 2-10) amino acids, preferably in a manner which does not substantially alter the biological activity of the CGTase. Guidance as to appropriate amino acid changes that do not affect biological activity of the CGTase may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be also made.
In particular, substitution of one hydrophobic amino acid such as isoleucine, valine, leucine or methionine for another may be made; or the substitution of one polar amino acid residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, may be made.
One or more (e.g. 1-5, 1-10) amino acids in the CGTase may be substituted by their corresponding D-amino acids, preferably at the N- and/or C-terminus.
In particular, the invention provides the use of a variant of the CGTase of SEQ ID NO: 1 or 3, wherein the amino acid sequence of the variant comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 1 or 3, preferably using the blastp method of alignment.
Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul et al. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BLAST). Preferably the standard or default alignment parameters are used.
Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. Preferably the standard or default alignment parameters are used. In some instances, the “low complexity filter” may be taken off.
BLAST protein searches may also be performed with the BLASTX program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs may be used.
The invention particularly relates to uses of CGTases of SEQ ID NO: 1 or 3 or to variants of the CGTases of SEQ ID NO: 1 or 3 as defined herein, wherein the amino acid which corresponds to the amino acid at position 223 is a small amino acid, e.g. glycine, alanine, leucine, serine, threonine or valine, preferably glycine. The size of the amino acid residue at position 223 may be involved in determining the size of any cyclodextrin products or ratio of any cyclodextrin products.
In general, a nucleic acid encoding the CGTase will be stably incorporated into the genome of the Clostridium sp.
The Clostridium sp. may be a wild-type Clostridium sp. or a mutant (e.g. genetically engineered) Clostridium sp. or a recombinantly-produced Clostridium sp. Preferably, the Clostridium sp. comprises an endogenous CGTase gene (i.e. incorporated into its genome). In other preferred embodiments, the Clostridium sp. does not comprise a vector encoding a heterologous CGTase.
The Clostridium sp. is one which produces or is capable of producing a CGTase. Preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum, more preferably a N1-strain. Even more preferably, the Clostridium sp. is Clostridium saccharoperbutylacetonicum N1-4(HMT) or N1-504. In some embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-4. In other embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-4(HMT). In other embodiments, the recombinant bacteria is not Clostridium saccharoperbutylacetonicum N1-504.
Suitable culture vessels and culture media are known in the art. Generally, the culture media will be an aqueous media. The Clostridium sp. is cultured under conditions such that it expresses its endogenous CGTase, thus enabling the digestion of the polysaccharide into a hydrolysate or other product. Suitable conditions are known in the art.
The process of the invention may be operated in any suitable manner. For example, it may be operated as a batch process, fed-batch process or any form of continuous process or perfusion process. Preferably, the process is allowed to proceed for a time which is sufficient for the CGTase to digest the polysaccharide, either partially or completely.
In some embodiments of the invention, no additional amylases and/or glucoamylases from non-Clostridial bacteria are present in the culture vessel. For example, Bacillus subtilis amylase is preferably not present in the culture vessel. Preferably, glucoamylase from Rhizopus niveus is not present in the culture vessel.
Genes encoding α-amylase and glucoamylase are not present in either C. saccharoperbutylacetonicum N1-4 (HMT) or N1-504. In some embodiments of the invention in which these bacteria or derivatives thereof are used, α-amylase and/or glucoamylase may be added to the culture media or the polysaccharide may be pre-treated with α-amylase and/or glucoamylase. In some embodiments, recombinant α-amylase and/or recombinant glucoamylase is produced by the Clostridium sp. (preferably recombinant C. saccharoperbutylacetonicum N1-4 (HMT) or N1-504) in the culture medium.
The Clostridium sp. is cultured in the culture vessel at a temperature which is optimal for the activity of the CGTase or, in processes for the production of a solvent or butyric acid or acid derivative thereof, which is optimal for the production of the aforementioned products. This temperature range has been found to be 32-47° C. Preferably, the temperature is 32-45° C., more preferably 32-40° C. or 33-40° C., and most preferably 32-36° C. or 33-36° C.
The initial pH of the culture medium is preferably between pH 5.3 and pH 6.8, more preferably between pH 5.5 and pH 6.2. In some embodiments, it is preferably pH 5.4-5.6 or pH 6.1-6.3, and most preferably about pH 5.5 or about pH 6.2. In other embodiments, the initial pH is 6.4-6.6. In some preferred embodiments, the culture temperature is 34-36° C. and the initial pH is pH 5.4-5.6 or pH 6.1-6.3. During acidogenic growth, the pH may be allowed to drop to about pH 5.3. It may then rise to pH 6.0-6.3 during the reassimilation and solventogenic phase.
The polysaccharide is one which is a substrate for the CGTase. The polysaccharide may be a lignocellulosic feedstock, i.e. a feedstock comprising hemicellulose, cellulose and lignin; or a cellulosic feedstock.
In one embodiment, the polysaccharide may be a glucose-containing or glucose-based polysaccharide. In such embodiments, the glucose molecules will in general be linked in the polysaccharide by α(1→4) and/or α(1→6) glycosidic bonds. In some embodiments, the substrate is a starch-based substrate, e.g. starch, amylopectin, amylose or glycogen. Most preferably, the polysaccharide is starch or a starch-based material, e.g. corn, corn starch, corn mash, potato, potato starch, potato mash, potato peeling, potato chips, cassava, cassava starch, cassava chips, sago, sago starch, or ‘soluble starch’ (e.g. as sold by Fisher/Sigma).
