Patent Publication Number: US-9410164-B2

Title: Biosynthesis pathway for prenol in a recombinant microorganism

Description:
The present invention concerns a method for the biological preparation of prenol comprising culturing a microorganism genetically modified for the bioproduction of prenol, wherein the microorganism comprises a metabolic pathway for conversion of 3-methylcrotonyl-CoA into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme. 
     INTRODUCTION 
     Prenol or 3-methyl-2-buten-1-ol (number CAS 556-82-1), has the following formula: 
     
       
         
         
             
             
         
       
     
     Prenol is a natural alcohol that occurs naturally in citrus fruits, cranberry, bilberry, currants, grapes, raspberry, blackberry, tomato, white bread, hop oil, coffee, arctic bramble, cloudberry and passion fruit. 
     Prenol is under the form of a clear, colorless oil that is reasonably soluble in water and miscible with most common organic solvents. It has a fruity odor and is used as an aroma compound, in pharmaceuticals and in perfumery. It is usually manufactured industrially, in particular by BASF. 
     Advantageously, prenol can be converted into isoprene, a compound of high importance for industry since it is the basis for the synthesis of synthetic rubber. 
     Isoprene (short for isoterpene) or 2-methyl-1,3-butadiene (Numero CAS 78-79-5) is an organic compound having the formula CH2=(CH3)CH═CH 2 . Under standard conditions it is a colorless liquid. However, this compound is highly volatile because of its low boiling point. Isoprene is the monomer of natural rubber and also a common structure motif to an immense variety of other naturally occurring compounds, collectively termed the isoprenoids. 
     About 95% of isoprene production is used to produce cis-1,4-polyisoprene—a synthetic version of natural rubber. Isoprene is also copolymerized for use as a synthetic elastomer in other products such as footwear, mechanical products, medical products, sporting goods, and latex. 
     Isoprene was first isolated by thermal decomposition of natural rubber. It is most readily available industrially as a by-product of the thermal cracking of naphtha or oil, as a side product in the production of ethylene. While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. 
     Isoprene is naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate pathway and the non-mevalonate pathway. However, the yield of isoprene from naturally-occurring organisms is commercially unattractive. 
     Thus, more economical methods for producing isoprene are needed. In particular, methods that produce isoprene at rates, titers, and purity that are sufficient to meet the demands of a robust commercial process are desirable. Also desired are systems for producing isoprene from inexpensive starting materials. 
     PRIOR ART 
     Although the main industrial way for producing prenol is chemical synthesis, some biosynthetic pathways have been identified recently. 
     The patent application WO 2009/006429 provides a method for producing prenol with a genetically modified cell expressing a first enzyme capable of catalysing the dephosphorylation of the intermediate products: isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). 
     Concerning the isoprene synthesis, methods for fermentative production have been described. In particular, WO 2009/076676 describes cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide. Two biosynthesis pathways are proposed. 
     The patent application WO 2010/031076 describes the conversion of prenyl derivatives, such as prenol, into isoprene. Said method comprises: a) culturing cells for producing prenol, wherein the cells comprise a heterologous isoprene synthase polypeptide, b) recovering prenol, and c) dehydrating or decarboxylating prenol to produce isoprene. The cells may further comprise: an IDI polypeptide, an MVA pathway enzyme, and a DXP pathway enzyme. 
     DESCRIPTION OF THE INVENTION 
     The present invention is related to a new biosynthesis pathway for prenol in a recombinant microorganism. This biosynthesis pathway is characterized by the intermediate product, that is 3-methylcrotonyl-CoA (number CAS 6247-62-7), and that is converted into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme. 
     In particular, the enzyme capable of converting 3-methylcrotonyl-CoA into prenol is the alcohol-aldehyde dehydrogenase enzyme AdhE, that is heterologous to the recombinant microorganism, and that is preferentially issued from  Clostridium acetobutylicum.    
     According to the invention, the intermediate product 3-methylcrotonyl-CoA can be obtained from two different metabolic pathways:
         1) From the degradation pathway of leucine, starting from a condensation of pyruvate and acetyl-CoA into 4-methyl-2-oxopentanoate, converted into 3-methylbutanoyl-CoA and then into 3-methylcrotonyl-CoA. Here, this pathway is named leucine pathway,   2) From the mevalonate biosynthesis pathway, the precursor 3-hydroxy-3-methylglutaryl-CoA being converted into 3-methylglutaconyl-CoA and then into 3-methylcrotonyl-CoA. Formation of 3-methylcrotonyl-CoA from two acetyl-CoA molecules has been suggested to be possible in the article from Gogerty and Bobik (Gogerty and Bobik, 2010). Here, this pathway is named HMG-CoA pathway.       

     According to a specific aspect of the invention, the obtained prenol is converted into isoprene by chemical dehydration. 
    
    
     
       DRAWINGS 
         FIG. 1 . Metabolic pathway for biosynthesis of prenol, from the leucine pathway. 
         FIG. 2 . Metabolic pathway for biosynthesis of prenol, from the HMG-CoA pathway. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting, which will be limited only by the appended claims. 
     All publications, patents and patent applications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors that are reported in the publications and that might be used in connection with the invention. 
     Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Prescott et al., (1999) and Sambrook et al., (1989) (2001). 
     It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a microorganism” includes a plurality of such microorganisms, and a reference to “an endogenous gene” is a reference to one or more endogenous genes, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described. 
     In the claims that follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. 
     DEFINITIONS 
     The term “prenol” as used herein refers to 3-methyl-2-buten-1-ol or to a 3,3-dimethylallyl alcohol or DMAPP-ol while the term “isoprenol” refers especially to 3-methyl-3-buten-1-ol or IPP-ol. 
     3-Methylcrotonyl-CoA or β-Methylcrotonyl-CoA (CAS number 6247-62-7) is an intermediate in the metabolism of leucine. It is usually formed from 3-methylbutanoyl-CoA (also called isovaleryl-coenzyme A) by isovaleryl-coenzyme A dehydrogenase. 
     The term “aldehyde dehydrogenase” in this invention designates the aldehyde dehydrogenase (CoA-acylating) enzyme which catalyzes the reaction of conversion of an acyl-CoA into an aldehyde. 
     The term “alcohol dehydrogenase” in this invention designates the enzyme which catalyzes the reaction of conversion of an aldehyde into an alcohol. 
     Here, the term “AdhE enzyme” refers to a bifunctional enzyme having the two activities aldehyde dehydrogenase and alcohol dehydrogenase. 
     The terms “activity” and “function” refer to a specific catalytic activity or function of an enzyme, i.e. the biochemical reaction(s) that is (are) catalyzed by this enzyme. 
     The term “microorganism”, as used herein, refers to a bacterium, yeast or fungus which is not modified artificially. 
     The term “recombinant microorganism” or “genetically modified microorganism”, as used herein, refers to a microorganism genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction, by deletion or by modification of genetic elements. It can also be transformed by forcing the development and evolution of new metabolic pathways in combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659). 
     A microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. A microorganism may be modified to modulate the expression level of an endogenous gene. The modification or “transformation” of microorganisms with exogenous DNA is a routine task for those skilled in the art. 
     The term “endogenous gene” means that the gene was present in the microorganism before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, down regulate and/or lower the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid. 
     The term “exogenous gene” means that the gene was introduced into a microorganism, by means well known by the man skilled in the art whereas this gene is not naturally occurring in the microorganism. Exogenous genes can be heterologous or not. A microorganism can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally by plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. These genes may be heterologous or homologous. The term “heterologous gene” means that the gene is derived from a species of microorganism different from the recipient microorganism that expresses it. It refers to a gene which is not naturally occurring in the microorganism. 
     In the present application, all genes are referenced with their common names and with references that give access to their nucleotidic sequences in the National Center for Biotechnology Information (NCBI) GenBank. 
     The man skilled in the art knows different means to modulate, and in particular up-regulate, the expression of endogenous genes. For example, a way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid. 
     Another way is to replace the endogenous promoter of a gene with a stronger promoter. These promoters may be homologous or heterologous. It is well within the ability of the person skilled in the art to select appropriate promoters, for example, the promoters Ptrc, Ptac, Plac or the lambda promoter cI are widely used. 
     Finally, the sequence of exogenous gene may be adapted for its expression in the host microorganism. Indeed, the man skilled in the art knows the notion of codon usage bias and how adapt nucleic sequence for a particular codon usage bias without modify the deduced protein. 
     The term ‘overexpression’ means in this context that the expression of a gene or an enzyme is increased compared to a non modified microorganism. Increase of expression of an enzyme is obtained by the increase of the expression of a gene encoding said enzyme. 
     The ‘activity’ of an enzyme is used interchangeably with the term ‘function’ and designates, in the context of the invention, the reaction that is catalyzed by the enzyme. 
     The terms “encoding” or “coding” refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence. 
     The gene(s) encoding the enzyme(s) can be exogenous or endogenous. 
     “Attenuation” of genes may be achieved by means and methods known to the man skilled in the art and contains gene deletion by homologous recombination, gene attenuation by insertion of an external element into the gene or gene expression under a weak promoter. The man skilled in the art knows a variety of promoters which exhibit different strength and which promoter to use for a weak genetic expression. 
     The “fermentation” is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used, containing at least one simple carbon source, and if necessary co-substrates. 
     An “appropriate culture medium” designates a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins. 
     As an example of known culture media for  E. coli , the culture medium can be of identical or similar composition to an M9 medium (Anderson, 1946), an M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al., (1999). 
     As another example of culture medium for  C. glutamicum , the culture medium can be of identical or similar composition to BMCG medium (Liebl et al., 1989) or to a medium such as described by Riedel et al., (2001). 
     Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention. In particular the bacteria are fermented at a temperature between 20° C. and 55° C., preferentially between 25° C. and 40° C., and more specifically about 30° C. for  C. glutamicum  and about 37° C. for  E. coli.    
     The term “carbon source” or “carbon substrate” or “source of carbon” according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a micro-organism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, hemicelluloses and combinations thereof. 
     Prenol Biosynthesis 
     The present invention is related to a method for the fermentative production of prenol, comprising culturing a recombinant microorganism in a culture medium comprising a source of carbon, wherein in said microorganism, the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA as intermediate product, that is converted into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme. 
     An alcohol dehydrogenase enzyme designates an enzyme catalyzing the following reactions, in one way or in the other:
 
an aldehyde(or a ketone)+NAD(P)H+H+ an alcohol+NAD(P)+
 
     This enzyme always functions with an ‘acceptor’ of hydrogen, such as NAD+, NADP+, or other specific acceptors. The activity of this enzyme is the conversion of an alcohol into an aldehyde, and/or the conversion of an aldehyde into an alcohol with a donor of hydrogen. 
     Other known names are: aldehyde reductase; ADH; NAD-dependent alcohol dehydrogenase; NADH-alcohol dehydrogenase; primary alcohol dehydrogenase; aldehyde reductase (NADPH); NADP-alcohol dehydrogenase; NADP-aldehyde reductase; NADP-dependent aldehyde reductase; NADPH-aldehyde reductase; NADPH-dependent aldehyde reductase; alcohol dehydrogenase (NADP); the common abbreviation is ADH. 
     In a specific aspect of the invention, the enzyme having alcohol dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 1: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene 
                   
                   
               
               
                 name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 adhA 
                 alcohol-dehydrogenase adhA from patent WO8704464-A- 
                 
                   A. niger 
                 
               
               
                   
                   Aspergillus niger  (EC: 1.1.1.1) 
               
               
                 ADH1 
                 ADH1 (ALCOHOL DEHYDROGENASE 1); alcohol 
                 
                   A. thaliana 
                 
               
               
                   
                 dehydrogenase 
               
               
                 adhA 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   B. cereus 
                 
               
               
                 gbsB 
                 choline dehydrogenase (EC: 1.1.1.—) 
                 
                   B. subtilis 
                 
               
               
                 adhB 
                 putative oxidoreductase (EC: 1.1.1.—) 
                 
                   B. subtilis 
                 
               
               
                 adhE1 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. acetobutylicum 
                 
               
               
                 adhE2 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. acetobutylicum 
                 
               
               
                 cgl2537 
                 putative Zn-NADPH:quinone dehydrogenase (EC: 1.1.1.1) 
                 
                   C. glutamicum 
                 
               
               
                 cgl0222 
                 Zn-dependent alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   C. glutamicum 
                 
               
               
                 cgl2807 
                 Zn-dependent alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   C. glutamicum 
                 
               
               
                 aad 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. kluyveri 
                 
               
               
                   
                 (EC: 1.1.1.—1.2.1.—) 
               
               
                 adhP 
                 ethanol-active dehydrogenase/acetaldehyde-active reductase 
                 
                   E. coli 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 yiaY 
                 predicted Fe-containing alcohol dehydrogenase, Pfam00465 
                 
                   E. coli 
                 
               
               
                   
                 family 
               
               
                 adhE 
                 fused acetaldehyde-CoA dehydrogenase/iron-dependent 
                 
                   E. coli 
                 
               
               
                   
                 alcohol dehydrogenase/pyruvate-formate lyase deactivase 
               
               
                   
                 (EC: 1.2.1.10 1.1.1.1) 
               
               
                 frmA 
                 alcohol dehydrogenase class III/glutathione-dependent 
                 
                   E. coli 
                 
               
               
                   
                 formaldehyde dehydrogenase (EC: 1.1.1.1 1.1.1.284) 
               
               
                 adhA 
                 alcohol dehydrogenase 
                 
                   E. faecalis 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   E. faecalis 
                 
               
               
                 adhC 
                 alcohol dehydrogenase class III 
                 
                   H. influenzae 
                 
               
               
                 adhP 
                 alcohol dehydrogenase 
                 
                   K. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   K. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   L. lactis 
                 
               
               
                   
                 (EC: 1.2.1.10) 
               
               
                 adhA 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   L. lactis 
                 
               
               
                 ypjA 
                 dehydrogenase 
                 
                   L. lactis 
                 
               
               
                 eutG 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   M. succiniciproducens 
                 
               
               
                 adhC 
                 AdhC protein 
                 
                   M. succiniciproducens 
                 
               
               
                 adhB 
                 alcohol dehydrogenase II (EC: 1.1.1.1) 
                 
