WO2009109580A2

WO2009109580A2 - Solvent production process

Info

Publication number: WO2009109580A2
Application number: PCT/EP2009/052512
Authority: WO
Inventors: Truke Widel Smoor
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2008-03-04
Filing date: 2009-03-03
Publication date: 2009-09-11
Anticipated expiration: 2010-09-04
Also published as: WO2009109580A3

Abstract

The present invention relates to a solvent production process, in particular to a fermentative solvent production process in which a fast growing microorganism is used. It also relates to the fast growing microorganism. The fast growing microorganism according to the invention has a high growth rate which is maintained over a wide range of solvent concentrations. It can therefore advantageously be used in solvent production processes to reach high productivity.

Description

SOLVENT PRODUCTION PROCESS

Field of the invention

The present invention relates to a solvent production process, in particular to a fermentative solvent production process in which fast growing microorganisms are used.

Background of the invention

The acetone/butanol/ethanol (ABE) fermentation process has received considerable attention in the recent years as a prospective process for the production of commodity chemicals, such as butanol and acetone from biomass.

The fermentation of carbohydrates to acetone, butanol, and ethanol by solventogenic Clostridia is well known since decades. Clostridia produce butanol by conversion of a suitable carbon source into acetyl-CoA. Substrate acetyl-CoA then enters into the solventogenesis pathway to produce butanol using six concerted enzyme reactions.

The formation of butanol requires the conversion of acetyl-CoA into acetoacetyl- CoA by acetyl transferase. This reaction is followed by the conversion of acetoacetyl- CoA into 3-hydroxylbutyryl-CoA by 3-hydroxyl-CoA dehydrogenase, which is followed by the conversion of 3-hydroxylbutyryl-CoA into crotonyl-CoA by 3-hydroxybutyryl-CoA dehydratase (also named crotonase) and the conversion of crotonyl-CoA into butyryl- CoA by butyryl-CoA dehydrogenase and followed by the conversion of butyryl-CoA to butyraldehyde by butyraldehyde dehydrogenase, with the final conversion of butyrylaldehyde to butanol by butanol dehydrogenase (Jones, DT. , Woods, D. R., 1986, Microbiol. Rev., 50, 484-524).

However, the production of butanol suffers from poor process economics, because the butanol produced is toxic for the microbial cells and thus titers are low. Many studies have been directed to increase the resistance of Clostridia strains against butanol and consequently achieve an increase in titers. In US 6,960,465, it is for instance shown that overexpression of the heat shock proteins in Clostridium acetobutylicum resulted in an increased butanol titer of about 17 g/l compared to the wild type strain (about 12 g/l). Similar results have been described for a Clostridium beijerinckii mutant in US 6,358,717. Despite the improvements that have been achieved

so far in the ABE fermentation in Clostridia, the maximum achievable titer of butanol in Clostridia is still considerably low (i.e. about 20 g/l) for a butanol production process on an industrial scale and the reliability of the process is still insufficient.

In addition to maximally achievable titer, the amount of butanol produced per hour (productivity) is an important feature. Traditionally butanol fermentations, using Clostridium as production organism, were operated batch wise. The solvent productivity in a batch reactor is limited to 0.5 g/L/h, (Blashek & Qureshi 2001 J. Ind. Microb. Biotech 27:292) despite research to improve the butanol production process using Clostridium sp.. However recent studies show that a continuous process with high cell density can improve the productivity to up to 7.5 g/l/h. (Tashiro et al. 2005 J Biotechnol 120:197). The concomitant advantage of such continuous processes at high cell density is an enormous decrease in reactor volume. The increase in productivity results from the higher amount of active biomass in the reactor. This is only possible if the growth rate in the process is higher than the death rate. Therefore, these continuous processes are often operated at low solvent concentration to minimize the death rate in the reactor. Therefore, there is a need for microorganisms which have high growth rates which are maintained at high solvent concentrations

Detailed description of the invention

In a first aspect, the present invention relates to a fast growing microorganism. The fast growing microorganism according to the invention has a relative growth rate of at least 0.65 at LC₅₀ Of a solvent selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms.