In some embodiments, butyric acid or an acid derivative thereof is harvested from the culture medium. This may be done by any suitable continuous or discontinuous process. Preferably, the harvested butyric acid or an acid derivative thereof is subsequently converted to a solvent, most preferably to butanol.
In other embodiments, solvent is harvested from the culture medium. This may be done by any suitable continuous or discontinuous process. Solvents may include butanol, ethanol and/or acetone, preferably butanol.
Isolation of butanol, acetone and/or ethanol from the culture medium may be carried out by any suitable means. Examples of include gas-stripping, pervaporation, distillation and solvent extraction.
Whilst it has been previously been reported that it has been necessary to sparge the culture media with oxygen-free nitrogen in order to achieve anaerobiosis (Thang et al., supra), it has now been found that the CGTase is not affected by the presence of oxygen and that growing cultures of Clostridium sp. (and C. saccharoperbutylacetonicum N1-4(HMT) and N1-504 in particular) may in fact be inoculated aerobically, with no special precautions necessary to exclude oxygen. Furthermore, the culture vessel may be operated with air in the head-space above the culture medium.
In some embodiments of the invention, therefore, step (i) comprises inoculating and growing Clostridium sp. (and C. saccharoperbutylacetonicum N1-4(HMT) and N1-504 in particular) in a culture medium in a culture vessel under aerobic conditions or conditions which are not oxygen-free.
In a further embodiment, the invention relates to a solvent, preferably butanol, acetone or ethanol, which is obtained by a process of the invention. Most preferably, the solvent is butanol.
The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
Freeze dried Clostridium saccharoperbutylacetonicum N1-4 (HMT) was grown up in RCM overnight before inoculating into CGM containing either 5% starch or 5% glucose. Cultures were grown at 32° C. for 8 hours before being subcultured a second time into CGM containing 5% starch or 5% glucose. These cultures were grown overnight at 32° C. before recovering the supernatant by centrifugation. This was filter sterilised using a 0.2 μm filter and sodium azide was added to a final concentration of 0.02%. 500 μl of supernatant was concentrated approx. 10-fold using 10 kDa MWCO Vivaspin columns according to manufacturer's instructions.
Starch agar plates were prepared using 1.5% agar and 1% soluble starch. pH was not adjusted and was approx. 6.5.
10 μl of unconcentrated supernatant sample or 2 μl of the concentrated samples were spotted onto the plates and left to soak in. As a control, B. amyloliquefaciens α-amylase was diluted 1000-fold and 2 μl was spotted onto the plate. 5 μl dH2O was used as a negative control. The plates were wrapped in foil and incubated at 33° C., 36° C., 40° C., 45° C., 50° C., 55° C. or 60° C. overnight.
Areas of starch degradation were visualised by adding iodine solution to the plate for 20-30 sec. Excess iodine was removed before imaging.
The results are shown in
Supernatant was prepared from Clostridium saccharoperbutylacetonicum N1-4 (HMT) cultures grown for 72 hours, 32° C. in CGM supplemented with 5% glucose or 5% soluble starch. The cultures were centrifuged at 4000×g, 10 min, RT and the supernatant was removed and filter sterilised using a 0.2 μm filter. Sodium azide was added to a final concentration of 0.02% before storage at 4° C. Supernatants were concentrated 10-fold using Vivaspin columns according to the manufacturer's instructions.
Experiments using growing cultures were all inoculated aerobically and subsequently maintained in an anaerobic environment.
Starch agar plates were prepared using 1.5% agar and 1% soluble starch. The pH of the agar was adjusted to pH 5.0, 5.3, 5.5, 5.8 or 6.2 with 10% HCl prior to autoclaving. The pH of the unadjusted starch agar was pH 6.8.
Supernatants were spotted onto starch agar in 5 μl volumes. Alpha amylase (from Bacillus amyloliquefaciens, Sigma) was diluted 100-fold in dH2O and 2 μl was plated as a positive control for starch degradation. A 5 μl volume of dH2O was plated as a negative control. The plates were wrapped in foil before being incubated for 18 h at 30° C., 33° C. or 35° C. Areas of starch degradation were visualised by adding iodine solution to the plate for 20-30 sec. Excess iodine was removed before imaging.
The results are shown in
At 33° C. optimal enzyme activity is between pH 5.5-6.8. As seen at 30° C., there is only slight differences in size or intensity of clearing zones under these conditions. Enzyme has very little activity below pH 5.3 when incubated at 33° C.
At 35° C. enzyme activity was observed between pH 5.5-6.8. Optimal activity (based on size and intensity of clearing zones) was seen at pH 5.5 and pH 6.2. The enzyme has very little activity below pH 5.3 when incubated at 35° C.
The above results show that the CGTase enzyme has most activity at about 35° C.; the optimal pHs were pH 5.5 and pH 6.2. Furthermore, CGTase activity appears to be lower at 30° C. across the pH range compared to the 33° C. and 35° C. plates. No CGTase enzyme activity was observed below pH 5.3.
The enzyme plate assays were all carried out aerobically. Therefore the enzyme is not affected by the presence of oxygen.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1302030.0 | Feb 2013 | GB | national |
| 1303595.1 | Feb 2013 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2014/050321 | 2/5/2014 | WO | 00 |