                   P. fluorescens 
                 
               
               
                 adh 
                 alcohol dehydrogenase, zinc-containing (EC: 1.1.1.1) 
                 
                   P. fluorescens 
                 
               
               
                 adhC 
                 alcohol dehydrogenase class III 
                 
                   P. fluorescens 
                 
               
               
                 adhA 
                 alcohol dehydrogenase 
                 
                   P. putida 
                 
               
               
                 FDH1 
                 Glutathione-dependent formaldehyde dehydrogenase (FDH) 
                 
                   P. stipitis 
                 
               
               
                   
                 (FALDH) (FLD) 
               
               
                 SAD1 
                 secondary alcohol dehydrogenase (SADH1) 
                 
                   P. stipitis 
                 
               
               
                 SAD2 
                 secondary alcohol dehydrogenase (SADH2) 
                 
                   P. stipitis 
                 
               
               
                 ADH1 
                 alcohol dehydrogenase 
                 
                   P. stipitis 
                 
               
               
                 IFR1 
                 Zinc-binding oxidoreductase alcohol dehydrogenase 
                 
                   P. stipitis 
                 
               
               
                   
                 (EC: 1.1.1.1 1.6.5.5) 
               
               
                 ADH2 
                 alcohol dehydrogenase 
                 
                   P. stipitis 
                 
               
               
                 ypch00392 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypf00190 
                 putative alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 adhA2 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypch01140 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypb00013 
                 iron alcohol dehydrogenase protein 
                 
                   R. etli 
                 
               
               
                 ypch00170 
                 zinc-binding oxidoreductase protein 
                 
                   R. etli 
                 
               
               
                 adhA1 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 adhCch 
                 formaldehyde dehydrogenase (glutathione)/alcohol 
                 
                   R. etli 
                 
               
               
                   
                 dehydrogenase protein (EC: 1.1.1.1 1.1.1.284) 
               
               
                 ypch00995 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypb00024 
                 putative alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypf00099 
                 Zn-dependent oxidoreductase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 ypch00832 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 adhE 
                 alcohol dehydrogenase protein (EC: 1.1.1.1) 
                 
                   R. etli 
                 
               
               
                 adhCf 
                 formaldehyde dehydrogenase (glutathione)/alcohol 
                 
                   R. etli 
                 
               
               
                   
                 dehydrogenase protein (EC: 1.1.1.1 1.1.1.284) 
               
               
                 adh 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   R. eutropha 
                 
               
               
                 rplK 
                 50S ribosomal protein L11 (EC: 1.1.1.1) 
                 
                   R. eutropha 
                 
               
               
                 adhC 
                 alcohol dehydrogenase, class III (EC: 1.1.1.284) 
                 
                   R. eutropha 
                 
               
               
                 adhP 
                 alcohol dehydrogenase, Zn-dependent (EC: 1.1.1.1) 
                 
                   R. eutropha 
                 
               
               
                 adh 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   R. opacus 
                 
               
               
                 adhP 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   S. aureus 
                 
               
               
                 adhA 
                 alcohol dehydrogenase 
                 
                   S. aureus 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. aureus 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 adh1 
                 alcohol dehydrogenase 
                 
                   S. aureus 
                 
               
               
                 adhC 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   S. aureus 
                 
               
               
                 adhA2 
                 alcohol dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 pteB 
                 dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 adhA1 
                 alcohol dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 adhA6 
                 alcohol dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 adhA3 
                 alcohol dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 ADH4 
                 Adh4p (EC: 1.1.1.190 1.1.1.1) 
                 
                   S. cerevisiae 
                 
               
               
                 ADH2 
                 Adh2p (EC: 1.1.1.190 1.1.1.1) 
                 
                   S. cerevisiae 
                 
               
               
                 ADH3 
                 Adh3p (EC: 1.1.1.190 1.1.1.1) 
                 
                   S. cerevisiae 
                 
               
               
                 ADH5 
                 Adh5p (EC: 1.1.1.190 1.1.1.1) 
                 
                   S. cerevisiae 
                 
               
               
                 SFA1 
                 Bifunctional enzyme containing both alcohol dehydrogenase 
                 
                   S. cerevisiae 
                 
               
               
                   
                 and glutathione-dependent formaldehyde dehydrogenase 
               
               
                   
                 activities, functions in formaldehyde detoxification and 
               
               
                   
                 formation of long chain and complex alcohols, regulated by 
               
               
                   
                 Hog1p-Sko1p (EC: 1.1.1.284 1.1.1.1) 
               
               
                 ADH1 
                 Adh1p (EC: 1.1.1.190 1.1.1.1) 
                 
                   S. cerevisiae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. enterica 
                 
               
               
                   
                 (EC: 1.1.1.1 1.2.1.10) 
               
               
                 adh2 
                 putative iron-containing alcohol dehydrogenase 
                 
                   S. enterica 
                 
               
               
                 adh3 
                 alcohol dehydrogenase class III 
                 
                   S. enterica 
                 
               
               
                 adhP 
                 alcohol dehydrogenase 
                 
                   S. enterica 
                 
               
               
                 adhA 
                 alcohol dehydrogenase 
                 
                   S. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. pneumoniae 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 adhP 
                 alcohol dehydrogenase (EC: 1.1.1.1) 
                 
                   S. pneumoniae 
                 
               
               
                 adhB 
                 alcohol dehydrogenase, zinc-containing 
                 
                   S. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   Y. pestis 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 adhC 
                 alcohol dehydrogenase 
                 
                   Y. pestis 
                 
               
               
                   
               
            
           
         
       
     
     According to the present invention, the alcohol dehydrogenase enzyme has specific activity for prenal or 3-methyl-2-butenal (number CAS 107-86-8), an aldehyde of formula: 
                         
and converts it into prenol with the hydrogen donor NADH. This enzyme is also named 3-methyl-2-butenal dehydrogenase in the invention.
 
     An aldehyde dehydrogenase (CoA-acylating) enzyme designates an enzyme catalyzing the following reaction, in one way or in the other:
 
an acyl-CoA+NAD(P)H+H+ an aldehyde+NAD++Coenzyme A
 
     Other known names are: aldehyde:NAD+ oxidoreductase (CoA-acylating), aldehyde dehydrogenase (acylating); acylating aldehyde dehydrogenase; Coenzyme A-acylating aldehyde dehydrogenase; aldehyde dehydrogenase (acetylating); aldehyde:NAD(P)+ oxidoreductase (CoA-acetylating); the common abbreviation is ALDH. 
     This enzyme always functions with an ‘acceptor’ of hydrogen, such as NAD+, NADP+, or other specific acceptors. The activity of this enzyme is the conversion of an acyl-CoA into an aldehyde, and/or the conversion of an aldehyde into an acyl-CoA with a donor of hydrogen. 
     In a specific aspect of the invention, the enzyme having aldehyde dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 2: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene 
                   
                   
               
               
                 name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 eutE 
                 acetaldehyde dehydrogenase (acetylating) (EC: 1.2.1.10) 
                 
                   A. hydrophila 
                 
               
               
                 mhpF 
                 acetaldehyde dehydrogenase (EC: 1.2.1.10) 
                 
                   B. megaterium 
                 
               
               
                 eutE 
                 acetaldehyde dehydrogenase (EC: 1.2.1.10) 
                 
                   B. megaterium 
                 
               
               
                 adhE2 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. acetobutylicum 
                 
               
               
                 adhE1 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. acetobutylicum 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. difficile 
                 
               
               
                   
                 (EC: 1.2.1.10 1.1.1.1) 
               
               
                 aad 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   C. kluyveri 
                 
               
               
                   
                 (EC: 1.1.1.—1.2.1.—) 
               
               
                 mhpF 
                 acetaldehyde-CoA dehydrogenase II, NAD-binding 
                 
                   E. coli 
                 
               
               
                   
                 (EC: 1.2.1.10) 
               
               
                 adhE 
                 fused acetaldehyde-CoA dehydrogenase/iron-dependent 
                 
                   E. coli 
                 
               
               
                   
                 alcohol dehydrogenase/pyruvate-formate lyase deactivase 
               
               
                   
                 (EC: 1.2.1.10 1.1.1.1) 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   E. faecalis 
                 
               
               
                 mhpF 
                 acetaldehyde dehydrogenase 
                 
                   K. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   K. pneumoniae 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   L. lactis 
                 
               
               
                   
                 (EC: 1.2.1.10) 
               
               
                 eutG 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   M. succiniciproducens 
                 
               
               
                 mhpF 
                 acetaldehyde dehydrogenase (EC: 1.2.1.10) 
                 
                   R. eutropha 
                 
               
               
                 cmtH 
                 acetaldehyde dehydrogenase (EC: 1.2.1.10) 
                 
                   R. opacus 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. aureus 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. enterica 
                 
               
               
                   
                 (EC: 1.1.1.1 1.2.1.10) 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   S. pneumoniae 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                 adhE 
                 metal binding site; other site (EC: 1.2.1.10) 
                 
                   S. typhimurium 
                 
               
               
                 adhE 
                 bifunctional acetaldehyde-CoA/alcohol dehydrogenase 
                 
                   Y. pestis 
                 
               
               
                   
                 (EC: 1.1.1.1) 
               
               
                   
               
            
           
         
       
     
     According to the present invention, the aldehyde dehydrogenase enzyme has specific activity for 3-methylcrotonyl-CoA, an acyl-CoA of formula: 
                         
and converts it into prenal with the hydrogen donor NADH. In the invention, this enzyme is also named 3-methyl-2-butanol dehydrogenase.
 
     In a preferred embodiment of the invention, the alcohol dehydrogenase and aldehyde dehydrogenase activities are catalyzed by the same enzyme, capable of both functions; in a specific embodiment, said enzyme capable of both functions is the alcohol-aldehyde dehydrogenase enzyme (AdhE enzyme). 
     Examples of enzymes possessing both activities are enzymes previously listed called “bifunctional acetaldehyde-CoA/alcohol dehydrogenase”. 
     Preferentially, the recombinant microorganism expresses an AdhE enzyme that is heterologous to the microorganism. 
     According to a specific embodiment, the AdhE enzyme has specificity for the substrate 3-methylcrotonyl-CoA. 
     The term “specificity” designates affinity of an enzyme for a precise substrate. According to this invention specificity of AdhE enzyme means that this enzyme recognizes the 3-methylcrotonyl-CoA as preferred substrate among all other substrates. 
     According to a preferred embodiment of the invention, the AdhE enzyme is AdhE1 from  Clostridium acetobutylicum.    
     According to a more preferred embodiment of the invention, the AdhE enzyme is encoded by the gene adhE2 from  Clostridium acetobutylicum  (listed in table 1 and table 2). 
     Prenol Bisosynthesis by Leucine Pathway 
     In this aspect of the invention, the biosynthesis pathway of 3-methylcrotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 4-methyl-2-oxopentanoate and 3-methylbutanoyl-CoA. 
     The whole pathway for the biosynthesis of prenol according to this embodiment of the invention is illustrated in  FIG. 1 , entitled “Metabolic pathway for biosynthesis of prenol, from the leucine pathway”. 
     The first reaction of the conversion of 4-methyl-2-oxopentanoate into 3-methylbutanoyl-CoA is catalysed by the branched-chain keto-acid dehydrogenase complex. This complex is composed of four subunits E1α, E1β, E2 and E3. This enzymatic complex has been identified in several species, and in particular in:
           Bacillus substilis  (genes bkdAA, bkdAB, bkdB, lpdV), for reference: Perham and Lowe, (1988).     Pseudomonas putida  (genes bkdA1, bkdA2, bkdB, lpdV): Sykes et al., (1987).     Streptomyces avermitilis , for reference: Skinner et al., (1995).     Enterococcus faecalis  (operon bkdABCD), for reference: Ward et al., (1999).     Saccharomyces cerevisiae , for reference: Sinclair et al., (1993).       

     In a specific aspect of the invention, the subunit E1 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 3: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene 
                   
                   
               
               
                 name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 bkdA2 
                 2-oxoisovalerate dehydrogenase beta subunit 
                 
                   A. tumefaciens 
                 
               
               
                 bkdA1 
                 2-oxoisovalerate dehydrogenase alpha subunit 
                 
                   A. tumefaciens 
                 
               
               
                 bkdA2 
                 2-oxoisovalerate dehydrogenase beta subunit (EC: 1.2.4.4) 
                 
                   B. brevis 
                 
               
               
                 bkdA1 
                 2-oxoisovalerate dehydrogenase alpha subunit (EC: 1.2.4.4) 
                 
                   B. brevis 
                 
               
               
                 bfmbAb 
                 3-methyl-2-oxobutanoate dehydrogenase, beta subunit 
                 
                   B. cereus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bfmbAa 
                 3-methyl-2-oxobutanoate dehydrogenase, alpha subunit 
                 
                   B. cereus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdAB 
                 2-oxoisovalerate dehydrogenase E1 component subunit beta 
                 
                   B. megaterium 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdAA 
                 2-oxoisovalerate dehydrogenase E1 component subunit alpha 
                 
                   B. megaterium 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdAA 
                 branched-chain alpha-keto acid dehydrogenase E1 subunit 
                 
                   B. subtilis 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdAB 
                 branched-chain alpha-keto acid dehydrogenase E1 subunit 
                 
                   B. subtilis 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase, E1 component, 
                 
                   E. faecalis 
                 
               
               
                   
                 beta subunit 
               
               
                 bkdA 
                 branched-chain alpha-keto acid dehydrogenase, E1 component, 
                 
                   E. faecalis 
                 
               
               
                   
                 alpha subunit 
               
               
                 BCKDHA 
                 branched chain keto acid dehydrogenase E1, alpha polypeptide 
                 
                   H. sapiens 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 BCKDHB 
                 branched chain keto acid dehydrogenase E1, beta polypeptide 
                 
                   H. sapiens 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdB 
                 TPP-dependent branched-chain alpha-keto acid 
                 
                   L. casei 
                 
               
               
                   
                 dehydrogenase, E1 beta subunit (branched-chain alpha-keto 
               
               
                   
                 acid dehydrogenase, E1 component, beta subunit) 
               