One advantage of a fast growing microorganism according to the invention is that it has a high growth rate which is maintained at high solvent concentrations. It can therefore advantageously be used in solvent production processes to reach high productivity.

The fast growing microorganism according to the invention has a relative growth rate of at least 0.65 at LC₅₀ of a solvent selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms. LC₅₀ is defined as 50% of the lethal concentration of a particular solvent for a particular microorganism. The lethal concentration is defined as the concentration at which the growth rate is below 0.01 h^"1. Preferably, the fast growing microorganism according to the invention has a relative growth rate of at least 0.70, of at least 0.75 or of at least 0.80, more preferably of at least 0.85, most preferably of at least 0.90, at least 0.95 or at least 1.0 at LC₅₀ Of a solvent selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms. More preferably, the solvent is a four carbon solvent, acetone or ethanol. Most preferably, the solvent is a four carbon aliphatic hydrocarbon, preferably a butanol. Preferably, a butanol is 1-butanol, 2-butanol, isobutanol, 2,3- butanediol or 2-butanone.

The skilled person will understand that the fermentation temperature is dependent on the fast growing microorganism involved. In general, the temperature will be a temperature in the range of 25 to 45 degrees C, preferably in the range of 28 to 40 degrees C, more preferably in the range of 30 to 40 degrees C. For yeast, the temperature is preferably in the range of 25 to 45 degrees C, more preferably in the range of 28 to 40 degrees C. For most bacteria, the temperature is preferably in the range of 25 to 40 degrees C, more preferably in the range of 28 to 40 degrees C, even more preferably in the range of 30 to 37 degrees C. For fungi, the temperature is preferably in the range of 25 to 40 degrees C, more preferably in the range of 28 to 40 degrees C, even more preferably in the range of 30 to 40 degrees C and most preferably in the range of 30 to 36 degrees C.

The pH is also dependent on the organism, but a suitable pH for fermentation is in the range of between pH 3.0 and pH 9.0, preferably between pH 3.0 and pH 7.0 for yeast and fungi, and between pH 5.0 and pH 9.0 for bacteria.

The fast growing microorganism according to the invention is preferably used for solvent production processes wherein the solvent is selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms. More preferably, the solvent is a four carbon solvent, acetone or ethanol. Most preferably, the solvent is a four carbon aliphatic hydrocarbon, preferably a butanol. Preferably, a butanol is 1-butanol, 2-butanol, isobutanol, 2,3-butanediol or 2-butanone. The fast growing microorganism according to the invention may be any microorganism but is preferably a well-established industrial microorganism. In one embodiment, the fast growing microorganism according to the invention is a eukaryotic microorganism. Preferably, it is a fungus or a yeast. More preferably, it is a filamentous fungus or a yeast. Suitable examples of such fungi and yeast belong to one of the genera Saccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Torulaspora, Trichosporon, Brettanomyces, Pachysolen or Yamadazyma. In particular, the species Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. glabrata, Hansenula polymorpha, Torulaspora delbrueckii, Brettanomyces bruxellensis, Zygosaccharomyces bailii, Saccharomyces uvarum, S. bayanus or S. cerevisiae species. Even more preferably, the fast growing organism is a Saccharomyces cerevisiae or a wine yeast. Most preferably, the fast growing microorganism is one with the identifying characteristics of CBS 122638.

