# Patent

## Title
Production of isopropanol by improved recombinant strains

## Abstract

The expression vector includes: the nucleic acids coding for the polypeptides forming a polypeptide complex having an enzyme activity allowing acetoacetyl-CoA to be converted to acetoacetate; optionally, at least one nucleic acid coding for a polypeptide having an enzyme activity allowing acetoacetate to be converted to acetone; and at least one nucleic acid coding for a polypeptide having an enzyme activity allowing acetone to be converted to isopropanol; the expression of the nucleic acids being controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids.

## Background
The present invention relates to a method for the production of isopropanol by improved recombinant strains.
BIBLIOGRAPHICAL RESEARCH
Isopropanol, also called 2-propanol, or isopropyl alcohol, is mainly used as a solvent for preparing inks and surfactants. Its other applications relate to the antiseptic and solvent properties of carboxymethylcellulose (CMC). Isopropanol is also used in the production of bases for cosmetics, as a solvent for pesticides or as a source of material in organic synthesis. As an indication, it may be mentioned that the production of isopropanol reached 45 million tonnes in the USA in 1990 (Papa A, 2005).
Nowadays, isopropanol is produced by an essentially chemical method consisting of hydration of propylene by a direct or indirect route. The indirect route uses two steps. In the first step, a mixture of mono- and di-isopropyl sulphuric esters is produced, which is hydrolysed to isopropanol in a second step. The direct route uses the hydration of propylene at high pressure and low temperature on a “fixed-bed” acid catalyst (Logsdon J. E. and Loke R. A., 2001).
From 1942 to 1958, isopropanol was produced in Taiwan by fermentation (Rogers et al. 2006). The substrates for the fermentation were sweet potato, cassava, and wheat starch, whereas the microorganism was a wild-type strain. Nowadays, this method for the production of isopropanol is no longer used industrially. In fact, the low yields obtained with wild-type strains (between 2 and 3 g/L of isopropanol for Clostridium beijerinckii) do not allow economically viable processes to be developed. Just like ABE (Acetone, Butanol, Ethanol) solventogenic fermentation, IBE (Isopropanol, Butanol, Ethanol) fermentation was abandoned at the beginning of the 1960s in parallel with the development of the petrochemical industry.
For ecological and economic reasons, research into the production of isopropanol by microbiological fermentation has experienced renewed interest in recent years. Isopropanol-producing fermentation is known as IBE fermentation. It differs from another solvent-producing fermentation, ABE fermentation, in that it produces isopropanol, solely or primarily, at the expense of acetone. The solvent-producing strains belong to the genus Clostridium. They have been reclassified into four species: C. acetobutylicum, C. beijerinckii, C. aurantibutyricum, C. saccharoperbutylacetonicum (Keis et al. 2001).
C. acetobutylicum is a strain that is often used in ABE fermentation. The main products from fermentation with C. acetobutylicum are therefore acetone, ethanol and butanol. Isopropanol is not produced by the wild-type strains of C. acetobutylicum. Isopropanol is a product that is characteristic of a microorganism formerly called “Clostridium butylicum” (Osburn et al. 1937; Langlykke et al. 1937). In 1983, George et al. showed that C. butylicum is similar to C. beijerinckii. In the current classification, the species C. beijerinckii therefore contains strains that were formerly classified as C. butylicum. However, not all strains of C. beijerinckii produce isopropanol. For example, the strain C. beijerinckii NRRL B593 does produce it, whereas the strain C. beijerinckii NRRL B592 does not.
The biosynthetic pathway of isopropanol in C. beijerinckii comprises the following steps:
(i) two molecules of acetyl-CoA are condensed to one molecule of acetoacetyl-CoA by acetyl-CoA transferase (EC 2.3.1.9), also called thiolase (Thl);
(ii) the CoA of acetylCoA is transferred to the acetate or to the butyrate by acetoacetyl-CoA transferase (Ca_CtfAB) (EC 2.8.3.9) to give the acetoacetate;
(iii) the acetoacetate is then converted to acetone and CO2 by acetate decarboxylase (Ca_Adc) (EC 4.1.1.4);
(iv) finally, the acetone is converted to isopropanol by secondary alcohol dehydrogenase (Cb_s-Adh) (EC 1.1.1.2).
Comparison of the enzyme activities in C. beijerinckii NRRL B593 and C. beijerinckii NRRL B592 (which produce and do not produce isopropanol, respectively) showed that the capacity for isopropanol excretion was linked to the activity of secondary alcohol dehydrogenase Cb_s-Adh (Yan et al. 1988). The enzyme responsible was purified from acellular extracts of C. beijerinckii NRRL B593 (Ismaiel et al. 1993). The dehydrogenase of C. beijerinckii NEST255 was also purified for purposes of comparison. The secondary alcohol dehydrogenases in the strains C. beijerinckii NRRL B593 and C. beijerinckii NEST255 are NADP-dependent. In vitro, they utilize secondary alcohols (isopropanol) as expected, but also primary alcohols, in particular butanol, but with lower activity. Kinetic studies of the enzyme confirmed that the physiological substrate of the secondary alcohol dehydrogenase was indeed acetone and not isopropanol (Chen et al. 1995). The secondary alcohol dehydrogenase in C. beijerinckii (Cb_s-Adh) is clearly distinct from the primary alcohol dehydrogenase originating from the same microorganism, which in vivo reduces butyraldehyde to butanol.
The gene coding for Cb_s-Adh has been sequenced (Petretz et al. 1997). The structure of the enzyme was determined by X-ray analysis. Cb_s-Adh is a homotetrameric zinc enzyme having four polypeptide chains of 351 amino acids. Each monomer is constituted by two domains. The first domain (residues 154-294) allows binding of the cofactor whereas the second domain incorporates the catalytic domain (residues 1-153 and 295-351) (Korkhin et al. 1998; Goihberg et al. 2010). Cb_s-Adh is similar to its homologue originating from the thermophilic bacterium Thermoanaeobacter brockii (Tb_s-Adh) with 75% sequence identity. In terms of properties, the two enzymes differ in their thermal stability (Korkhin et al. 1999). Replacement, in Cb_s-Adh, of Gln100 with Pro100 in the amino acid sequence significantly increases the thermal stability of Cb_s-Adh (Goihberg et al. 2007), which is originally lower.
The maximum capacity for producing isopropanol is relatively low (2 to 3 g/L) with the wild-type strains of C. beijerinckii (Chen et al., 1986). Patent application US2009/246842 describes a method for the production of isopropanol in a strain of E. coli transformed by an expression vector comprising the gene coding for acetyl-CoA transferase of C. acetobutylicum (Ca_thl), the genes coding for acetoacetyl-CoA transferase of C. acetobutylicum (Ca_CtfAB), the gene coding for an acetate decarboxylase (Ca_adc) of C. acetobutylicum and the gene coding for the secondary alcohol dehydrogenase (Cb_s-adh) of C. beijerinckii. In this vector, expression of the secondary alcohol dehydrogenase is under the control of the promoter PLlacO-1 which can be induced by IPTG, whereas expressions of the genes Ca_thl, Ca_CtfAB and Ca_adc are under the control of the native promoter of the Ca_thl gene of C. acetobutylicum. The drawback of this method is that it is necessary to use a rich production medium, in particular yeast extract (5 g·L−1), and an inducer, such as isopropyl-β-thiogalactopyranoside or IPTG. Moreover, the secondary alcohol dehydrogenase is expressed only when IPTG is added to the culture medium, which delays the production of isopropanol and limits the fermentation productivity.
International application WO 11/037414 describes another method capable of producing isopropanol. This method consists of using microorganisms of the genus Clostridium transformed by an expression vector comprising the gene coding for the acetate decarboxylase (Ca_adc) of C. acetobutylicum, the genes coding for the acetoacetyl-CoA transferase of C. acetobutylicum (Ca_CtfAB) and the gene coding for the secondary alcohol dehydrogenase (Cb_s-adh) of C. beijerinckii. This vector requires two promoters: a constitutive promoter Ca_thlp which only controls the expression of the secondary alcohol dehydrogenase and an inducible promoter Ca_adcp which controls the expression of the proteins Ca_CtfAB and Ca_Adc. Given that the proteins Ca_CtfAB and Ca_Adc are expressed only during the solventogenesis phase, the secondary alcohol dehydrogenase does not have a substrate (acetone) before this phase. As a result, isopropanol, the end product of fermentation, is produced only after 15 hours of culture, which limits the isopropanol productivity of this method of production.
Consequently, there is still a need to develop a method for the production of isopropanol with a better productivity and a better yield.
The objective of the present invention is to rectify the drawbacks of the prior art and to propose a method for the production of isopropanol by fermentation having a better productivity and a better yield.
DESCRIPTION OF THE INVENTION
The inventors have discovered that it is possible, advantageously, to produce isopropanol using recombinant microorganisms into which vectors have been introduced comprising the genes coding for the enzymes of the metabolic pathway leading from acetoacetyl-CoA to acetone, namely CoA transferase subunits A and B and acetoacetate decarboxylase, as well as the gene coding for a secondary alcohol dehydrogenase of C. beijerinckii NRRL B593.
The first objective of the present invention is to provide an expression vector comprising:

    
    
        the nucleic acids coding for the polypeptides forming a polypeptide complex having an enzyme activity allowing acetoacetyl-CoA to be converted to acetoacetate,
        at least one nucleic acid coding for a polypeptide having an enzyme activity allowing acetone to be converted to isopropanol, and
        optionally, at least one nucleic acid coding for a polypeptide having an enzyme activity allowing acetoacetate to be converted to acetone,

the expression of said nucleic acids being controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids.

    
    


By “expression vector” is meant an exogenous DNA molecule of circular shape, which is capable of autonomous and independent replication of the chromosomal DNA of the host cells, allowing transcription and translation of the genes contained in said DNA molecule by means of the host cells.
In the present invention, the term “vector” and the term “plasmid” are interchangeable.
By “promoter” is meant a region of DNA where RNA polymerase binds and orients this enzyme towards the site where transcription of the gene will begin.
The promoters can be constitutive or inducible.
By “constitutive promoter” is meant an unregulated promoter allowing continuous transcription of the associated gene.
In contrast to a constitutive promoter, an inducible promoter allows transcription of the associated gene in response to the presence of a particular compound, for example the presence of IPTG, or to a defined external condition, for example a high temperature.
By “a promoter . . . located upstream of the . . . nucleic acids” is meant a promoter located before the 5′ ends of the nucleic acids.
The conversion of acetoacetyl-CoA to acetoacetate is carried out by acetoacetyl-CoA transferase. In most microorganisms, this enzyme is formed by two subunits encoded by two different genes. Thus, the acetoacetyl-CoA transferase of E. coli (Ato AD) is formed by the units A and D, and the acetoacetyl-CoA transferase of C. acetobutylicum (Ca_CtfAB) is formed by the units A and B.
In a particular embodiment, the present invention relates to a vector comprising:

    
    
        the nucleic acids coding for acetoacetyl-CoA transferase (Ca_CtfAB), and
        at least one nucleic acid coding for the gene of the secondary alcohol dehydrogenase of C. beijerinckii (Cb_s-adh); and
        optionally at least one nucleic acid coding for the gene of acetoacetate decarboxylase (Ca_adc).
    