               
                 bkdA 
                 branched-chain alpha-keto acid dehydrogenase, E1 component, 
                 
                   L. casei 
                 
               
               
                   
                 alpha subunit 
               
               
                 Bckdha 
                 branched chain ketoacid dehydrogenase E1, alpha polypeptide 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 Bckdhb 
                 branched chain ketoacid dehydrogenase E1, beta polypeptide 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bkdA1 
                 2-oxoisovalerate dehydrogenase (alpha subunit) 
                 
                   P. aeruginosa 
                 
               
               
                 bkdA2 
                 2-oxoisovalerate dehydrogenase (beta subunit) 
                 
                   P. aeruginosa 
                 
               
               
                 bkdA2 
                 2-oxoisovalerate dehydrogenase, beta subunit 
                 
                   P. putida 
                 
               
               
                 bkdA1 
                 3-methyl-2-oxobutanoate dehydrogenase 
                 
                   P. putida 
                 
               
               
                 bkDa1 
                 2-oxoisovalerate dehydrogenase alpha subunit protein 
                 
                   R. etli 
                 
               
               
                 bkDa2 
                 2-oxoisovalerate dehydrogenase beta subunit protein 
                 
                   R. etli 
                 
               
               
                 Bckdhb 
                 branched chain keto acid dehydrogenase E1, beta polypeptide 
                 
                   R. norvegicus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 Bckdha 
                 branched chain ketoacid dehydrogenase E1, alpha polypeptide 
                 
                   R. norvegicus 
                 
               
               
                   
                 (EC: 1.2.4.4) 
               
               
                 bfmBAA 
                 2-oxoisovalerate dehydrogenase alpha subunit (EC: 1.2.4.4) 
                 
                   S. aureus 
                 
               
               
                 bfmBAB 
                 2-oxoisovalerate dehydrogenase beta subunit (EC: 1.2.4.4) 
                 
                   S. aureus 
                 
               
               
                   
               
            
           
         
       
     
     In a specific aspect of the invention, the subunit E2 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 4: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   A. tumefaciens 
                 
               
               
                 bkdB 
                 lipoamide acyltransferase component of branched-chain 
                 
                   B. brevis 
                 
               
               
                   
                 alpha-keto acid dehydrogenase complex (EC: 2.3.1.168) 
               
               
                 bfmbB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   B. cereus 
                 
               
               
                   
                 (EC: 2.3.1.—) 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase complex 
                 
                   B. megaterium 
                 
               
               
                   
                 lipoamide acyltransferase E2 component (EC: 2.3.1.168) 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   B. subtilis 
                 
               
               
                   
                 (EC: 2.3.1.—) 
               
               
                 bkdC 
                 branched-chain alpha-keto acid, E2 component, 
                 
                   E. faecalis 
                 
               
               
                   
                 dihydrolipoamide acetyltransferase 
               
               
                 DBT 
                 dihydrolipoamide branched chain transacylase E2 
                 
                   H. sapiens 
                 
               
               
                   
                 (EC: 2.3.1.168) 
               
               
                 bkdC 
                 branched-chain alpha-keto acid, E2 component, 
                 
                   L. casei 
                 
               
               
                   
                 dihydrolipoamide acetyltransferase 
               
               
                 Dbt 
                 dihydrolipoamide branched chain transacylase E2 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 2.3.1.168) 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   P. aeruginosa 
                 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   P. putida 
                 
               
               
                 bkdB 
                 branched-chain alpha-keto acid dehydrogenase subunit E2 
                 
                   R. etli 
                 
               
               
                   
                 (EC: 2.3.1.12) 
               
               
                 Dbt 
                 dihydrolipoamide branched chain transacylase E2 
                 
                   R. norvegicus 
                 
               
               
                   
                 (EC: 2.3.1.168) 
               
               
                 bfmB 
                 lipoamide acyltransferase component of branched-chain 
                 
                   S. aureus 
                 
               
               
                   
                 alpha-keto acid dehydrogenase complex (EC: 2.3.1.—) 
               
               
                   
               
            
           
         
       
     
     In a specific aspect of the invention, the subunit E3 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 5: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 lpdA 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   A. tumefaciens 
                 
               
               
                 pdhD 
                 dihydrolipoyl dehydrogenase (EC: 1.8.1.4) 
                 
                   B. brevis 
                 
               
               
                 acoL 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. brevis 
                 
               
               
                 lpd 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. brevis 
                 
               
               
                 acoL 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. cereus 
                 
               
               
                 pdhD 
                 pyruvate dehydrogenase complex E3 component, 
                 
                   B. megaterium 
                 
               
               
                   
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
               
               
                 lpdV 
                 branched-chain alpha-keto acid dehydrogenase 
                 
                   B. megaterium 
                 
               
               
                   
                 complex dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
               
               
                 acoL 
                 acetoin dehydrogenase E3 component 
                 
                   B. megaterium 
                 
               
               
                   
                 (dihydrolipoamide dehydrogenase) (EC: 1.8.1.4) 
               
               
                 pdhD 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. subtilis 
                 
               
               
                 acoL 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. subtilis 
                 
               
               
                 lpdV 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   B. subtilis 
                 
               
               
                 bkdD 
                 branched-chain alpha-keto acid dehydrogenase, E3 
                 
                   E. faecalis 
                 
               
               
                   
                 component, dihydrolipoamide dehydrogenase 
               
               
                 lpdA 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   E. faecalis 
                 
               
               
                 DLD 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   H. sapiens 
                 
               
               
                 bkdD 
                 dihydrolipoyl dehydrogenase (EC: 1.8.1.4) 
                 
                   L. casei 
                 
               
               
                 pdhD 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   L. casei 
                 
               
               
                 Dld 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   M. musculus 
                 
               
               
                 lpd3 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. aeruginosa 
                 
               
               
                 lpdG 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. aeruginosa 
                 
               
               
                 lpdV 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. aeruginosa 
                 
               
               
                 lpdG 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. putida 
                 
               
               
                 lpd3 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. putida 
                 
               
               
                 lpdV 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   P. putida 
                 
               
               
                 lpdAc 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   R. etli 
                 
               
               
                 lpdAch1 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   R. etli 
                 
               
               
                 lpdAch2 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   R. etli 
                 
               
               
                 pdhD 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   S. aureus 
                 
               
               
                 lpdA 
                 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) 
                 
                   S. aureus 
                 
               
               
                   
               
            
           
         
       
     
     Preferentially, the enzymatic complex having branched-chain keto acid dehydrogenase activity is the complex from  P. putida.    
     The second reaction of the conversion of 3-methylbutanoyl-CoA into 3-methylcrotonyl-CoA is catalysed by an acyl-CoA dehydrogenase, in particular an isovaleryl-CoA dehydrogenase. This enzyme has been identified in several species, and in particular in:
           Pseudomonas aeruginosa  (gene liuA), for reference: Förster-Fromme and Jendrossek (2008).     Streptomyces coelicolor  and  Streptomyces avermitilis  (acdH), for reference: Zhang et al., (1999).     Caenorhabditis elegans  (ivd) Mohsen et al., (2001).     Arabidopsis thaliana  (ivd): Däschner et al., (2001).       

     In a specific aspect of the invention, the enzyme having acyl-CoA dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 6: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 IBR3 
                 IBR3 (IBA-RESPONSE 3); acyl-CoA dehydrogenase/ 
                 
                   A. thaliana 
                 
               
               
                   
                 oxidoreductase (EC: 1.3.99.3) 
               
               
                 IVD 
                 IVD (ISOVALERYL-CoA-DEHYDROGENASE); ATP 
                 
                   A. thaliana 
                 
               
               
                   
                 binding/isovaleryl-CoA dehydrogenase (EC: 1.3.99.12) 
               
               
                 acd 
                 acyl-CoA dehydrogenase 
                 
                   A. tumefaciens 
                 
               
               
                 acd 
                 acyl-CoA dehydrogenase 
                 
                   A. tumefaciens 
                 
               
               
                 acdA 
                 acyl-CoA dehydrogenase, short-chain specific 
                 
                   B. cereus 
                 
               
               
                   
                 (EC: 1.3.99.3) 
               
               
                 bcd 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   B. cereus 
                 
               
               
                 acdA 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   B. megaterium 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   B. megaterium 
                 
               
               
                 mmgC 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   B. megaterium 
                 
               
               
                 acdh-10 
                 Acyl CoA DeHydrogenase 
                 
                   C. elegans 
                 
               
               
                 acdh-7 
                 Acyl CoA DeHydrogenase 
                 
                   C. elegans 
                 
               
               
                 acdh-8 
                 Acyl CoA DeHydrogenase 
                 
                   C. elegans 
                 
               
               
                 ivd-1 
                 IsoValeryl-CoA Dehydrogenase 
                 
                   C. elegans 
                 
               
               
                 fadE 
                 acyl coenzyme A dehydrogenase (EC: 1.3.99.3) 
                 
                   E. coli 
                 
               
               
                 ACADM 
                 acyl-CoA dehydrogenase, C-4 to C-12 straight chain 
                 
                   H. sapiens 
                 
               
               
                   
                 (EC: 1.3.99.3) 
               
               
                 IVD 
                 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) 
                 
                   H. sapiens 
                 
               
               
                 fadE1 
                 acyl-CoA dehydrogenase FadE1 (EC: 1.3.99.—) 
                 
                   M. bovis 
                 
               
               
                 fadE13 
                 acyl-CoA dehydrogenase FadE13 (EC: 1.3.99.—) 
                 
                   M. bovis 
                 
               
               
                 fadE2 
                 acyl-CoA dehydrogenase FadE2 (EC: 1.3.99.—) 
                 
                   M. bovis 
                 
               
               
                 fadE20 
                 acyl-CoA dehydrogenase FadE20 (EC: 1.3.99.—) 
                 
                   M. bovis 
                 
               
               
                 fadE23 
                 acyl-CoA dehydrogenase FadE23 
                 
                   M. bovis 
                 
               
               
                 fadE4 
                 acyl-CoA dehydrogenase FadE4 (EC: 1.3.99.—) 
                 
                   M. bovis 
                 
               
               
                 ibd2 
                 isobutyryl-CoA dehydrogenase (EC: 1.3.99.10) 
                 
                   M. extorquens 
                 
               
               
                 Acadm 
                 acyl-Coenzyme A dehydrogenase, medium chain 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 1.3.99.3) 
               
               
                 Ivd 
                 isovaleryl coenzyme A dehydrogenase (EC: 1.3.99.10) 
                 
                   M. musculus 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   P. aeruginosa 
                 
               
               
                 liuA 
                 putative isovaleryl-CoA dehydrogenase 
                 
                   P. aeruginosa 
                 
               
               
                 acd-6 
                 acyl-CoA dehydrogenase family protein 
                 
                   P. fluorescens 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   P. fluorescens 
                 
               
               
                 fadE1 
                 acyl-CoA dehydrogenase family protein 
                 
                   P. fluorescens 
                 
               
               
                 fadE13 
                 hypothetical protein 
                 
                   P. fluorescens 
                 
               
               
                 fadE20 
                 acyl-CoA dehydrogenase 
                 
                   P. fluorescens 
                 
               
               
                 ivd 
                 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) 
                 
                   P. fluorescens 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   P. putida 
                 
               
               
                 ivd 
                 acyl-CoA dehydrogenase domain containing protein 
                 
                   P. putida 
                 
               
               
                 acd1 
                 acyl-CoA dehydrogenase protein 
                 
                   R. etli 
                 
               
               
                 acd2 
                 acyl-CoA dehydrogenase protein 
                 
                   R. etli 
                 
               
               
                 ivdH 
                 isovaleryl-CoA dehydrogenase protein 
                 
                   R. etli 
                 
               
               
                 abmD 
                 putative acyl-CoA dehydrogenase (EC: 1.3.99.—) 
                 
                   R. eutropha 
                 
               
               
                 acaD 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   R. eutropha 
                 
               
               
                 aidB 
                 acyl-CoA dehydrogenase, short-chain specific (AidB 
                 
                   R. eutropha 
                 
               
               
                   
                 protein) (EC: 1.3.99.3) 
               
               
                 ivd1 
                 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) 
                 
                   R. eutropha 
                 
               
               
                 ivd2 
                 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) 
                 
                   R. eutropha 
                 
               
               
                 fadE15 
                 acyl-CoA dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 fadE17 
                 acyl-CoA dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 fadE7 
                 acyl-CoA dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 acdH 
                 acyl-CoA dehydrogenase 
                 
                   S. avermitilis 
                 
               
               
                 acdH2 
                 acyl-CoA dehydrogenase 
                 
                   S. coelicolor 
                 
               
               
                 acdH3 
                 acyl-CoA dehydrogenase 
                 
                   S. coelicolor 
                 
               
               
                 acdC 
                 putative acyl-CoA dehydrogenase 
                 
                   S. enterica 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. enterica 
                 
               
               
                 acdA 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 acdA-3 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 fadE1 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 fadE13 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 fadE21 
                 putative acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 fadE22 
                 putative acyl-CoA dehydrogenase 
                 
                   S. erythraea 
                 
               
               
                 fadE31 
                 putative acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   S. erythraea 
                 
               
               
                 fadE 
                 domain of unknown function (DUF1974); region: 
                 
                   S. typhimurium 
                 
               
               
                   
                 DUF1974; pfam09317 (EC: 1.3.99.3) 
               
               
                 acdA 
                 acyl-CoA dehydrogenase 
                 
                   X. oryzae 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   X. oryzae 
                 
               
               
                 fadE 
                 acyl-CoA dehydrogenase (EC: 1.3.99.3) 
                 