In another embodiment, the fast growing microorganism according to the invention is a bacterium, be it an aerobic, a strictly anaerobic or facultative anaerobic bacterium, a Gram positive or Gram negative bacterium. Suitable examples belong to one of the genera selected from the group consisting of Escherichia, Streptomyces, Clostridium, Alcaligenes, Azoarcus, Thauera, Brady ryzobium, Brevibacterium, Shewanella, Staphylococcus, Mycobacterium, Brucella, Bordetella, Fusobacterium, Rhodococcus, Geotrichum, Corynebacterium, Bacillus, Lactobacillus, Streptococcus, Pseudomonas, and Zymomonas. More preferably, the solvent tolerant microorganism is selected from the group consisting of Escherichia coli, Streptomyces lividans, Staphylococcus cohnii, Bacillus subtilis, B. cereus, B. coagulans, B. sphericus, B. licheniformis, B. amyloliquefaciens, B. megaterium, B. pumilus, B. thuringiensis, Clostridium acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum, C. thermobutyricum, C. tyrobutyricum, C. butyricum, C. saccharolyticum , C. saccherobutylicum, C. tetanomorphum, C. pasteurianum, C. aurantibutyricum, Lactobacillus homohiochii, L. plantarum, L. heterohiochii, L. fructivorans, Streptococcus lactis, Streptococcus amoris, Pseudomonas butanovora, Pseudomonas putida, and Zymomonas mobilis. Preferably, the bacterium is a lactic acid bacterium or a Clostridium. Most preferably, the fast growing microorganism is a Clostridium beijerinckii with the identifying characteristics of CBS 124166.

The fast growing microorganism may be obtained by any method known in the art or combinations of methods known in the art. The fast growing capacity may have been naturally present or may have been introduced or accomplished by techniques available in the art, such as classical strain improvement, selective pressure by exposure to solvents, genetic engineering, genome shuffling, adaptive evolution, or by any combination of techniques, resulting in a mutant from the wild type strain.

Preferably, a microorganism according to the present invention is obtained by growing the microorganism in a continuous culture at increasing solvent concentrations during a period of between 1 day and 6 months, preferably between 1 week and 4 months, preferably between 2 weeks and 2 months. Increasing solvent concentrations is used herein to indicate a continuous culture comprising a concentration increasing from 0.1 v/v% to 5 v/v% of solvent, preferably a concentration increasing from 0.2 to 4 v/v%, preferably a from 0.3 to 3 v/v%, more preferably from 0.5 to 2 v/v% of solvent. Alternatively, the fast growing microorganism may be obtained by growing the microorganism in the presence of a solvent under conditions that increase selective pressure on growth rate by increasing the dilution rate in a continuous culture during a period of between 1 day and 6 months, preferably between 1 week and 4 months, preferably between 2 weeks and 2 months. An increasing dilution rate is used herein to indicate a continuous culture with an increased dilution rate from 0.01 h^"1 to 0.5 h^"1, preferably from 0.015 h^"1 to 0.4 h^"1, preferably from 0.02 to 0.3 h^"1. Preferably, a mutagens is applied in the continuous culture. The skilled person in the art knows how to execute a continous culture. In another aspect, the present invention relates to the use of a fast growing microorganism according to the invention in a solvent production process.

In another aspect, the present invention relates to the use of a fast growing microorganism according to the invention in a solvent production process, wherein the solvent selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms.

A great advantage of the solvent production process according to the present invention is that smaller fermentors may be used, since the biomass per fermentor will be higher than in conventional processes, because the growth of the microorganisms will continue, even at high levels of the solvent product. As a consequence, downstream processing will also be more efficient.

The solvent production process according to the invention comprises i) culturing a fast growing microorganism according to claims 1 -7 in an appropriate culture medium and under appropriate conditions to produce the solvent, and optionally, ii) separating the solvent from the culture medium. Preferably, the solvent produced is an aliphatic hydrocarbon comprising two, three or four carbon atoms. More preferably, the solvent is a four carbon solvent, acetone or ethanol. Most preferably, the hydrocarbon comprising four carbon atoms is a butanol, such as 1-butanol, 2-butanol, isobutanol, 2,3-butanediol or 2-butanone.

If the fast growing microorganism according to the invention does not produce the desired solvent, the microorganism may be genetically engineered to obtain a microorganism which produces the solvent. For example, by introducing vectors or plasmids containing genes encoding enzymes of one of the solvent biosynthetic pathways together with regulatory elements into a solvent tolerant microorganism according to the invention.