    


Expression of the gene of the secondary alcohol dehydrogenase of C. beijerinckii NRRL B 593 makes it possible to ensure conversion of acetone to isopropanol and excretion of the latter. A recombinant strain expressing this enzyme can reduce almost all of the acetone and acetoin (3-hydroxy-2-butanone) to isopropanol and 2,3-butanediol respectively. Although it is known that the enzyme Cb_s-Adh, from the strain C. beijerinckii NRRL B 593, can reduce acetone and butanone, the activity of this enzyme on acetoin has been found for the first time by the inventors.
In a more particular embodiment, the present invention relates to a vector comprising:

    
    
        a nucleic acid represented by the sequence SEQ ID NO: 1, or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 1, and
        a nucleic acid represented by the sequence SEQ ID NO: 2 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 2, and
        a nucleic acid represented by the sequence SEQ ID NO: 3 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 3, and
        optionally a nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 4.
    
    


The nucleic acid represented by the sequence SEQ ID NO: 1 codes for subunit A of the acetoacetyl-CoA transferase of C. acetobutylicum ATCC 824 (Ca_ctfA).
The nucleic acid represented by the sequence SEQ ID NO: 2 codes for subunit B of the acetoacetyl-CoA transferase of C. acetobutylicum ATCC 824 (Ca_ctfB).
The nucleic acid represented by the sequence SEQ ID NO: 3 codes for the secondary alcohol dehydrogenase of C. beijerinckii (Cb_s-adh).
The nucleic acid represented by the sequence SEQ ID NO: 4 codes for the acetoacetate decarboxylase of C. acetobutylicum (Ca_adc).
The percentage identity between two peptide sequences or between two nucleic acid sequences can be calculated from the following formula:




  
    number
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    identical
    ⁢
    
      
    
    ⁢
    residues
    ×
    100
  
  
    number
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    residues
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    the
    ⁢
    
      
    
    ⁢
    shorter
    ⁢
    
      
    
    ⁢
    sequence
  




The combinations of the abovementioned nucleic acids (or genes) form the “ipa” operons. Expression of the genes of the abovementioned ipa operons is controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids (or genes). A constitutive promoter, which only controls the expression of the ipa operons, allows constitutive expression of the latter, which allows a recombinant strain containing one of the vectors of the invention to produce isopropanol as from the first hours of fermentation.
Owing to the early expression of the genes that lead to the production of isopropanol, the mechanism of solventogenesis is triggered earlier in a recombinant strain containing a vector according to the invention than in a wild-type strain. Consequently, two fermentation phases, namely, acidogenesis and solventogenesis, take place at the same time in a recombinant strain containing a vector according to the invention, which makes it possible to reduce the fermentation time by at least 20 hours.
The constitutive promoter used in the construction of the vector of the present invention can be the promoter of the gene coding for the thiolase of C. acetobutylicum ATCC 824, represented by the sequence SEQ ID NO: 5, or the promoter of the gene of the phosphotransbutyrylase of C. acetobutylicum ATCC 824, represented by the sequence SEQ ID NO: 6.
In a particular embodiment, the expression vector of the present invention comprising:

    
    
        a nucleic acid represented by the sequence SEQ ID NO: 1, or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 1, coding for subunit A of the acetoacetyl-CoA transferase of C. acetobutylicum and
        a nucleic acid represented by the sequence SEQ ID NO: 2 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 2, coding for subunit B of the acetoacetyl-CoA transferase of C. acetobutylicum, and
        optionally a nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 4, coding for the acetoacetate decarboxylase of C. acetobutylicum, and
        a nucleic acid represented by the sequence SEQ ID NO: 3 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 3, coding for the secondary alcohol dehydrogenase of C. beijerinckii, the expression of said nucleic acids being controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids, said promoter being selected from the promoter represented by the sequence SEQ ID NO: 5 (thl promoter) and the promoter represented by the sequence SEQ ID NO: 6.
    
    


In a particular embodiment, the vector of the present invention comprises:

    
    
        a nucleic acid represented by the sequence SEQ ID NO: 1,
        a nucleic acid represented by the sequence SEQ ID NO: 2,
        a nucleic acid represented by the sequence SEQ ID NO: 3,
        a nucleic acid represented by the sequence SEQ ID NO: 4, and
        a promoter represented by the sequence SEQ ID NO: 5.
    
    


This vector designated pFC007 contains, in the order from the 5′ end to the 3′ end, the gene coding for the secondary alcohol dehydrogenase (Cb_s-adh) of C. beijerinckii NRRL B 593 as well as the gene coding for the acetoacetate decarboxylase (Ca_adc) and the genes coding for subunits A and B of the coenzyme A transferase (Ca_CtfAB) of C. acetobutylicum ATCC 824. The vector pFC007 allows overexpression of the genes of the “ipa7” operon under the control of just one constitutive promoter, that of the gene of the acetyl-CoA transferase (thiolase, Ca_thl) of C. acetobutylicum ATCC 824. Consequently, production of their respective enzymes (Cb_s-Adh, Ca_Adc and Ca_CtfAB) begins as from the first hours of fermentation (after approximately 4 and 10 hours).
In another particular embodiment, the vector of the present invention comprises:

    
    
        a nucleic acid represented by the sequence SEQ ID NO: 1,
        a nucleic acid represented by the sequence SEQ ID NO: 2,
        a nucleic acid represented by the sequence SEQ ID NO: 3, and
        a promoter represented by the sequence SEQ ID NO: 5.
    