                   Y. pestis 
                 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment of the invention, the acyl-CoA dehydrogenase is encoded by the gene acdH from  S. avermitilis.    
     In a specific embodiment of the invention, in the recombinant microorganism, at least one of the following enzymes is overexpressed: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase. 
     In a more specific aspect of the invention, in the recombinant microorganism, branched chain keto acid dehydrogenase complex and the enzyme acyl-CoA dehydrogenase, that are overexpressed in the genetically modified microorganism, are heterologous. 
     In particular, the branched chain keto acid dehydrogenase is encoded by one or several genes issued from  Bacillus subtilis  (bkdAA, bkdAB, bkdB, lpdV),  Pseudomonas putida  (bkdA1, bkdA2, bkdB, lpdV),  Streptomyces avermitilis, Enterococcus faecalis  (bkdABCD) or  Saccharomyces cerevisiae.    
     In particular the acyl-CoA dehydrogenase is encoded by a gene from  Pseudomonas aeruginosa  (liuA),  Streptomyces coelicolor, Streptomyces  avermitilis (acdH),  Caenorhabditis elegans  (ivd) or  Arabidopsis thaliana  (ivd). 
     In another aspect of the invention, the microorganism is further modified to improve the availability of at least one biosynthesis intermediate chosen among pyruvate, 3-methyl-2-oxobutanoic acid and 4-methyl-2-oxopentanoate. 
     In order to optimize pyruvate availability, the microorganism is modified to overexpress at least one gene involved in pyruvate biosynthesis pathway, chosen among gene coding for phosphoglycerate mutase (gpmA and pgmI in  E. coli  or homologous gene), enolase (eno in  E. coli  or homologous gene) or pyruvate kinase (pykA and pykF in  E. coli  or homologous gene). Alternatively or in combination, at least one gene involved in pyruvate degradation pathway is attenuated. This gene is chosen among pyruvate oxidase (poxB in  E. coli  or homologous gene), phosphate acetyltransferase (pta in  E. coli  or homologous gene), acetate kinase (ackA in  E. coli  or homologous gene), aldehyde/alcohol dehydrogenase (adhE in  E. coli  or homologous gene), pyruvate dehydrogenase operon repressor (pdhR in  E. coli  or homologous gene) or lactate dehydrogenase (pfl, lldD, ldhA or did in  E. coli  or homologous gene). 
     In order to optimize 3-methyl-2-oxobutanoic acid availability, the microorganism is modified to overexpress at least one gene involved in the L-valine biosynthesis pathway, chosen among gene coding for acetolactate synthase (ilvl, ilvH, ilvN and ilvB in  E. coli  or homologous genes), keto-acid reductoisomerase (ilvC in  E. coli  or homologous gene) and dihydroxy-acid dehydratase (ilvD in  E. coli  or homologous gene). Alternatively or in combination, the gene encoding the branched chain amino acid transaminase (ilvE in  E. coli  or homologous gene) is deleted in the microorganism of the invention. In another embodiment of the invention, the ilvN gene is modified so as to produce an IlvN protein which is feedback deregulated. Such mutations of ilvN are disclosed in Park et al., 2011. 
     In order to optimize 4-methyl-2-oxopentanoate availability, the microorganism is modified to overexpress at least one gene chosen among the genes coding for 3-isopropylmalate dehydratase (leuC, leuD in  E. coli  or homologous genes), 3-isopropylmalate dehydrogenase (leuB in  E. coli  or homologous gene) or 2-isopropylmalate synthase (leuA in  E. coli  or homologous gene). Alternatively or in combination, the gene encoding the branched chain amino acid transaminase (ilvE in  E. coli  or homologous gene) is deleted in the microorganism of the invention. In another embodiment of the invention, the leuA gene is modified so as to produce a LeuA protein which is feedback deregulated. Such mutations of leuA are disclosed in patent application U.S. Pat. No. 6,403,342. 
     Prenol Bisosynthesis by HMG-CoA Pathway 
     In this aspect of the invention, the biosynthesis pathway of 3-methylcrotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and 3-methylglutaconyl-CoA. 
     The whole pathway for the biosynthesis of prenol according to this embodiment of the invention is illustrated in  FIG. 2 , entitled “Metabolic pathway for biosynthesis of prenol, from an intermediate of the HMG-CoA pathway”. 
     The first reaction of condensation of two acetyl-CoA molecules into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) is catalysed successively by two enzymes: (1) an acetyl-CoA acetyltransferase, and (2) a 3-hydroxy-3-methylglutaryl-CoA synthase. 
     Genes coding for enzymes having an acetyl-CoA acetyltransferase activity have been identified in several species, and in particular in  Escherichia coli  (gene atoB),  Clostridium acetobutylicum  (thlA) and  Saccharomyces cerevisiae  (ERG10). 
     In a specific aspect of the invention, the enzyme having acetyl-CoA acetyltransferase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 7: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene 
                   
                   
               
               
                 name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 ACAT2 
                 ACAT2 (ACETOACETYL-CoA THIOLASE 2); acetyl- 
                 
                   A. thaliana 
                 
               
               
                   
                 CoA C-acetyltransferase/catalytic (EC: 2.3.1.9) 
               
               
                 mmgA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   B. brevis 
                 
               
               
                 thl 
                 acetyl-CoA acetyltransferase 
                 
                   B. cereus 
                 
               
               
                 atoB 
                 acetyl-CoA acetyltransferase 
                 
                   B. cereus 
                 
               
               
                 thiL 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. acetobutylicum 
                 
               
               
                 ERG10 
                 similar to Acetoacetyl-CoA Thiolase A; mevalonate/sterol 
                 
                   C. albicans 
                 
               
               
                   
                 pathway 
               
               
                 thlA1 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. difficile 
                 
               
               
                 thlA2 
                 putative acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. difficile 
                 
               
               
                 kat-1 
                 3-Ketoacyl-CoA Thiolase 
                 
                   C. elegans 
                 
               
               
                 thlA1 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. kluyveri 
                 
               
               
                 thlA2 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. kluyveri 
                 
               
               
                 thlA3 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   C. kluyveri 
                 
               
               
                 atoB 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   E. coli 
                 
               
               
                 yqeF 
                 predicted acyltransferase 
                 
                   E. coli 
                 
               
               
                 ACAT1 
                 acetyl-CoA acetyltransferase 1 (EC: 2.3.1.9) 
                 
                   H. sapiens 
                 
               
               
                 ACAT2 
                 acetyl-CoA acetyltransferase 2 (EC: 2.3.1.9) 
                 
                   H. sapiens 
                 
               
               
                 yqeF 
                 acetyl-CoA acetyltransferase 
                 
                   K. pneumoniae 
                 
               
               
                 thiL 
                 acetyl coenzyme A acetyltransferase (EC: 2.3.1.9) 
                 
                   L. lactis 
                 
               
               
                 fadA 
                 acetyl coenzyme A acetyltransferase (EC: 2.3.1.9) 
                 
                   L. lactis 
                 
               
               
                 fadA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 fadA6 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 fadA3 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 fadA4 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 fadA2 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 fadA5 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   M. bovis 
                 
               
               
                 phaA 
                 beta-ketothiolase (EC: 2.3.1.16) 
                 
                   M. dichloromethanicum 
                 
               
               
                 Acat1 
                 acetyl-Coenzyme A acetyltransferase 1 (EC: 2.3.1.9) 
                 
                   M. musculus 
                 
               
               
                 Acat2 
                 acetyl-Coenzyme A acetyltransferase 2 (EC: 2.3.1.9) 
                 
                   M. musculus 
                 
               
               
                 atoB 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   P. aeruginosa 
                 
               
               
                 atoB 
                 acetyl-CoA acetyltransferase 
                 
                   P. putida 
                 
               
               
                 fadAx 
                 acetyl-CoA acetyltransferase 
                 
                   P. putida 
                 
               
               
                 ERG10 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   P. stipitis 
                 
               
               
                 fadA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   R. etli 
                 
               
               
                 phbAch 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   R. etli 
                 
               
               
                 phbAf 
                 acetyl-CoA acetyltransferase (beta-ketothiolase) protein 
                 
                   R. etli 
                 
               
               
                   
                 (EC: 2.3.1.9) 
               
               
                 phaA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   R. eutropha 
                 
               
               
                 bktB 
                 beta-ketothiolase (EC: 2.3.1.9) 
                 
                   R. eutropha 
                 
               
               
                 fadA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.16) 
                 
                   R. opacus 
                 
               
               
                 pcaF 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.174) 
                 
                   R. opacus 
                 
               
               
                 fadA5 
                 3-ketoacyl-CoA thiolase/acetyl-CoA acetyltransferase 
                 
                   S. avermitilis 
                 
               
               
                 fadA1 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   S. avermitilis 
                 
               
               
                 fadA7 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   S. avermitilis 
                 
               
               
                 fadA4 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   S. avermitilis 
                 
               
               
                 fadA2 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   S. avermitilis 
                 
               
               
                 fadA3 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   S. avermitilis 
                 
               
               
                 ERG10 
                 Acetyl-CoA C-acetyltransferase (acetoacetyl-CoA 
                 
                   S. cerevisiae 
                 
               
               
                   
                 thiolase), cytosolic enzyme that transfers an acetyl group 
               
               
                   
                 from one acetyl-CoA molecule to another, forming 
               
               
                   
                 acetoacetyl-CoA; involved in the first step in mevalonate 
               
               
                   
                 biosynthesis (EC: 2.3.1.9) 
               
               
                 atoB 
                 acetoacetyl-CoA thiolase 
                 
                   X. oryzae 
                 
               
               
                 yfcY 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   X. oryzae 
                 
               
               
                 fadA 
                 acetyl-CoA acetyltransferase (EC: 2.3.1.9) 
                 
                   X. oryzae 
                 
               
               
                 acat1 
                 acetyl-Coenzyme A acetyltransferase 1 (EC: 2.3.1.9) 
                 
                   X. tropicalis 
                 
               
               
                 acat2 
                 acetyl-Coenzyme A acetyltransferase 2 (EC: 2.3.1.9) 
                 
                   X. tropicalis 
                 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment of the invention, the acetyl-CoA acetyltransferase is encoded by the gene atoB from  E. coli.    
     Genes coding for enzymes having a 3-hydroxy-3-methylglutaryl-CoA synthase activity have been identified in several species, and in particular in  Enterococcus faecalis  (gene mvaS), and  Saccharomyces cerevisiae  (ERG13). 
     In a specific aspect of the invention, the enzyme having 3-hydroxy-3-methylglutaryl-CoA synthase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 8: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 MVA1 
                 MVA1; acetyl-CoA C-acetyltransferase/ 
                 
                   A. thaliana 
                 
               
               
                   
                 hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) 
               
               
                 pksG 
                 acetyl-S-AcpK beta-ketothioester polyketide intermediate 
                 
                   B. subtilis 
                 
               
               
                   
                 transferase 
               
               
                 HMGCS1 
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 
                 
                   B. taurus 
                 
               
               
                   
                 (soluble) 
               
               
                 HMGCS2 
                 3-hydroxy-3-methylglutaryl-CoA synthase 2 
                 
                   B. taurus 
                 
               
               
                   
                 (mitochondrial) (EC: 2.3.3.10) 
               
               
                 ERG13 
                 3-hydroxy-3-methylglutaryl coenzyme A synthase 
                 
                   C. albicans 
                 
               
               
                 HMGCS1 
                 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble) 
                 
                   E. caballus 
                 
               
               
                 mvaS 
                 3-hydroxy-3-methylglutaryl-CoA synthase 
                 
                   E. faecalis 
                 
               
               
                 HMGCS1 
                 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble) 
                 
                   H. sapiens 
                 
               
               
                   
                 (EC: 2.3.3.10) 
               
               
                 HMGCS2 
                 3-hydroxy-3-methylglutaryl-CoA synthase 2 
                 
                   H. sapiens 
                 
               
               
                   
                 (mitochondrial) (EC: 2.3.3.10) 
               
               
                 hmcM 
                 hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) 
                 
                   L. lactis 
                 
               
               
                 Hmgcs1 
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 2.3.3.10) 
               
               
                 Hmgcs2 
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 2.3.3.10) 
               
               
                 mvaS 
                 3-hydroxy-3-methylglutaryl coenzyme A synthase 
                 
                   S. aureus 
                 
               
               
                 ERG13 
                 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, 
                 
                   S. cerevisiae 
                 
               
               
                   
                 catalyzes the formation of HMG-CoA from acetyl-CoA and 
               
               
                   
                 acetoacetyl-CoA; involved in the second step in mevalonate 
               
               
                   
                 biosynthesis (EC: 2.3.3.10) 
               
               
                   
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 
                 
                   S. avermitilis 
                 
               
               
                 pksG 
                 hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) 
                 
                   S. erythraea 
                 
               
               
                 hmgcs1 
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 
                 
                   X. tropicalis 
                 
               
               
                   
                 (soluble) (EC: 2.3.3.10) 
               
               
                 pksG 
                 putative hydroxymethylglutaryl-coenzyme A synthase 
                 
                   Y. pestis 
                 
               
               
                   
               
            
           
         
       
     
     Preferentially the 3-hydroxy-3-methylglutaryl-CoA synthase is encoded by the gene mvaS from  Enterococcus faecalis.    
     The second reaction of the conversion of HMG-CoA into 3-methylcrotonyl-CoA is catalysed successively by two enzymes: (1) 3-methylglutaconyl-CoA hydratase, and (2) 3-methylglutaconyl-CoA decarboxylase. 
     Enzymes having a 3-methylglutaconyl-CoA hydratase activity have been identified in several species and in particular in  Galactomyces reessi  (See for reference: Dhar et al., 2002). 
     In a specific aspect of the invention, the enzyme having 3-methylglutaconyl-CoA hydratase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 9: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Gene name 
                 Enzyme name 
                 Organism 
               
               
                   
               
             
            
               
                 mgh 
                 3-methylglutaconyl-CoA hydratase (EC: 4.2.1.18) 
                 
                   A. baumannii 
                 
               
               
                 fadB1 
                 enoyl-CoA hydratase 
                 
                   A. radiobacter 
                 
               
               
                 paaG 
                 enoyl-CoA hydratase (EC: 4.2.1.17) 
                 
                   B. multivorans 
                 
               
               
                 AUH 
                 AU RNA binding protein/enoyl-CoA hydratase (EC: 4.2.1.18) 
                 
                   B. taurus 
                 
               
               
                 AUH 
                 AU RNA binding protein/enoyl-CoA hydratase 
                 
                   C. familiaris 
                 
               
               
                 paaF 
                 methylglutaconyl-CoA hydratase (EC: 4.2.1.18) 
                 
                   C. hutchinsonii 
                 
               
               
                 auh 
                 AU RNA binding protein/enoyl-Coenzyme A hydratase 
                 
                   D. rerio 
                 
               
               