In one embodiment, a fast growing microorganism according to the invention which produces a butanol may be obtained by introducing vectors or plasmids containing genes encoding enzymes of one of a butanol biosynthetic pathways together with regulatory elements into this fast growing microorganism, which then acts as a host cell. Methods for constructing butanol producing microorganisms are well- known in the art, for example from WO2007/041269 or WO2008/052991 disclosing n- butanol producing microorgnisms, and will generally involve transformation of the host cell with one or more nucleic acid constructs comprising one or more nucleotide sequences encoding acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or acetaldehyde dehydrogenase or NAD(P)H-dependent butanol dehydrogenase. Methods for constructing isobutanol producing microorganisms are known from WO2007/050671 and WO2008/098227 and will generally involve transformation of a host cell with one ore more nucleic acid constructs comprising one or more nucleotide sequences encoding encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of: i) pyruvate to acetolactate ii) acetolactate to 2,3-dihydroxyisovaferate iii) 2,3-dihydroxyisovalerate to [alpha]-ketoisovalerate iv) [alpha]-ketoisovalerate to isobutyraldehyde, and v) isobutyraldehyde to isobutanol. Alternative isobutanol pathways are also disclosed in

WO2007/050671.

Therefore, a fast growing microorganism according to the invention which has been transformed with genetic information for encoding enzymes of a solvent biosynthetic pathway is also encompassed by the present invention.

EXAMPLES

Example 1 Preparation of fast growing strains Saccharomyces cerevisiae cells were cultured in YEPD in a 1 liter applikon fermentor with a working volume of 500 ml. From this fermentor, cells were grown for one month in a continuous culture system to which increasing amounts of butanol were added in order to select for mutants with high growth rates at high solvent concentrations. At least once a week a sample was plated on a YEPD agar plate. One or more colonies were collected, resuspended in physiological saline and used for growth rate experiments. In this way, mixed populations of fast growing organisms V370 CC3, CC4 and CC5 were isolated. One single isolate from the CC5 strain population was deposited with the CBS in Baarn on 26 February 2008 under Accession No. CBS 122638.

Example 2 Growth rate of baker's yeast

Growth rate in the absence and in the presence of butanol was measured in microtiter plates (MTP). The MTP medium was based on YEPD medium, but some water was eliminated in order to compensate for the butanol to be added. The media contained 0-5-10-15-20 and 25 g/L of butanol respectively prior to inoculation. After inoculation, every 20 minutes the optical density at 620 nm was measured in the wells of the microtiter plate using a Multiscan Ascent photometer (Thermo Fisher Scientific, Inc.), while the temperature was controlled at 30 degrees C. Total time of measurement was maximally 24 hours. Before measurements were taken, the microtiter plates were shaken for 30 seconds to homogenize any settled biomass.

For retrieving kinetic data, the incubation period was limited to 5-10 hours at 30 degree C to keep the error in butanol concentrations due to butanol evaporation as low as possible in the sealed microtiter plates. Growth was monitored by measuring the optical density at 620 nm. The maximum growth rate was calculated by a linear fit through the logarithmic value of the optical density. The relative growth rate was calculated using the reference at 0 g/L as 1.00. In Table 1 the growth rate of three different strain populations according to the invention are given. The wild type strain is included as a reference.

Table 1. Growth rate and relative growth rates at LC50

The results in Table 1 show that the growth rate of the fast growing strains according to the invention (V370 CC3, CC4 and CC5) decreases only slowly in reaction to increasing butanol concentrations. At 20 g/l butanol, growth is lower than 0.01 h ^"1 and therefore, LC₅O of butanol for yeast is 10 g/l. The relative growth rate at the LC50 of the fast growing microorganisms of the invention is higher than the LC50 of the wild type, which indicates that their growth rates are less affected by butanol than the growth rate of the wild type.

Example 3

Preparation of fast growing strains

Clostridium beijerinckii cells were cultured in TGY medium in a 1 liter applikon fermentor with a working volume of 500 ml. From this fermentor, cells were grown for two months in a continuous culture system to which increasing amounts of butanol were added in order to select for mutants with high growth rates at high solvent concentrations. At least once a week a sample was plated on a TGY agar plate. One or more single colonies were collected and used for growth rate experiments. In this way, fast growing organisms of ATTCC 35702 were identified.. The single isolate from ATCC 35702 CC4 strain population was deposited with the CBS in Baarn on 27 February 2009 under Accession No. CBS 124166.