    


This vector designated pFC006 contains the gene coding for the secondary alcohol dehydrogenase (Cb_s-adh) of C. beijerinckii NRRL B 593 (DSMZ 6423) as well as the genes coding for subunits A and B of the coenzyme A transferase (Ca_CtfAB) of C. acetobutylicum ATCC 824. These genes are under the control of just one constitutive promoter of the gene of the acetyl-CoA transferase (or thiolase or Ca_thl) of C. acetobutylicum ATCC 824.
The expression vectors according to the present invention can further comprise any other element necessary for the transcription and translation of the aforementioned enzymes, such as an RBS (ribosome binding site) region, one or more selection genes.
An RBS region allows binding of the ribosome to the messenger RNA molecule before the start codon of a gene. The sequence of RBS region used in the vectors of the present invention can be any sequence of RBS region of prokaryotic origin known to a person skilled in the art.
The presence of a selection gene in a vector according to the invention makes it possible to select the microorganisms that have been transformed successfully. A selection gene in a vector allows the microorganisms comprising said gene to survive under certain particular conditions, or can lead to the presence of a molecule that can be easily identified. The selection gene used in the vectors of the present invention can be any selection genes known to a person skilled in the art, in particular a gene endowing the microorganisms with antibiotic resistance, such as the gene Amp, or ErmB.
The expression vectors also comprise replication origins, which allow initiation of the replication of said vectors in the host microorganisms. The replication origin used in the vectors of the present invention can be any replication origin known to a person skilled in the art.
The present invention also aims to provide a recombinant microorganism for producing isopropanol with a better productivity and a better yield.
Said microorganism comprises an expression vector as described in the present invention.
In particular, said microorganism comprises a vector comprising:

    
    
        a nucleic acid represented by the sequence SEQ ID NO: 1, or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 1, and
        a nucleic acid represented by the sequence SEQ ID NO: 2 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 2, and
        a nucleic acid represented by the sequence SEQ ID NO: 3 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 3, and
        optionally a nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85%, particularly 90%, in particular 95% with the sequence SEQ ID NO: 4,

the expression of said nucleic acids being controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids, said promoter being selected from the promoter represented by the sequence SEQ ID NO: 5 (thl promoter) and the promoter represented by the sequence SEQ ID NO: 6.

    
    


In an advantageous embodiment, said microorganism comprises the vector pFC007.
In another advantageous embodiment, said microorganism comprises the vector pFC006.
Said microorganism of the present invention can be selected from bacteria of the genus Clostridium, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, bacteria of the genus Bacillus, such as Bacillus subtilis, or enterobacteria, such as E. coli. 
In a particular embodiment, the microorganism according to the invention is C. acetobutylicum. 
In a more particular embodiment, the microorganism according to the invention is the strain C. acetobutylicum ATCC 824.
In a particularly advantageous embodiment, the invention relates to a recombinant strain of C. acetobutylicum ATCC 824 comprising the vector pFC007. The recombinant strain is designated ATCC 824(pFC007).
In another particularly advantageous embodiment, the invention relates to a recombinant strain of C. acetobutylicum ATCC 824 comprising the vector pFC006. The recombinant strain is designated ATCC 824(pFC006).
For the two recombinant strains (ATCC 824(pFC007) and ATCC 824(pFC006)), production of isopropanol can be detected as from the first 10 hours of culture, whereas no trace of solvents (ABE) is detectable in the wild-type strain of C. acetobutylicum ATCC 824 or the recombinant strain of C. acetobutylicum ATCC 824 into which a vector is inserted that only comprises the gene coding for the secondary alcohol dehydrogenase (Cb_s-adh) of C. beijerinckii NRRL B 593.
Another aim of the present invention is to provide a method for the production of isopropanol, and/or of D and L 2,3-butanediol, with a better productivity and a better yield.
Said method comprises at least the following steps:

    
    
        cultivating a microorganism according to the invention in a culture medium comprising at least one carbon source and one nitrogen source, under conditions allowing the production of isopropanol, for example the culture medium described by Gapes et al. (Gapes, 1996)
        recovering the isopropanol, and/or D and L 2,3-butanediol, from the culture medium.
    
    


The culture media are known to a person skilled in the art.
The carbon source can be starch, glucose, xylose, lignocellulosic hydrolysates.
The nitrogen source can be ammonia, yeast extract, peptone, urea.
The culture conditions for microorganisms of the genus Clostridium, Bacillus or the E. coli bacteria are known to a person skilled in the art.
The recovery of the isopropanol, and/or of D and L 2,3-butanediol, from the culture medium is performed by distillation (Mujiburohman et al. 2006).
The examples and figures presented below illustrate the present invention in more detail. However, the scope of the present invention must in no case be limited to these examples or figures.



FIGURES
FIG. 1: this figure illustrates construction of the vector pFC007 from the plasmid pMTL500E.
FIG. 2: this figure gives the restriction sites of the plasmid pMTL500E.
FIG. 3: this figure illustrates the restriction map of the vector pFC007.



EXAMPLES
Materials and Methods
Equipment and Materials
The kits used in the operations of molecular biology (construction of the plasmid vectors) are presented in Table 1.