                   
                 (EC: 4.2.1.18) 
               
               
                 mvaA 
                 enoyl-CoA hydratase (EC: 4.2.1.18) 
                 
                   D. shibae 
                 
               
               
                   
                 3-methylglutaconyl-CoA hydratase 
                 
                   G. reessi 
                 
               
               
                 AUH 
                 AU RNA binding protein/enoyl-CoA hydratase (EC: 4.2.1.18) 
                 
                   H. sapiens 
                 
               
               
                 caiD 
                 enoyl-CoA hydratase/carnithine racemase (EC: 4.2.1.17) 
                 
                   H. seropedicae 
                 
               
               
                 AUH 
                 AU RNA binding protein/enoyl-CoA hydratase 
                 
                   M. mulatta 
                 
               
               
                 Auh 
                 AU RNA binding protein/enoyl-coenzyme A hydratase 
                 
                   M. musculus 
                 
               
               
                   
                 (EC: 4.2.1.18) 
               
               
                 MXAN_3757 
                 methylglutaconyl-CoA hydratase 
                 
                   M. xanthus 
                 
               
               
                 liuC 
                 gamma-carboxygeranoyl-CoA hydratase 
                 
                   P. aeruginosa 
                 
               
               
                 gnyH 
                 gamma-carboxygeranoyl-CoA hydratase 
                 
                   P. aeruginosa 
                 
               
               
                 echA7 
                 gamma-carboxygeranoyl-CoA hydratase 
                 
                   P. fluorescens 
                 
               
               
                 AUH 
                 AU RNA binding protein/enoyl-CoA hydratase 
                 
                   P. troglodytes 
                 
               
               
                 menB 
                 naphthoate synthase (EC: 4.1.3.36) 
                 
                   R. capsulatus 
                 
               
               
                 Auh 
                 AU RNA binding protein/enoyl-coenzyme A hydratase 
                 
                   R. norvegicus 
                 
               
               
                   
                 (EC: 4.2.1.18) 
               
               
                 eccH2 
                 enoyl-CoA hydratase (EC: 4.2.1.17) 
                 
                   S. meliloti 
                 
               
               
                 fadB 
                 probable enoyl-CoA hydratase (EC: 4.2.1.17) 
                 
                   S. ruber 
                 
               
               
                 auh 
                 AU RNA binding protein/enoyl-Coenzyme A hydratase 
                 
                   X. laevis 
                 
               
               
                   
                 (EC: 4.2.1.18) 
               
               
                 auh 
                 AU RNA binding protein/enoyl-Coenzyme A hydratase 
                 
                   X. tropicalis 
                 
               
               
                   
                 (EC: 4.2.1.18) 
               
               
                   
               
            
           
         
       
     
     Preferentially the 3-methylglutaconyl-CoA hydratase is encoded by the gene MXAN_3757 from  Myxococcus xanthus.    
     At least one gene coding for an enzyme having a 3-methylglutaconyl-CoA decarboxylase activity has been identified in  Myxococcus xanthus  (See for reference: Bode et al., 2009). 
     Preferentially the 3-methylglutaconyl-CoA decarboxylase is encoded by the genes MXAN_4264 and MXAN_4265 from  Myxococcus xanthus , each of them coding for one subunit of the enzyme. 
     In a specific embodiment of the invention, in the recombinant microorganism, at least one of the following enzymes is overexpressed: an acetyl-CoA acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, and a 3-methylglutaconyl-CoA decarboxylase. 
     In another aspect of the invention, the microorganism is further modified to improve the availability of acetyl-CoA by:
         overexpressing at least one gene encoding the pyruvate dehydrogenase chosen among aceE, aceF or lpd. In a specific aspect of the invention, inactivation of pdhR coding for a repressor leads to an overexpression of aceE and aceF.   modifying the lpd gene so as to produce a mutant, feedback deregulated Lpd enzyme. Such mutations of lpd are disclosed in patent application WO2005073364.   attenuating at least one gene chosen among aldehyde/alcohol dehydrogenase (adhE), phosphate acetyltransferase (pta), acetate kinase (ackA) or citrate Synthase (gltA).       

     Preferentially, the microorganism of the invention is selected among  Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Corynebacteriaceae  and  Saccharomycetaceae . More preferentially the microorganism is a species of  Escherichia, Clostridium, Bacillus, Klebsiella, Pantoea, Salmonella, Pseudomonas, Corynebacterium  or  Saccharomyces.    
     According to a specific aspect of the invention, the microorganism is from the species  Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, Saccharomyces cerevisiae, Corynebacterium glutamicum  or  Bacillus subtilis.    
     An especially preferred simple carbon source is glucose. Another preferred simple carbon source is sucrose. 
     In some embodiments of the invention, the culture medium comprises a carbon source being a by-product of another process using biomass as starting material, or eventually, the product of mechanical and/or chemical and/or enzymatic, and in such instance in vitro or in vivo, degradation of biomass, such as degradation of cellulose. 
     According to a specific aspect of the invention, the fermentative production of prenol comprises a step of isolation of the prenol from the culture medium. Recovering the prenol from the culture medium is a routine task for a man skilled in the art. It may be achieved by a number of techniques well known in the art including but not limiting to distillation, gas-stripping, pervaporation or liquid extraction. The expert in the field knows how adapt parameters of each technic dependant of the characteristics of the material to be separated. 
     Distillation may involve an optional component different from the culture medium in order to facilitate the isolation of prenol by forming azeotrope and notably with water. This optional component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, trichloroethylene, octane, diethylether or a mixture thereof. 
     Gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen or mixture thereof. 
     Liquid extraction is achieved with organic solvent as the hydrophobe phase such as pentane, hexane, heptane, dodecane. 
     Conversion of Prenol into Isoprene: 
     In a specific embodiment, the invention is also related to a method for the production of isoprene from prenol. Said method comprises the following successive steps: 
     
         
         
           
             culturing a recombinant microorganism in a culture medium comprising a source of carbon, wherein in said microorganism, the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA as intermediate product, that is converted into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme, and 
             performing a step of chemical dehydratation of the bioproduced prenol into isoprene. 
           
         
       
    
     This conversion of “bioproduced prenol”, i.e. prenol produced from a simple source of carbon by fermentation, into isoprene can be achieved by means and methods known to the man skilled in the art. In particular, this conversion may be achieved by chemical way involving dehydrogenation and dehydration by acid catalyst (chemical dehydratation) such as disclosed in patent application US20100216958. 
     Another way of conversion is the enzymatic conversion of prenol by action of a prenol kinase, an isopentenyl-diphosphate delta isomerase and an isoprene synthase polypeptide such as disclosed in patent application WO2010031076. These three enzymes may be produced in a different strain than that producing prenol or in the same strain. If they are produced independently from the prenol production, the expressed enzymes or the strains expressing these enzymes can be directly mixed, partly or not, with the fermentation medium of the strain producing prenol or with the culture supernatant wherein prenol is accumulated. 
     In a specific aspect of the invention, the bioproduced prenol is purified before the step of chemical dehydration. 
     In another embodiment of the invention, isoprene is purified by standard methods well known in the art. For examples, isoprene can be recovered by gas stripping, extractive distillation with an alcohol such as ethanol, methanol, propanol, or a combination thereof, liquid extraction or solid separation (adsorption, desorption) or combination thereof. 
     The present invention is also related to a genetically modified microorganism for the fermentative production of prenol such as described above. Specifically, said microorganism overexpresses at least one enzyme chosen among the group consisting of: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase, an acetyl-CoA C-acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, and a 3-methylglutaconyl-CoA decarboxylase. In particular, the genetically modified microorganism comprises a heterologous enzyme AdhE, which has specificity for the substrate 3-methylcrotonyl-CoA. 
     In said genetically modified microorganism, endogenous sequences may also be knocked out or deleted, to favour the new metabolic pathway for producing prenol. 
     All techniques for transforming the microorganisms, and regulatory elements used for enhancing the production of prenol, are well known in the art and available in the literature, including the applicant&#39;s own patent applications on the modification of biosynthesis pathways in various microorganisms, including WO 2008/052973, WO 2008/052595, WO 2008/040387, WO 2007/144346, WO 2007/141316, WO 2007/077041, WO 2007/017710, WO 2006/082254, WO 2006/082252, WO 2005/111202, WO 2005/073364, WO 2005/047498, WO 2004/076659, the content of which is incorporated herein by reference. 
     EXAMPLES 
     The present invention is further defined in the following examples. It should be understood that these example, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the man skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modifying the essentials means of the invention. 
     In particular, examples show modified  Escherichia coli  ( E. coli ) strains, but these modifications can easily be performed in other microorganisms of the same family. 
       E. coli  belongs to the Enterobacteriaceae family, which comprises members that are Gram-negative, rod-shaped, non-spore forming and are typically 1-5 μm in length. Most members have flagella used to move about, but a few genera are non-motile. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants.  E. coli  is one of the most important model organisms, but other important members of the Enterobacteriaceae family include  Klebsiella , in particular  Klebsiella terrigena, Klebsiella planticola  or  Klebsiella oxytoca , and  Salmonella.    
     Protocoles 
     Several protocols are used to construct prenol producing strains and are described in the following examples. 
     Protocol 1: 
     Chromosomal modifications by homologous recombination and selection of recombinants (Datsenko and Wanner, 2000) 
     Allelic replacement or gene disruption in specified chromosomal loci is carried out by homologous recombination as described by Datsenko and Wanner (2000). The chloramphenicol (Cm) resistance cat or the kanamycin (Km) resistance kan flanked by Flp recognition sites, are amplified by PCR by using pKD3 or pKD4 plasmids as template respectively. The resulting PCR products are used to transform the recipient  E. coli  strain harbouring plasmid pKD46 that expresses the λ, Red (γ, β, exo) recombinase. Antibiotic-resistant transformants are then selected and the thermo-sensitive pKD46 plasmid is removed by cultivating the strain at 42° C. following by an isolation of the strain&#39;s culture on LB plates. Single clones are verified for the loss of ampicillin resistance and by PCR analysis with appropriate primers listed in Table 2. 
     The cat and kan-resistance genes are removed by using plasmid pCP20 as described by Datsenko &amp; Wanner (2000). Antibiotic sensitive clones are then verified by PCR using primers listed in Table 2. 
     Protocol 2: Transduction of Phage P1 
     Chromosomal modifications are transferred to a given  E. coli  recipient strain by P1 transduction. The protocol is composed of 2 steps: (i) preparation of the phage lysate on a donor strain containing the resistance associated chromosomal modification and (ii) infection of the recipient strain by this phage lysate. 
     Preparation of the Phage Lysate
         Inoculate 100 μl of an overnight culture of the strain MG1655 with the chromosomal modification of interest in 10 ml of LB+Cm 30 μg/ml or Km 50 μg/ml+glucose 0.2%+CaCl 2  5 mM.   Incubate 30 min at 37° C. with shaking.   Add 100 μl of P1 phage lysate prepared on the donor strain MG1655 (approx. 1×10 9  phage/ml).   Shake at 37° C. for 3 hours until the complete lysis of cells.   Add 200 μl of chloroform, and vortex.   Centrifuge 10 min at 4500 g to eliminate cell debris.   Transfer of supernatant to a sterile tube.   Store the lysate at 4° C.       

     Transduction
         Centrifuge 10 min at 1500 g 5 ml of an overnight culture of the  E. coli  recipient strain cultivated in LB medium.   Suspend the cell pellet in 2.5 ml of MgSO 4  10 mM, CaCl 2  5 mM.   Infect 100 μl cells with 100 μl P1 phage of strain MG1655 with the modification on the chromosome (test tube) and as a control tubes 100 μl cells without P1 phage and 100 μl P1 phage without cells.   Incubate 30 min at 30° C. without shaking.   Add 100 μl sodium citrate 1 M in each tube, and vortex.   Add 1 ml of LB.   Incubate 1 hour at 37° C. with shaking.   Centrifuge 3 min at 7000 rpm.   Plate on LB+Cm 30 μg/ml or Km 50 μg/ml.       

     Incubate at 37° C. overnight. 
     The antibiotic-resistant transductants are then selected and the chromosomal structure of the mutated locus was verified by PCR analysis with appropriates primers listed in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Describe the genotype and corresponding number of intermediate 
               
               
                 strains and producing strains that appear in the following examples. 
               