Example 4 Growth rate of Clostridium

Growth rate in the absence and in the presence of butanol was measured in anaerobic flasks. The medium that was used was Reinforced Clostridial medium (Merck

KGaA). The media contained 0-6-12 and 16 g/L of butanol respectively prior to inoculation. The flasks were incubated in a kelvitron incubator (Hereaus Instruments), while the temperature was controlled at 37 degrees C.

Growth was monitored by measuring the optical density at 600 nm. The maximum growth rate was calculated by a linear fit through the logarithmic value of the optical density. The relative growth rate was calculated using the reference at 0 g/L as 1.00.

In Table 2 the growth rate of three different strain populations according to the invention are given. The wild type strain is included as a reference.

Table 2. Growth rate and relative growth rates at LC50

The results in Table 2 show that the growth rate of the fast growing strains according to the invention (ATCC CC4) decreases only slowly in reaction to increasing butanol concentrations. At 12 g/l butanol, growth is lower than 0.01 h -1 and therefore, LC50 of butanol for Clostridium beijerinckii ATCC 35702 under these conditions is 6 g/l. The relative growth rate at the LC50 of the fast growing microorganisms of the invention is higher than the LC50 of the wild type, which indicates that their growth rates are less affected by butanol than the growth rate of the wild type.

I Applicant's or agent's file reference number 26459 WO International application No.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule \3bιs)

A. The indications made below relate to the microorganism referred to in the description first mentioned on page 4 line 8 .

B. EJENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet X

Name of depositary institution

CENTRAAL BUREAU VOOR SCfflMMELCULTURES

Address of depositary institution (including postal code and country) Uppsalalaan 8 P.O. Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 26 February 2008 Accession Number CBS 122638

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated

States)

E. SEPARATE FURNB HING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications eg , "Accession Number of Deposit")

For receiving Office use only

This sheet was received with the international application

Authorized officer

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

A. The indications made below relate to the microorganism referred to in the description first mentioned on page 4 line 27.

B. roENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Date of deposit 27 February 2009 Accession Number CBS 124166

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g , "Accession Number of Deposit")

For receiving Office use only

This sheet was received with the international application

Authorized officer

Foπn PCT/RO/134 (July 1992)

Claims

1. A microorganism which has a relative growth rate of at least 0.65 at the LC₅o θf a solvent selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms.

2. A microorganism according to claim 1 , wherein the aliphatic hydrocarbon comprising two, three or four carbon atoms is a four carbon solvent, acetone or ethanol.

3. A microorganism according to claim 1 or 2 wherein the aliphatic hydrocarbon comprising four carbon atoms is a butanol.

4. A microorganism according to any one of the claims 1 to 3 wherein the microorganism is a eukaryotic microorganism, preferably a fungus or a yeast.

5. A microorganism according to any one of the claims 1 to 3, wherein the microorganism is a prokaryotic organism, preferably a bacterium, more preferably a lactic acid bacterium or a Clostridium.

6. A microorganism as deposited under number CBS 122638 or CBS 124166.

7. A microorganism according to claims 1-6 which is transformed with genetic information for encoding enzymes of a solvent biosynthetic pathway, preferably of a butanol biosynthetic pathway.

8. Use of a microorganism according to claims 1-7 in a solvent production process, wherein the solvent is selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms.

9. Use according to claim 8, wherein the aliphatic hydrocarbon comprising four carbon atoms is a butanol.

10. A solvent production process, which process comprises: i) culturing a microorganism according to claims 1-7 in an appropriate culture medium and under appropriate conditions to produce the solvent, and optionally, ii) separating the solvent from the culture medium, wherein the solvent is selected from the group consisting of an aliphatic hydrocarbon comprising two, three or four carbon atoms.