TABLE 1







Kits and materials used in the operations of molecular biology









Name of the kit
Use
Supplier





Restriction enzymes: apaI, speI,
Enzymatic digestion of plasmid DNAs
New England


sphI, xbaI and xhoI

Biolabs (NEB)


High Fidelity PCR Master Mix TM
PCR amplification
Roche


DreamTaq TM
PCR amplification
Fermentas


Oligonucleotides (Table 3)
Primers for the PCR reactions, created by
BaseClear



the inventors


Alkaline phosphatase
dephosphorylation of the 5′ ends of the
New England



linearized vectors preventing self-ligation
Biolabs


T4 DNA ligase
ligation of DNA fragment
New England




Biolabs (NEB)


GenJET TM plasmid miniprep
Extraction of plasmid DNA from cultures of
Fermentas



E. coli


GenElute TM Bacterial Genomic
Extraction of genomic DNA from bacteria
Sigma-Aldrich


DNA
(Gram-positive and Gram-negative)


Z-Competent TM E. coli
Production of chemocompetent bacteria E. coli
Zymo Research


transformation
XL1 blue and E. coli BW25113









The strains used in the examples of the present invention are:

    
    
        C. acetobutylicum ATCC 824, originating from the ATCC collection
        C. beijerinckii NRRL B 593, originating from the DSMZ collection (number 6423)
        E. coli XL1 blue, used for plasmid cloning and maintenance
        E. coli DH10B (PAN2), used for methylation of the plasmids

Assaying of Solvents (Acetone, Acetoin, Isopropanol, Butanol, Butanediol) and Carboxylic Acids (Butyric and Acetic Acids)

    
    


The fermentation samples (2 mL) are centrifuged for 5 minutes at 20,000 g and the supernatants are used for assaying the fermentation metabolites (glucose, acetic and butyric acid, 2,3-butanediol, acetone, ethanol, isopropanol and butanol). For each sample, an equivolume of an acid solution (0.5 mM H2SO4) containing the internal standard (30 mM of 4-methylvaleric acid) is added, the mixture is then homogenized and filtered (0.2 μm, Whatman). 10 μL of sample is then injected and analyzed in a Shodex lonpack KC-811 column (RP). The assayed compounds are detected at column outlet by two detectors: a refractometer (Waters 2487) and a spectrophotometer (Waters 2487) measuring the absorbance at 210 nm. The eluent used is a 3 mM solution of sulphuric acid, the flow rate of the eluent is 1 mL/min and the column temperature is fixed at 85° C.
Construction of the Vectors
The genetic structures of the vectors used or constructed in the examples of the present invention are summarized in Table 2 below.







TABLE 2







Genetic structures of the vectors











Clostridium/





E. coli



selective


Plasmid
marker
Description
Reference





pMTL500E
ermB/ampR
Empty vector
Oultram et al., 1988


pFC002
ermB/ampR
Ca_thlp [Cb_s-adh]
This document


pFC005
ermB/ampR
Ca_thlp [Cb_s-adh;
This document




Ca_adc]


pFC006
ermB/ampR
Ca_thlp [Cb_s-adh;
This document




Ca_ctfAB]


pFC007
ermB/ampR
Ca_thlp [Cb_s-adh;
This document




Ca_adc; Ca_ctfAB]


pAN2
ttc

(Mermelstein, 1993;





Heap, 2009)





ermB: erythromycin resistance gene;


ampR: ampicillin resistance gene;


ttc: tetracycline resistance gene;


Ca: this prefix indicates that the gene originates from C. acetobutylicum ATCC 824;


Cb: this prefix indicates that the gene originates from C. beijerinckii NRRL B593;


Ca_thl: thiolase gene;


Cb_s-adh: gene of the secondary dehydrogenase of C. beijerinckii NRRL B593;


Ca_adc: gene of the acetate decarboxylase of C. acetobutylicum ATCC 824;


Ca_CtfAB: gene of the CoA transferase of C. acetobutylicum ATCC 824.






Construction of the vector pFC007 from the plasmid pMTL500E (FIG. 2) requires at least 3 steps (FIG. 1).
Step 1: Construction of the Vector pFC002
The vector pFC002 is constructed from the plasmid pMTL500E. The latter is digested with the restriction enzymes sphI and xhoI on the lacZ gene. The digestion product is then dephosphorylated. The restriction sites sphI and apaI are added respectively at 5′ and 3′ of the DNA sequence of the promoter Pthl, by the PCR technique using the primers Ca_thlp-for and Ca_thlp_rev derived from the genomic DNA of C. acetobutyliccum ATCC 824 (Nolling, J. et al., 2001) (NCBI reference: NC_003030.1 (http://www.ncbi.nlm.nih.gov/nuccore/NC_003030) (Table 3). The restriction sites apaI and xhoI are added at 5′ and 3′ respectively of the DNA sequence of the gene Cb_s-adh of C. beijerinckii NRRL B593, by the PCR technique using the primers Cb_s-adh-for and Cb_s-adh-rev (Table 3).
The PCR product thus obtained is digested with the restriction enzymes apaI and xhoI. The digested plasmid, the digested promoter and the digested gene originating from the PCR are ligated together to give a vector called pFC002 (FIG. 2). This vector is maintained and multiplied in the E. coli strain XL1 blue.
Step 2: Construction of the Vector pFC006 from the Plasmid pFC002
The vector pFC002 is digested with the restriction enzymes xhoI and XbaI. The genes coding for Ca_CtfAB are amplified together starting from the genomic DNA of C. acetobutylicum ATCC 824, using the primers Ca_CtfAB for and Ca_CtfAB rev (Table 3). The PCR product is digested with the restriction enzymes xhoI and speI. The digested vector pFC002 and the digested DNA fragment containing the gene Ca_CtfAB are ligated together. The vector thus obtained is designated pFC006 (FIG. 1). The vector is maintained and multiplied in the E. coli strain XL1 blue.
Step 3: Construction of the Vector pFC007 from the Plasmid pFC006
The vector pFC006 is digested with the restriction enzymes xhoI and XbaI. The gene coding for Ca_Adc is amplified from the genomic DNA of C. acetobutylicum ATCC 824, using the primers Ca_adc for and Ca_adc rev (Table 3). The PCR product is digested with the enzymes xhoI and SpeI. The digested vector pFC006 and the digested DNA fragment containing the gene Ca_adc are ligated together. The vector thus obtained is designated pFC007 (FIGS. 1 and 3). The vector is maintained and multiplied in the E. coli strain XL1 blue.