            
           
           
               
               
            
               
                 Strain 
                   
               
               
                 number 
                 Genotype 
               
               
                   
               
               
                 1 
                 DH5α (pNSTLL-factorXA-adhE2ca) 
               
               
                 2 
                 MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20- 
               
               
                   
                 22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) 
               
               
                   
                 (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq- 
               
               
                   
                 lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07- 
               
               
                   
                 Ptrc30/RBS01-adhE2ca-TT02) 
               
               
                 3 
                 MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) 
               
               
                 4 
                 MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/ 
               
               
                   
                 OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) 
               
               
                   
                 (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)- 
               
               
                   
                 TT07) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Primers used for PCR verifications of chromosomal modifications 
               
               
                 described in the following examples 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Location of the  
                   
               
               
                 Genes 
                   
                 SEQ 
                 homology with the 
                   
               
               
                 name 
                 Primers name 
                 ID N o   
                 chromosomal region 
                 Sequences 
               
               
                   
               
               
                 atoB 
                 atoB F 
                 17 
                 2323586-2323603 
                 CCATCAATACCGTCAACC 
               
               
                   
                 atoB R 
                 18 
                 2324536-2324554 
                 CATAAACCTGTCCGTCTCC 
               
               
                   
               
               
                 pdhR 
                 pdhR F 
                 21 
                 121197-121217 
                 GAATGTATTTACCCACGGCAG 
               
               
                   
                 pdhR R 
                 22 
                 123095-123114 
                 GCACGCTCAACACCTTCTTC 
               
               
                   
               
               
                 lpd* 
                 Lpd*(A55V) ver F 
                 25 
                 127731-127750 
                 GGTGGTGGTTAGGGTATTAC 
               
               
                 (A55V) 
                 Lpd*(A55V) cer FR 
                 26 
                 129415-129433 
                 CGTGGAGCAAGAAGACTGG 
               
               
                   
               
            
           
         
       
     
     Example 1 
     Calculation of Maximum Yields for Prenol Production on Glucose and Sucrose 
     1.1—Parameters Used for Simulations 
     Simulations were performed with the METEX proprietary software METOPT™. A simplified metabolic network of  E. coli  was used including a central metabolic network, metabolic pathways for all biomass precursors and specific production pathways as described in  FIG. 1  for the leucine pathway and  FIG. 2  for the HMG-CoA pathway. A classical biomass composition for  E. coli  was used. Simulations were performed using either glucose or sucrose carbon source. For sucrose utilization, both the PTS system and the non-PTS system were modelled. As there were no differences on maximal yields calculated, only one yield on sucrose is reported. Maximum prenol yields were calculated from the “leucine pathway” and the “HMG-CoA pathway”. Calculation of a theoretical maximum yield was performed, taking into account no growth and no maintenance. Calculation of a practical maximum yield was performed, taking into account a growth rate of 0.11 h −1  and maintenance energy of 5 mmol ATP ·g DW   −1 ·h −1 . All simulations were performed with a specific uptake rate of glucose of 3 mmol·g DW   −1 ·h −1 , or a specific uptake rate of sucrose of 1.5 mmol·g DW   −1 ·h −1 . Simulations were performed under aerobic conditions. 
     1.2—Simulation Results 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Prenol on 
                   
                   
                   
               
               
                   
                 glucose 
                 Prenol on 
                 Prenol on 
                 Prenol on 
               
               
                   
                 by the 
                 glucose by the 
                 sucrose by the 
                 sucrose by the 
               
               
                   
                 “leucine 
                 “HMG-CoA 
                 “leucine 
                 “HMG-CoA 
               
               
                   
                 pathway” 
                 pathway” 
                 pathway” 
                 pathway” 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Maximum 
                 0.32 
                 0.32 
                 0.34 
                 0.34 
               
               
                 theoretical 
               
               
                 yield (g/g) 
               
               
                 Maximum 
                 0.24 
                 0.24 
                 0.26 
                 0.26 
               
               
                 practical 
               
               
                 yield (g/g) 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Demonstration of the 3-Methyl-2-Butenal Dehydrogenase and 3-Methyl-2-Butenol Dehydrogenase Activities Encoded by the Gene adhE2 of  Clostridium acetobutylicum    
     2.1—Construction of Strain DH5α (pNSTLL-Factor XA-adhE2ca) 
     pNSTLL-factor XA-adhE2ca plasmid was derived from pSO595 (genbank accession No AY187686) and RBS-Strep-Tag-Linker-factor XA-adhE2ca fragment with the adhE2 gene from  Clostridium acetobutylicum  ATCC-824 coding for the aldehyde/alcohol dehydrogenase. 
     In this plasmid, expression of the adhE2ca gene is driven by the promoter of the thiolase gene from  Clostridium acetobutylicum . The RBS-Strep-Tag-Linker-factor XA-adhE2ca fragment was amplified by PCR with primers ST_LL_XA_adhE2ca F (SEQ ID No01) and adhE2ca R (SEQ ID No02) using  Clostridium acetobutylicum  genomic DNA. The PCR product was digested and cloned between the BamHI and SfoI sites of the pSO595. The ligation product was introduced in DH5α strain, the resulting strain DH5α (pNSTLL-factor XA-adhE2ca) is called strain 1 (table 1). The resulting plasmid was verified by DNA sequencing and called: pNSTLL-factor XA-adhE2ca. 
     
       
         
           
               
            
               
                 ST_LL_XA_adhE2ca F 
               
               
                 (SEQ ID No 1) 
               
               
                 
                   TAGGATCC 
                   
                     atcaaaatttaggaggttagttagaatg 
                     tggtcacatcct 
                   
                 
               
               
                   
               
               
                     caatttgaaaaa ggtagtggtggtggtagtggtggtggtagt CCCGGG 
               
               
                   
               
               
                 
                   atcgaagggcgc 
                   atgaaagttacaaatcaaaaag 
                 
               
            
           
         
       
         
         
           
             sequence (bold upper case) for BamHI restriction site and extrabases 
             sequence (underlined italic lower case) corresponding to RBS sequence 
             sequence (underlined bold lower case) corresponding to Strep-tag sequence (Strep-Tag® II, IBA-GmbH) 
             sequence (underlined lower case) corresponding to linker sequence (artificial sequence) 
             sequence (upper case) for SmaI restriction site 
             sequence (italic lower case) corresponding to factor XA sequence (Nagai and Thorgersen, 1984) 
             sequence (bold lower case) homologous to the adhE gene of  Clostridium acetobutylicum  (36298-36277 (pSOL1), reference sequence on the NCBI GenBank) 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 adhE2ca R 
               
               
                   
                 (SEQ ID No 2) 
               
               
                   
                 
                   TAAGTGGCGCC 
                   TTAAAATGATTTTATATAGATATCC 
                 
               
            
           
         
       
         
         
           
             sequence (bold upper case) for SfoI restriction site and extrabases, 
             sequence (italic upper case) homologous to the adhE gene of  Clostridium acetobutylicum  (33722-33746 (pSOL1), reference sequence on the NCBI GenBank) 
           
         
       
    
     2.2—Overproduction of the Protein AdhE2ca Protein production was realised in a 1 L Schott bottle. The production strain was inoculated in fifteen precultures of 5 mL LB medium (Sigma 25%) with 2.5 g·L −1  glucose. The temperature was maintained at 37° C. and agitation at 200 RPM. 
     Theses precultures were used to inoculate an 800 mL culture of MAC medium to an OD 600 nm  of 0.3. The temperature of the culture was maintained at 37° C. and the agitation at 150 RPM. Ampicilin was added at concentration of 50 mg·L −1  in preculture and culture. When the culture had reached an OD 600  of 0.8 (approximately 7 hours), the culture was centrifuged and the cell pellet conserved. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 MAC Medium composition. 
               
            
           
           
               
               
               
            
               
                   
                 Compound 
                 Concentration (g · L −1 ) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 Glycerol 
                 20.000 
               
               
                   
                 Tryptone 
                 10.000 
               
               
                   
                 NaCl 
                 5.000 
               
               
                   
                 NaNO 3   
                 0.085 
               
               
                   
                 Yeast extract 
                 5.000 
               
               
                   
                 K 2 HPO 4   
                 0.500 
               
               
                   
                 FeSO 4 •7H 2 O 
                 0.050 
               
               
                   
                 HEPES 
                 23.000 
               
               
                   
                 Nitrilotriacetic acid 
                 0.200 
               
               
                   
                 H 2 SO 4  (96%) 
                 Adjusted to pH 7.3 
               
               
                   
                   
               
            
           
         
       
     
     2.3—Purification of the Protein adhE2 
     All the purification steps were performed under anaerobic conditions. 
     Step 1: Preparation of Cell-Free Extracts 
     About 200 mg of  E. coli  biomass was suspended in 30 ml of 100 mM Tris HCl, 150 mM NaCl, 1 mM EDTA pH 8 and a protease inhibitor cocktail. The cell suspension was sonicated on ice (Sonics and Materials, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with 30 sec intervals. After sonication, cell debris was removed by centrifugation at 12000 g for 30 min at 4° C. The crude extract was incubated with 0.16 g/L Avidin during 30 min at 4° C. The crude extract was centrifuged at 12000 g for 5 min and filtered through a 0.45 μm filter. 
     Step 2: Affinity Purification 
     After Avidin treatment, the crude extract was loaded on a 1 ml StrepTrap HP column (GE Healthcare) equilibrated with 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH8. The column was washed with 10 column volumes of the same buffer. The protein was eluted from the column with 6 column volumes of 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2.5 mM Desthiobiotin pH8. The fractions containing the protein were pooled. For the storage of the protein; the buffer was exchanged using a desalting column (Econo-Pac, Bio-Rad) against 100 mM Hepes pH7.5. 
     2.4—3-Methyl-2-Butenal Dehydrogenase Assay 
     3-Methyl-2-butenal dehydrogenase activity was assayed by measuring the initial rate of NADH oxidation under anaerobic conditions with a spectrophotometer at a wavelength of 340 nm and a constant temperature of 30° C. The reaction mixture using 2.5 mM 3-methyl-crotonyl-coA as substrate was carried out in 200 mM HEPES, 144 mM Semicarbazine buffer pH 7.5, 2 mM DTT, 0.2 mM NADH, and about 8 μg of purified enzyme (adhE2) in a final volume of 1 ml. Control assay (blank), lacking the substrate was run in parallel, and the value measured for the control is subtracted to the value measured for the assay in order to take into account the non-specific oxidation of NADH (Epsilon 340 nm=6290 M−1 cm-1). 
     One unit of enzyme activity was defined as the amount of enzyme that consumed 1 μmol substrate per mM under the conditions of the assay. Specific enzyme activity was expressed as units per mg of protein. 
     2.5—3-Methyl-2-Butenol Dehydrogenase Assay 
     3-Methyl-2-butenol dehydrogenase activity was assayed by measuring the initial rate of NADH oxidation under anaerobic conditions with a spectrophotometer at a wavelength of 340 nm and a constant temperature of 30° C. The reaction mixture using 5 mM 3-Methyl-2-butenal as substrate was carried out in 100 mM HEPES buffer pH 7.5, 2 mM DTT, 0.2 mM NADH, and about 12 μg of purified enzyme (adhE2) in a final volume of 1 ml. Control assay (blank), lacking the substrate was run in parallel and the value measured for the control is subtracted to the value measured for the assay in order to take into account non-specific oxidation of NADH (Epsilon 340 nm=6290 M−1 cm-1). 
     One unit of enzyme activity was defined as the amount of enzyme that consumed 1 μmol substrate per min under the conditions of the assay. Specific enzyme activity was expressed as units per mg of protein. 
     2.6—Activity of Purified Enzyme AdhE2 
     
       
         
           
               
               
             
               
                   
                   
               
               
                   
                 Activity of purified 
               
               
                   
                 enzyme (mUI/mg) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 3-Methyl-2-butenal dehydrogenase assay 
                 335 
               
               
                   
                 3-Methyl-2-butenol dehydrogenase assay 
                 56 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
     Construction of Strain 2 MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02) 
     3.1—Construction of pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 Plasmid 
     3.1.1—Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 
     Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 is derived from plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C described in patent applications EP11306306.9 and U.S. 61/544,748 to which the ilvD gene from  Escherichia coli  coding for the dihydroxy-acid dehydratase is added. 
     In this plasmid, expression of the ilvD gene is driven by a constitutive Ptrc promoter, the ilvD gene is expressed in the operon with ilvBN*(GVM20-22DDF)C and a transcriptional terminator is added downstream of the gene. The ilvD gene is amplified by PCR with primers ilvD F (SEQ ID No03) and ilvD R (SEQ ID No04) using pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C-PilvE-ilvED-TT07 described in patent applications EP11306306.9 and US61/544,748. The PCR product is digested and cloned between the BamHI and NheI sites of the pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C described in patent applications EP11306306.9 and US61/544,748. The resulting plasmid is verified by DNA sequencing and called: pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07. 
     
       
         
           
               
               
            
               
                   
                 ilvD F 
               
               
                   
                 (SEQ ID No 3) 
               
               
                   
                 TACTGGCTAGC atacaaaaaatgggacggc   
               
            
           
         
       
     
     with
         sequence (upper case) for NheI restriction site and extrabases   sequence (bold lower case) homologous to the region upstream of ilvD gene (3951437-3951455, reference sequence in the MySql relational database (EcoGene.org))       

     
       
         
           
               
               
            
               
                   
                 ilvD R 
               
               
                   
                 (SEQ ID No 4) 
               
               
                   
                 agcaaggatcc GCAGAAAGGCCCACCCGAAGG   
               
            
           
         
       
     
     with
         sequence (lower case) for BamHI restriction site and extrabases   sequence (bold upper case) for T7Te transcriptional terminator sequence from T7 phage (Harrington et al., 2001).       

     3.1.2—Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 
     Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 is derived from pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 described above and the synthetic gene acdH from  Streptomyces avermitilis  optimized for  Escherichia coli  described below. 
     Synthetic Gene acdHsaO1ec 
     A synthetic gene of the  Streptomyces avermitilis  acdH gene coding for the acyl-CoA dehydrogenase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to  Escherichia coli  according to the supplier&#39;s matrix. The construct is cloned into supplier&#39;s pM vectors and verified by sequencing. 
     acdH gene sequence from  Streptomyces avermitilis  (AF143210) optimized for  Escherichia coli : acdHsaO1ec contains the following sequence (SEQ ID No35): 
     
       
         
           
               
            
               
                 atggatcatcgtctgacaccggaactggaagaactgcgtcgtaccgttga 
               
               
                   
               
               
                 agaatttgcacatgatgagagcaccgaaaatcggcgatttctatgaacgt 
               
               
                   
               
               
                 catgaattcccgtatgaaattgtgcgtgaaatgggtcgtatgggtctgtt 
               
               
                   
               
               
                 tggtctgccgtttccggaagaatatggtggtatgggtggtgattatctgg 
               
               
                   
               
               
                 cactgggtattgccctggaagaactggcacgtgagatagcagcgagcaat 
               
               
                   
               
               
                 taccctggaagccggtgttagcctgggtgcaatgccgattcacctgtttg 
               
               
                   
               
               
                 gcaccgatgcacagaaagcagaatggctgcctcgtctgtgtagcggtgaa 
               
               
                   
               
               
                 attctgggtgcataggtctgaccgaaccggatggtggtagtgatgccggt 
               
               
                   
               
               
                 gcaacccgtaccaccgcacgtctggatgaaagcaccaatgaatgggttat 
               
               
                   
               
               
                 taatggcaccaaatgcttcattaccaatagcggcaccgatatcaccggtc 
               
               
                   
               
               
                 tggttaccgttaccgcagttaccggtcgtaaacctgatggtaaaccgctg 
               
               
                   
               
               
                 attagcagcattattgaccgagcggtacaccgggattaccgagcagcacc 
               
               
                   
               
               
                 gtatagcaaagaggaggaatgcaagcgatacccgtgaactgagctagcag 
               
               
                   
               
               
                 atgacgtgaccggcagcaaatctgctgggtgaacagggtcgtggttatgc 
               
               
                   
               
               
                 acagtttctgcgtatcctggatgaaggtcgtattgcaattagcgcactgg 
               
               
                   
               
               
                 caacaggtctggcacagggttgtgagatgaaagcgttaaatatgcaggcg 
               
               
                   
               
               
                 aacgccatgcctaggtcgtaatattggtgcatatcaggcaatccagataa 
               
               
                   
               
               
                 aatcgcagatatggaaatgaaagcccatatggcacgcgaggaggcgtgat 
               
               
                   
               
               
                 gcagcaagccgtctggagccggtgaaccgttcaaaaaagaagcagcaatt 
               
               
                   
               
               
                 gcaaaactgtatagcagtaccgagccgttgataatgcacgtgaagcaacc 
               
               
                   
               
               
                 cagattcatggtggttatggattatgaatgaatatccggagcacgtatgt 
               
               
                   
               
               
                 ggcgtgatagcaaaattctggaaattggtgaaggcaccagcgaagttcag 
               
               
                   
               
               
                 cgtatgctgattgcacgcgaactgggtctggtgggttaa 
               
            
           
         
       
     
     Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-IT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 
     In this plasmid, expression of the synthetic gene is driven by a constitutive Ptrc promoter and a transcriptional terminator is added downstream of the acdHsaO1ec synthetic gene. The acdHasaO1ec synthetic gene is amplified by PCR with primers Ptrc01-acdHsaO1ec F (SEQ ID No05) and Ptrc01-acdHsaO1ec R (SEQ ID No06) using the pM vector harbouring the acdHsaO1ec synthetic gene provided by the supplier. The PCR product is digested and cloned between the BamHI and XbaI sites of the plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 described above. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02. 
     