TABLE 3 







Primers used for constructing the ipa operons








Primers
Sequence (5′-3′)





Ca_thlp-for
GCATGCGAATTTAGAATGAAGTTTCTTATGCA



(SEQ ID NO: 7)


 


Ca_thlp_rev
GGGCCCCCATAGTTTATCCCTAATTTATACG



(SEQ ID NO: 8)


 


Cb_s-adh-for
GGGCCCTTAGACTATTAAAGGAATATTTTTAAGG



(SEQ ID NO: 9)


 


Cb_s-adh-rev
TTTTCTCGAGGTATAATCCTCCATGATCTATTATG



(SEQ ID NO: 10)


 


Ca_ctf A &B
CAACTACTCGAGATAATTTTTCTAGAGAATTTAAA


for
AGGAGGGATTAAAATG (SEQ ID NO: 11)


 


Ca_ctf A &B
AATGGTACTAGTTATTTTTTGTCGACTGTTTCATA


rev
GTATTTCTTTCTAAACAGCC (SEQ ID NO: 12)


 


Ca_adc for
AAACAACTCGAGTTATAATCTAGATATAAATAAATA



GGACTAGAGGCG (SEQ ID NO: 13)


 


Ca_adc rev
AAAAATACTAGTTACCATTTAAGTCGACTCTTATTT



TTATTACTTAAG (SEQ ID NO: 14)









The added restriction sites (sphI, apaI, xhoI, SalI, xbaI and speI) are underlined. The ribosome binding sites are shown in bold.
The prefix “Ca” means that the primer is used on the genomic DNA of C. acetobutylicum 
The prefix “Cb” means that the primer is used on the genomic DNA of C. beijerinckii NRRL B 593
Using similar methods, the gene coding for Ca_Adc can be inserted into the vector pFC002 to obtain the vector pFC005.
According to similar methods, the genes coding for Ca_CtfAB can then be inserted into the vector pFC005 to obtain the vector pFC008.
Transformation of C. Acetobutylicum 
The vectors pFC002, pFC006 and pFC007 are first methylated respectively by cotransformation with the plasmid pAN1 in the E. coli strain DH10B.
The wild-type strain of C. acetobutylicum ATCC 824 is then transformed respectively by the methylated vectors pFC002, pFC006 and pFC007 according to the protocol described by Oultram (1988). The strains of C. acetobutylicum ATCC 824 transformed by pFC002, pFC006 and pFC007 are called ATCC 824(pFC002), ATCC 824(pFC006) and ATCC 824(pFC007), respectively.
Genetic Characterization of the Recombinant Strains
In order to confirm the presence of one of the vectors described above in the strain C. acetobutylicum ATCC 824 after transformation, a PCR specific to the plasmid is carried out with a different primer pair. The results shown in Table 4 confirm the presence of vectors in the recombinant strains analyzed.







TABLE 4







Identification of the vectors pFC005, pFC006 or pFC007 in C. acetobutylicum ATCC


824 by PCR









Size of PCR fragments expected [kbp] for the primer pairs:













Cb_thlp for
Cb_s-adh for
Cb_s-adh for
Ca_adc for
Ca_ctfAB for


PCR mix
Cb_s-adh rev
Ca_adc rev
Ca_ctfAB rev
Ca_ctfAB rev
Ca_adc rev





pFC002
1.4






pFC005
1.4
2.0
No
No
No


pFC006
1.4
No
2.5
No
No


pFC007
1.4
2.0
3.4
2.3
No


pFC008
1.4
3.4
2.5
No
2.3









Fermentation of the Recombinant Strains

Fermentations were carried out in a culture medium described by Gapes et al. (GAPES et al., 1996), containing 90 g/L of glucose with wild-type strains, C. beijerinckii NRRL B 593 and C. acetobutylicum ATCC 824, and the recombinant strains namely C. acetobutylicum ATCC 824 transformed respectively by the vectors pFC006 and pFC007. The fermentation results are presented in Table 5.







TABLE 5







Final performances on glucose by wild-type and recombinant strains of C. beijerinckii


NRRL B 593 and of C. acetobutylicum ATCC 824













NRRL B







593
ATCC 824
ATCC 824
ATCC 824
ATCC 824


Characteristic
wild-type
wild-type
(pFC002)
(pFC006)
(pFC007)















Glucose consumed [g/L]
34.30
61.99
54.82
69.26
67.79


Lactic acid [g/L]
0.55
0.00
−0.16
0.17
0.19


Acetic acid [g/L]
0.83
2.86
4.61
3.24
1.61


Acetoin [g/L]
0.00
0.59
0.10
0.06
0.05


2,3-Butanediol [g/L]
0.00
0.08
0.48
0.91
0.88


Butyric acid [g/L]
0.21
2.02
2.51
1.08
1.06


Acetone [g/L]
0.17
5.70
0.05
0.35
0.09


Ethanol [g/L]
0.12
1.26
0.31
1.34
1.71


Isopropanol [g/L]
4.47
0.10
4.25
7.27
8.37


Butanol [g/L]
8.08
8.98
8.22
11.80
12.95


IBE or ABE [g/L]
12.83
16.04
12.82
20.76
23.12


Fermentation time [h]*
33.5
49.5
>45
29.9
28.0


Maximum productivity for
0.41
0.41
0.33
0.69
0.81


IBE or ABE [g/L · h]


Yield of IBE or ABE [g/g
0.37
0.26
0.24
0.30
0.34


substrate]


Selectivity for isopropanol
0.35
0.01
0.33
0.35
0.36


[g/g solvents]


Selectivity for acetone
0.01
0.36
<0.01
0.02
0.00


[g/g solvents]





*time necessary to reach 95% of the final production of solvent.






Fermentation of Glucose by the Wild-type Strain C. Acetobutylicum ATCC 824

Approximately 50 hours of fermentation are needed to reach 95% of the final production of solvents. The presence of solvent (butanol) can only be detected 11 hours after inoculation. Neither isopropanol nor 2,3-butanediol is produced by the wild-type strain C. acetobutylicum ATCC 824. The production of acetone and that of acetoin reach 5.7 g/L and 0.6 g/L respectively. The final productions of butanol and of ethanol reach 9.0 g/L and 1.3 g/L respectively.
Fermentation of Glucose by the Recombinant Strain C. Acetobutylicum ATCC 824(pFC002)
C. acetobutylicum ATCC 824(pFC002) is a recombinant strain that has received the vector pFC002. The vector pFC002 allows its host constitutive expression of the gene of the secondary alcohol dehydrogenase of C. beijerinckii NRRL B 593 (Cb_s-adh) coding for the enzyme Cb_s-Adh. The production of Cb_s-Adh in the recombinant strain ATCC 824(pFC002) makes it possible to hydrogenate acetone and acetoin to isopropanol and 2,3-butanediol respectively. Acetoin is produced naturally by the wild-type strain of C. acetobutylicum ATCC 824. However, this recombinant strain is not capable of producing more isopropanol than the wild-type strain of C. beijerinckii NRRL B593. Moreover, the strain ATCC 824(pFC002) displays mediocre fermentation performances, thus, the production of isopropanol, and of solvents in general, of the strain ATCC 824(pFC002) is 25% lower than the production of acetone and 20% lower than the production of solvents of the wild-type strain of C. acetobutylicum ATCC 824. Acetate and butyrate remain the majority products at the end of the fermentation. The significant production of acids causes the drop in yield of solvents, as a proportion of the carbohydrates is used for the production of acids.
Fermentation of Glucose by the Recombinant Strain C. Acetobutylicum ATCC 824(pFC006)
Like the recombinant strain ATCC 824(pFC002), the recombinant strain ATCC 824(pFC006) produces isopropanol and 2,3-butanediol instead of acetone and acetoin.
However, in contrast to the profile of production for the recombinant strain ATCC 824(pFC002), the recombinant strain ATCC 824(pFC006) can reach a high level of production of solvents over a shorter period of time. The time necessary to reach 95% of the final production of solvent for this strain is approximately 30 hours. The consumption of butyric acid is more effective than that of the wild-type strain or of the recombinant strain ATCC 824(pFC002), but remains incomplete. The production of solvents in this strain begins earlier than for the wild-type strain or for the recombinant strain ATCC 824(pFC002). The production of isopropanol can be detected as from the first 10 hours of culture of the strain ATCC 824(pFC006), whereas traces of solvents are not yet detectable in the wild-type strain or in the strain ATCC 824(pFC002).
Moreover, the incorporation of acetate and of butyrate in the strain ATCC 824(pFC006) are both improved relative to those in the wild-type strain, which makes it possible to have an isopropanol yield better than the acetone yield of the wild-type strain. For the strain ATCC 824(pFC007), the respective titres of isopropanol, butanol and ethanol are 7.3 g/L, 11.8 g/L and 1.37 g/L, in other words 28%, 31% and 6% higher than those of the wild-type strain. The productivity is increased from 0.41 g/L·h for a wild-type strain to 0.69 g/L·h for the strain ATCC 824(pFC007).
The strain ATCC 824(pFC006) produces twice as much 2,3-butanediol than the strain ATCC 824(pFC002), and this increase in production of 2,3-butanediol may be due to an overall improvement in metabolic flux.
It is found that the improvement in the incorporation of acids allows the strain ATCC 824(pFC006) to produce less acids and to increase its production of solvents.
However, despite overexpression of the enzyme Ca_CtfAB, the final production of acetate is higher in the strain ATCC 824(pFC006) than in the wild-type strain, which may be due to increased production of acids as a result of an overall improvement in metabolic flux.
Fermentation of Glucose by the Recombinant Strain C. Acetobutylicum ATCC 824 Transformed by pFC007
The strain ATCC 824(pFC007) has a fermentation profile similar to that of the strain ATCC 824(pFC006). The time to reach 95% of the final production of solvents is less than 30 hours. Acidogenesis begins normally. The production of solvents in the strain ATCC 824(pFC007) begins during the exponential phase and earlier than in the wild-type strain. The presence of isopropanol can be detected in the culture medium of the strain ATCC 824(pFC007), only 4 hours after inoculation, whereas acetone is not yet detectable in the culture medium of a wild-type strain.
Moreover, the incorporation of acetate and of butyrate in the strain ATCC 824(pFC007) are both improved relative to those in the strain ATCC 824(pFC006), which makes it possible to have a better isopropanol yield relative to that of the strain ATCC 824(pFC006), or of other strains described previously. In the strain ATCC 824(pFC007), the respective titres of isopropanol, butanol and ethanol are 8.4 g/L, 13.0 g/L and 1.71 g/L, in other words 46%, 44% and 36% higher than those of the wild-type strain. Productivity is increased from 0.41 g/L·h for a wild-type strain to 0.81 g/L·h for the strain ATCC 824(pFC007).
The production of 2,3-butanediol, of the strain ATCC 824(pFC007), is similar to that of the strain ATCC 824(pFC006). As for the strain ATCC 824(pFC006), this increase in the production of 2,3-butanediol may be due to an overall improvement in metabolic flux.
Fermentation of Glucose by the Wild-type Strain C. Beijerinckii NRRL B 593
The wild-type strain C. beijerinckii NRRL B 593 is cultivated under the same conditions as the wild-type or recombinant strain C. acetobutylicum ATCC 824.
After 34 hours of fermentation, the wild-type strain of C. beijerinckii reaches 95% of the final production of solvents. However, the final productions of this strain are only 4.5 g/L for isopropanol, 8.1 g/L for butanol and 0.1 g/L for ethanol, which is lower than for the wild-type strain C. acetobutylicum ATCC 824 and for the recombinant strains C. acetobutylicum ATCC 824 (strains ATCC 824(pFC006) and ATCC 824(pFC007).
As expected, the wild-type strain C. beijerinckii NRRL B 593 does not produce acetoin or butanediol.
CONCLUSION
The recombinant strains ATCC 824(pFC006) and ATCC 824(pFC007) both exhibit better solvents productivity than the wild-type strain C. acetobutylicum ATCC 824 or than the wild-type strain C. beijerinckii NRRL B 593. The solvents productivity is multiplied respectively by a factor of 1.8 with the strain ATCC 824(pFC006) and by a factor of 2 with the strain ATCC 824(pFC007). These results show that these two strains can produce more solvents for a shorter time than the wild-type strains. Moreover, the strain ATCC 824(pFC007) has been found to be very suitable for continuous single-stage culture (a single fermenter). The productions of isopropanol, butanol and ethanol observed were to those known for the strain C. beijerinckii NRRL B 593 (Shrikant A., 2011) and C. acetobutylicum ATCC824(pFC007) (Godin et al 1990)
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## Claims