       
         
           
               
            
               
                 Ptrc01-acdHsaO1ec F 
               
               
                 (SEQ ID No 5) 
               
               
                 TTCTGCGGATCC gagctgttgacaattaatcatccggctcgtataatgt   
               
               
                   
               
               
                 
                   gtggaa 
                   GTCGACGTTAACCCTAGG 
                   taaggaggttataa 
                   atggatcatcg 
                 
               
               
                   
               
               
                 
                   tctgacaccgg 
                 
               
            
           
         
       
     
     with
         sequence (upper case) for BamHI restriction site and extrabases,   sequence (bold lower case) for the trc promoter sequence (Amann et al., 1983 and Amann et al., 1988),   sequence (italic upper case) for SalI, HpaI and AvrII restriction sites,   sequence (underlined lower case) corresponding to RBS consensus sequence with a PsiI restriction site,   sequence (italic lower case) homologous to the beginning of acdHsaO1ec synthetic gene sequence,       

     
       
         
           
               
            
               
                 Ptrc01-acdHsaO1ec R 
               
               
                 (SEQ ID No 6) 
               
               
                 ggtcgactctaga AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAG   
               
               
                   
               
               
                 
                   CCTTTCGTTTTATTTGATG 
                   agatct 
                   TTAACCCACCAGACCCAGTTCGCG 
                 
               
            
           
         
       
     
     with
         sequence (lower case) for the XbaI restriction site and extrabases,   sequence (bold upper case) for T 1  transcriptional terminator sequence from the  Escherichia coli  rrnB gene (Orosz et al., 1991),   sequence (underlined lower case) for BglII restriction site,   sequence (italic upper case) homologous to the end of the acdHsaO1ec synthetic gene sequence.       

     3.2—Construction of Plasmid pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07 
     Plasmid pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07 is derived from pBBR1MCS5 (Kovach et al., 1995) and the bkdA1-bkdA2-bkdB-lpdV operon from  Pseudomonas putida  ATCC-23287 coding for the branched-chain keto-acid dehydrogenase complex. 
     In this plasmid, expression of the bkdA1-bkdA2-bkdB-lpdV operon is driven by a constitutive Ptrc promoter and a transcriptional terminator is added downstream of the operon. The bkdA1-bkdA2-bkdB-lpdV operon is amplified by PCR with primers RBSbkdA1 F (SEQ ID No07) and lpdV-TT07 XhoI R (SEQ ID No08) using  Pseudomoans putida  ATCC-23287 genomic DNA. The PCR product is digested and cloned between the XbaI and XhoI sites of the pBBR1MCS5. The resulting plasmid is verified by DNA sequencing and called: pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07. 
     
       
         
           
               
            
               
                 RBSbkdA1 F 
               
               
                 (SEQ ID No 7) 
               
               
                 GCCGCTCTAGAACTAGT gagctgttgacaattaatcatccggctcgtat   
               
               
                   
               
               
                   aatgtgtggaa gtcgac GTTAAC   caaatacccgagcgagcg   
               
            
           
         
       
     
     with
         sequence (upper case) for XbaI and SpeI restriction sites and extrabases,   sequence (italic lower case) for the trc promoter sequence (Amann et al., 1983 and Amann et al., 1988),   sequence (italic upper case) for the HpaI restriction site,   sequence (underlined lower case) homologous to the region upstream of the bkdA1 gene.       

     
       
         
           
               
            
               
                 lpdV-TT07 XhoI R 
               
               
                 (SEQ ID No 8) 
               
               
                 taccgggcccctcgag GCAGAAAGGCCCACCCGAAGGTGAGCCAG   TCAGA   
               
               
                   
               
               
                 
                   TATGCAGGGCGTGGCCC 
                 
               
            
           
         
       
     
     with
         sequence (lower case) for ApaI and XhoI restriction sites and extrabases,   sequence (bold upper case) for T7Te transcriptional terminator sequence from T7 phage (Harrington et al., 2011),   sequence (underlined upper case) homologous to the end of lpdV gene.       

     3.3—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02 
     3.3.1—Construction of Plasmid pSCB-RBS28-leuABCD-TT07-Ptrc30 
     Plasmid pSCB-RBS28-leuABCD-TT07-Ptrc30 is derived from pSCB (Agilent) and the leuA-leuB-leuC-leuD operon from  Escherichia coli  coding for 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase and two 3-isopropylmalate dehydratase respectively. 
     The leuA-leuB-leuC-leuD operon is amplified by PCR with primers RBS28-leuA F (SEQ ID No09) and Ptrc30-TT07-leuD R (SEQ ID No10) using  E. coli  MG1655 genomic DNA. The PCR product is cloned in the pSCB (Agilent). The resulting plasmid is verified by DNA sequencing and called pSCB-RBS28-leuABCD-TT07-Ptrc30. 
     
       
         
           
               
            
               
                 RBS28-leuA F 
               
               
                 (SEQ ID No 09) 
               
               
                 TAACAATTTACGTAGCTCAGCCGGCACTAGTGAATTC attaaagaggaga   
               
               
                   
               
               
                 
                   aaGGTACC 
                   atgagccagcaagtcattattttcg 
                 
               
            
           
         
       
     
     with
         sequence (upper case) for SnaBI, BlpI, SpeI and EcoRI restriction sites and extrabases,   sequence (bold lower case) corresponding to RBS sequence of the pZE12-luc (Lutz et al., 1997),   sequence (bold upper case) for KpnI restriction site,   sequence (underlined lower case) homologous to the beginning of leuA gene (83529-83505, reference sequence in the MySql relational database (EcoGene.org))       

     
       
         
           
               
            
               
                 Ptrc30-TT07-leuD R 
               
               
                 (SEQ ID No 10) 
               
               
                 tccttatacgta TTCCACACAGTATACGAGCCGGATGATTAATCGTCAAC   
               
               
                   
               
               
                 
                   AGCTCgggccc 
                   GCAGAAAGGCCCACCCGAAGGTGAGCCAGgtcgac 
                   TTAA 
                 
               
               
                   
               
               
                 
                   TTCATAAACGCAGGTTGTTTTGC 
                 
               
            
           
         
       
     
     with
         sequence (lower case) for SnaBI restriction site and extrabases,   sequence (bold upper case) corresponding to modified trc promoter sequence (Amann et al., 1983 and Amann et al., 1988),   sequence (bold lower case) for ApaI restriction site,   sequence (upper case) for T7Te transcriptional terminator sequence from T7 phage (Harrington et al., 2011),   sequence (underlined lower case) for Sail restriction site,   sequence (italic upper case) homologous to the end of leuD gene (78848-78874, reference sequence in the MySql relational database (EcoGene.org))       

     3.3.2—Construction of Plasmid pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 
     Plasmid pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 is obtained from an oligonucleotide-directed mutagenesis on the pSCB-RBS28-leuABCD-TT07-Ptrc30 with primers leuA*(G462D) F (SEQ ID No11) and leuA*(G462D) R (SEQ ID No12) using pSCB-RBS28-leuABCD-TT07-Ptrc30 as template. The PCR product is digested with DpnI and transformed in competent cell. The resulting plasmid is verified by DNA sequencing and called pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30. 
     The mutant leuA described above confer leucine resistance in  E. coli   
     
       
         
           
               
            
               
                 leuA*(G462D) F 
               
               
                 (SEQ ID No 11) 
               
               
                 ggccacggtaaagatgcgcttgA t caggtggatatcgtcgctaactac 
               
            
           
         
       
     
     with
         base modification (upper case) to introduce the amino acid substitution,   base modification (bold lower case) to introduce a BclI restriction site.       

     
       
         
           
               
            
               
                 leuA*(G462D) R 
               
               
                 (SEQ ID No 12) 
               
               
                 GTAGTTAGCGACGATATCCACCTG A tCAAGCGCATCTTTACCGTGGCC 
               
            
           
         
       
     
     with
         base modification (lower case) to introduce the amino acid substitution,   base modification (bold upper case) to introduce a BclI restriction site.       

     3.3.3—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02 
     Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02 is derived from pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703 and the PlacIq-lacI from pTRC99A (Amersham). 
     In this plasmid, expression of the lacI gene is driven by its natural promoter and a transcriptional terminator is added downstream of the gene. The PlacIq-lacI is amplified by PCR with primers PlacIq F (SEQ ID No13) and lad R (SEQ ID No14) using pTRC99A (Amersham). The PCR product is digested and cloned between the BamHI and SacI sites of the pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703. The resulting plasmid is verified by DNA sequencing and called: pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02. 
     PlacIq F (SEQ ID No13) 
     tcgggcccggatcccatttacgttgacaccatcgaatgg 
     with
         sequence (lower case) for ApaI and BamHI restriction sites and extrabases,   sequence (underlined bold lower case) for the lacIq promoter sequence       

     
       
         
           
               
            
               
                 lacI R 
               
               
                 (SEQ ID No 14) 
               
               
                 ACTTAAGGAGCTC   AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGA     
               
               
                   
               
               
                     GCCTTTCGTTTTATTTGATG   TACG TCACTGCCCGCTTTCCAGTCGGG   
               
            
           
         
       
     
     with
         sequence (upper case) for Sad restriction site and extrabases,   sequence (underlined bold upper case) for T 1  transcriptional terminator sequence from the  Escherichia coli  rrnB gene (Orosz et al., 1991),   sequence (italic upper case) homologous to the end of the lad gene.       

     3.3.4—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02 
     Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)-TT07-Ptrc30/RBS01-adhE2ca-TT02 is derived from pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 and pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02 described above. 
     In this plasmid, expression of the leucine operon is driven by an IPTG-inductible Ptrc promoter and expression of the adhE2ca gene is driven by a constitutive Ptrc promoter. A transcriptional terminator is added downstream the leucine operon. The pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 is digested and cloned in the SnaBI site of the pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02. The resulting plasmid is verified by DNA sequencing and called: pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02. 
     3.4—Construction of the Strain 2: MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02) 
     Construction of a strain with increased prenol pathway flux express the ilvBN*(GMV20-22DDF)CD operon to produce 3-methyl-2-oxobutanoic acid, the leuA*(G462D)BCD operon to produce 4-methyl-2-oxopentanoate, the bkdA12B+lpdV operon from  Pseudomonas putida  to produce 3-methylbutanoyl-CoA, the optimized acdH gene from  Streptomyces avermitilis  to produce 3-methylcrotonyl-CoA and the adhE2ca gene from  Clostridium acetobutylicum  to produce prenol. 
     The pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02, pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07 and pUC19-PlacIq-lacI-TT02-Ptre01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02 plasmids are introduced by electroporation into the MG1655 strain. The presence of the three plasmids is verified and the resulting strain MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02) is called strain 2 (Table 1). 
     Example 4 
     Construction of Strain 4: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07) 
     4.1—Construction of the Strain 3: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) 
     4.1.1—Construction of Strain MG1655 Ptrc30-atoB:Cm 
     To increase the expression level of acetyl-CoA acetyltransferase atoB, a constitutive artificial trc promoter is added upstream atoB gene into the strain MG1655 pKD46 according to Protocol 1, except that primers Ptrc01/OP01-atoB F (SEQ ID No15) and Ptrc30-atoB R (SEQ ID No16) are used to amplify the chloramphenicol resistance cassette from pKD3 plasmid. 
     Chloramphenicol resistant recombinants are selected. The presence of the artificial promoter Ptrc30 and the insertion of the resistance cassette are verified by PCR with primers atoB F (SEQ ID No17) and atoB R (SEQ ID No18) (Table 2) and by DNA sequencing. The resulting strain is called MG1655 Ptrc30-atoB:Cm. 
     