1. A vector comprising:
a nucleic acid represented by the sequence SEQ ID NO: 1, or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 1, coding for subunit A of the acetoacetyl-CoA transferase of C. acetobutylicum and
a nucleic acid represented by the sequence SEQ ID NO: 2 or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 2, coding for subunit B of the acetoacetyl-CoA transferase of C. acetobutylicum, and
optionally a nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 4, coding for the acetoacetate decarboxylase of C. acetobutylicum, and
a nucleic acid represented by the sequence SEQ ID NO: 3 or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 3, coding for the secondary alcohol dehydrogenase of C. beijerinckii, 
the expression of said nucleic acids being controlled by a single constitutive promoter located upstream of the abovementioned nucleic acids, said promoter being selected from the promoter represented by the sequence SEQ ID NO: 5(thl promoter) and the promoter represented by the sequence SEQ ID NO: 6.

2. The expression vector according to claim 1, wherein said vector comprises:
a nucleic acid represented by the sequence SEQ ID NO: 1,
a nucleic acid represented by the sequence SEQ ID NO: 2,
a nucleic acid represented by the sequence SEQ ID NO: 3,
a nucleic acid represented by the sequence SEQ ID NO: 4, and
a promoter represented by the sequence SEQ ID NO: 5.

3. The expression vector according to claim 1, wherein said vector comprises:
a nucleic acid represented by the sequence SEQ ID NO: 1,
a nucleic acid represented by the sequence SEQ ID NO: 2,
a nucleic acid represented by the sequence SEQ ID NO: 3, and
a promoter represented by the sequence SEQ ID NO: 5.

4. A microorganism comprising the expression vector according to claim 1.

5. The microorganism according to claim 4, wherein said microorganism is a bacteria of the genera selected from the group consisting of Clostridium, Bacillus, and enterobacteria.

6. The microorganism according to claim 5, wherein said microorganism is selected from the group consisting of Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, and Clostridium saccharobutylicum.

7. The microorganism according to claim 5, wherein said microorganism is E. coli.

8. A method for the production of isopropanol, and/or of D and L 2,3-butanediol, said method comprising:
cultivating a microorganism according to claim 4, in a culture medium comprising at least one carbon source and one nitrogen source, under conditions allowing the production of isopropanol, and
recovering the isopropanol, and/or D and L 2,3-butanediol, from the culture medium.

9. The expression vector according to claim 1, wherein the expression vector does comprise the nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 4, coding for the acetoacetate decarboxylase of C. acetobutylicum.

10. The expression vector according to claim 1, wherein the expression vector does not comprise the nucleic acid represented by the sequence SEQ ID NO: 4 or a nucleic acid having a sequence identity of at least 85% with the sequence SEQ ID NO: 4, coding for the acetoacetate decarboxylase of C. acetobutylicum.