       
         
           
               
            
               
                 Ptrc01/OP01-atoB F 
               
               
                 (SEQ ID No 15) 
               
               
                 GCATCACTGCCCTGCTCTTCTCCGGTGTCATTTTCGTCATTGGTTTAA 
               
               
                   
               
               
                 CGCTGTTCTGACGGCACCCCTACAAACAGAAGGAATATAAA CTGGCTC   
               
               
                   
               
               
                 
                   ACCTTCGGGTGGGCCTTTCTGC 
                   TGTAGGCTGGAGCTGCTTC 
                 
               
            
           
         
       
     
     with
         sequence (upper case) homologous to sequence upstream of the atoB gene (2324042-2324130 reference sequence in the MySql relational database (EcoGene.org))   sequence (underlined upper case) for T7Te transcriptional terminator sequence from phage T7 (Harrington et al., 2001)   sequence (italic upper case) corresponding to the primer site 2 of plasmid pKD3 (Datsenko and Wanner, 2000)       

     
       
         
           
               
            
               
                 Ptrc30-atoB R 
               
               
                 (SEQ ID No 16) 
               
               
                 
                   CCGATAGCAGTACGTACCGCACTGACGATGACACAATTTTTCATT 
                   TAT 
                 
               
               
                   
               
               
                 
                   AACCTCCTTA 
                   TTCCACACAGTATACGAGCCGGATGATTAATCGTCAAC 
                 
               
               
                   
               
               
                   AGCTC CATGGTCcatatgaatatcctccttag 
               
            
           
         
       
     
     with
         sequence (bold upper case) homologous to sequence of the atoB gene (2324131-2324174, reference sequence in the MySql relational database (EcoGene.org))   sequence (italic upper case) corresponding to RBS consensus sequence with a PsiI restriction site,   sequence (underlined upper case) for the trc promoter sequence (Amann et al., 1983 and Amann et al., 1988),   sequence (lower case) corresponding to the primer site 1 of plasmid pKD3 (Datsenko and Wanner, 2000).       

     4.1.2—Construction of Strain MG1655 ΔpdhR:Km 
     To delete the pdhR gene, which encodes a pyruvate dehydrogenase operon repressor, into the strain MG1655 pKD46, Protocol 1 is used except that primers DpdhR F (SEQ ID No19) and DpdhR R (SEQ ID No20) are used to amplify the kanamycin resistance cassette from pKD4 plasmid. 
     
       
         
           
               
            
               
                 DpdhR R 
               
               
                 (SEQ ID No 20) 
               
               
                 gaccaattgacttcggcaagtggcttaagacaggaactcatg attc   
               
               
                   
               
               
                 
                   cggggatccgtcgacctgcagttcgaagttcctattctcta 
                   gaaag 
                 
               
               
                   
               
               
                 
                   tataggaacttcttcaagatcccctcacgctgccgc 
                 
               
            
           
         
       
     
     with
         sequence (lower case) homologous to sequence of pdhR gene (122053-122094, reference sequence in the MySql relational database (EcoGene.org)),   sequence (italic lower case) corresponding to the upstream region of pKD13-Km gene (Datsenko and Wanner, 2000),   sequence (bold lower case) corresponding to the FRT site and downstream region of pKD4 plasmid (Datsenko and Wanner, 2000),       

     
       
         
           
               
            
               
                 DpdhR F 
               
               
                 (SEQ ID N o  19) 
               
               
                 CATCTTCTGGATAATTTTTACCAGAAAAATCACTAATTCTTTCGTTGCT 
               
               
                   
               
               
                 CCAG TGTAGGCTGGAGCTGCTTC   GAAGTTCCTATACTTTCTAGAGAATA   
               
               
                   
               
               
                 
                   GGAACTTCAGAGCGCTTTTGAAGCTGGGG 
                 
               
            
           
         
       
         
         
           
             sequence (upper case) homologous to sequence of pdhR gene (122839-122888, reference sequence in the MySql relational database (EcoGene.org)), 
             sequence (italic upper case) corresponding to the downstream region of pKD13-Km gene (Datsenko and Wanner, 2000), 
             sequence (bold upper case) corresponding to the FRT site and upstream region of pKD4 plasmid (Datsenko and Wanner, 2000). 
           
         
       
    
     Kanamycin resistant recombinants are selected. The insertion of the resistance cassette is then verified by PCR with primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) (Table 2) and by DNA sequencing. The verified and selected strain is called MG1655 ΔpdhR:Km. 
     4.1.3—Construction of Strain MG1655 Ptrc30-atoB ΔpdhR:Km 
     The ΔpdhR:Km chromosomal modification is transduced into the strain MG1655 Ptrc30-atoB:Cm with a P1 phage lysate from strain MG1655 ΔpdhR:Km described above, according to Protocol 2. 
     Kanamycin resistant transductants are selected and the presence of ΔpdhR:Km chromosomal modification was verified by PCR with primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) (Table 2). The resulting strain is called MG1655 Ptrc30-atoB:Cm ΔpdhR:Km. 
     The kanamycin and chloramphenicol resistances of the above strain are removed according to Protocol 1. The loss of the kanamycin and chloramphenicol resistant cassettes is verified by PCR by using the primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) and atoB F (SEQ ID No17) and atoB R (SEQ ID No18) respectively (Table 2). The resulting strain is called MG1655 Ptrc30-atoB ΔpdhR. 
     4.1.4—Construction of Strain MG1655 lpd*(A55V):Km 
     To transfer the lpd*(A55V) mutation (described in WO2005073364 patent application), into the strain MG1655 Ptrc30-atoB ΔpdhR, a kanamycin resistant cassette is inserted downstream of the lpd*(A55V) gene according to Protocol 1 except that primers lpd*(A55V)_Cm F (SEQ ID No23) and lpd*(A55V)_Cm R (SEQ ID No24) are used to amplify the kanamycin resistance cassette from pKD4 plasmid. 
     
       
         
           
               
            
               
                 Lpd*(A55V)_Cm F 
               
               
                 (SEQ ID N o  23) 
               
               
                 cccgaaagcgaagaagaagtaatttttcgtttgccggaacatccggca 
               
               
                   
               
               
                 attaaaaaagcggctaaccacgccgctttttttacgtctgcaag tgta   
               
               
                   
               
               
                 
                   ggctggagctgcttcg 
                 
               
            
           
         
       
     
     with:
         sequence (lower case) homologous to sequence downstream of the lpd*(A55V) gene (129315-129405 reference sequence in the MySql relational database (EcoGene.org)),   sequence (bold lower case) corresponding to the primer site 2 of plasmid pKD4 (Datsenko and Wanner, 2000),       

     
       
         
           
               
            
               
                 Lpd*(A55V)_Cm R 
               
               
                 (SEQ ID N o  24) 
               
               
                 CCATACTGTCAGGCTGAATAACGAGCAACGGTCAGCAGTATGCGAACG 
               
               
                   
               
               
                 TCTCTCTGAACGTGGAGCAAGAAGACTGGAAAGGTAAA CATATGAATA   
               
               
                   
               
               
                 
                   TCCTCCTTAG 
                 
               
            
           
         
       
     
     with:
         sequence (upper case) homologous to sequence downstream of the lpd*(A55V) gene (129491-129406 reference sequence in the MySql relational database (EcoGene.org)),   sequence (bold upper case) corresponding to the primer site 1 of plasmid pKD4 (Datsenko and Wanner, 2000).       

     Kanamycin resistant recombinants are selected. The insertion of the resistance cassette is then verified by PCR with primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) and by DNA sequencing. The verified and selected strain is called MG1655 lpd*(A55V):Km. 
     4.1.5—Construction of Strain MG1655 Ptrc30 ΔpdhR lpd*(A55V):Km 
     The lpd*(A55V) chromosomal modification is transduced into the strain MG1655 Ptrc30-atoB ΔpdhR with a P1 phage lysate from strain MG1655 lpd*(A55V):Km described above, according to Protocol 2. 
     Kanamycin resistant transductants are selected and the presence of lpd*(A55V):Km chromosomal modification was verified by PCR with primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) (Table 2). The resulting strain is called MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V):Km. 
     The kanamycin resistance of the above strain is removed according to Protocol 1. The loss of the kanamycin resistant cassette is verified by PCR using the primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) (Table 2). The resulting strain MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) is called strain 3 (Table 1). 
     4.2—Construction of pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 Plasmid 
     The pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 plasmid is derived from pUC19 plasmid (Norrander et al., 1983), adhE2 gene from  Clostridium acetobutylicum  coding for the bifunctional aldehyde/alcohol dehydrogenase described in patent application U.S. Ser. No. 13/169,703 and the synthetic gene mvaS from  Enterococcus faecalis  coding for an hydroxymethylglutaryl-CoA synthase optimized for  Escherichia coli  described below. 
     In this plasmid, expressions of both genes are driven by a constitutive trc promoter with an operator site, and a transcriptional terminator is added downstream the entire construct. 
     Synthetic Gene mvaSefO1ec 
     A synthetic gene of the  Enterococcus faecalis  mvaS gene coding for an 3-hydroxy-3-methylglutaryl-CoA synthase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to  Escherichia coli  according to the supplier&#39;s matrix. The construct is cloned into supplier&#39;s pM vectors and verified by sequencing. 
     mvaS gene sequence from  Enterococcus faecalis  (AF290092) optimized for  Escherichia coli : mvaSefO1ec contains the following sequence (SEQ ID No36): 
     
       
         
           
               
            
               
                 ATGACCATTGGCATCGACAAAATCAGCTTTTTTGTTCCGCCTTACTAT 
               
               
                   
               
               
                 ATCGACATGACCGCACTGGCCGAAGCACGTAATGTTGATCCGGGTAAA 
               
               
                   
               
               
                 TTTCATATTGGTATTGGTCAGGATCAGATGGCCGTTAATCCGATTAGC 
               
               
                   
               
               
                 CAGGATATTGTTACCTTTGCAGCAAATGCAGCAGAAGCAATTCTGACC 
               
               
                   
               
               
                 AAAGAAGATAAAGAAGCCATCGATATGGTTATTGTTGGCACCGAAAGC 
               
               
                   
               
               
                 AGCATTGATGAAAGCAAAGCAGCCGCAGTTGTTCTGCATCGTCTGATG 
               
               
                   
               
               
                 GGTATTCAGCCGTTTGCACGTAGCTTTGAAATTAAAGAAGCATGTTAC 
               
               
                   
               
               
                 GGCGCAACCGCAGGTCTGCAGCTGGCAAAAAATCATGTTGCACTGCAT 
               
               
                   
               
               
                 CCGGATAAAAAAGTTCTGGTTGTTGCAGCAGATATCGCCAAATATGGT 
               
               
                   
               
               
                 CTGAATAGCGGTGGTGAACCGACCCAGGGTGCCGGTGCAGTTGCAATG 
               
               
                   
               
               
                 CTGGTTGCAAGCGAACCGCGTATTCTGGCACTGAAAGAGGATAATGTT 
               
               
                   
               
               
                 ATGCTGACGCAGGATATCTATGATTTTTGGCGTCCGACCGGTCATCCG 
               
               
                   
               
               
                 TATCCGATGGTTGATGGTCCGCTGAGCAATGAAACCTATATTCAGAGC 
               
               
                   
               
               
                 TTTGCACAGGTGTGGGATGAACATAAAAAACGTACCGGTCTGGATTTC 
               
               
                   
               
               
                 GCAGATTATGATGCACTGGCCTTTCATATTCCGTACACCAAAATGGGT 
               
               
                   
               
               
                 AAAAAAGCACTGCTGGCCAAAATTAGCGATCAGACCGAAGCCGAACAA 
               
               
                   
               
               
                 GAACGTATCCTGGCACGTTATGAAGAAAGCATTATCTATAGCCGTCGT 
               
               
                   
               
               
                 GTGGGTAATCTGTACACCGGTAGCCTGTATCTGGGTCTGATTAGCCTG 
               
               
                   
               
               
                 CTGGAAAATGCAACCACCCTGACCGCTGGTAATCAGATTGGTCTGTTT 
               
               
                   
               
               
                 AGCTATGGTAGCGGTGCCGTTGCAGAATTTTTCACAGGTGAACTGGTT 
               
               
                   
               
               
                 GCAGGTTATCAGAATCATCTGCAGAAAGAAACCCATCTGGCCCTGCTG 
               
               
                   
               
               
                 GATAATCGTACCGAACTGAGCATTGCAGAATATGAAGCAATGTTTGCA 
               
               
                   
               
               
                 GAAACCCTGGATACCGATATTGATCAGACCCTGGAAGATGAACTGAAA 
               
               
                   
               
               
                 TATAGCATTAGCGCCATTAATAACACCGTGCGTAGCTATCGTAACTAA 
               
            
           
         
       
     
     The mvaSefO1ec synthetic gene is amplified by PCR with primers RBS01*2-mvaSefO1ec-XhaI F (SEQ ID No27) and mvaSefO1ec-NheI R (SEQ ID No28) using the pM vector harbouring the mvaSefO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the XbaI and NheI sites of the pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703. The resulting plasmid is verified by DNA sequencing and called pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02. 
     
       
         
           
               
            
               
                 RBS01*2-mvaSefO1ec-XhaI F 
               
               
                 (SEQ ID N o  27) 
               
               
                 gctctagaTAAGGAGGTTATAA atgaccattggcatcgac   
               
            
           
         
       
     
     with
         sequence (lower case) for XbaI restriction site and extrabases,   sequence (upper case) for the RBS consensus sequence with PsiI restriction site,   sequence (underlined lower case) homologous to the beginning of mvaSefO1ec synthetic gene sequence,       

     
       
         
           
               
            
               
                 mvaSefO1ec-NheI R 
               
               
                 (SEQ ID N o  28) 
               
               
                   CTAGCTAGC TTAGTTACGATAGCTACGCAC 
               
            
           
         
       
         
         
           
             sequence (underlined upper case) for NheI restriction site and extrabases, 
             sequence (upper case) homologous to the end of the mvaSefO1ec synthetic gene sequence. 
           
         
       
    
     4.3—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07 Plasmid 
     The pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3457-4264-4265)-TT07 plasmid is derived from plasmid pCL1920 (Lerner and Inouye, 1990), MXAN_3757, MXAN_4264 and MXAN_4265 synthetic genes from  Myxococcus Xanthus , strain DK 1622, coding for a 3-methylglutaconyl-CoA hydratase, a 3-methylglutaconyl-CoA decarboxylase subunit A and B respectively, optimized for  Escherichia coli  described below. 
     In this plasmid, these genes are organized in operon and their expressions are driven by a constitutive trc promoter with an operator site, and a transcriptional terminator is added downstream the entire construct. 
     4.3.1—Construction of pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec Plasmid 
     MXAN_3757mxO1ec Synthetic Gene 
     A synthetic gene of the  Myxococcus xanthus  MXAN_3757 gene coding for a 3-methylglutaconyl-CoA hydratase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to  Escherichia coli  according to the supplier&#39;s matrix. The construct is cloned into supplier&#39;s pM vectors and verified by sequencing. 
     MXAN_3757 gene sequence from  Myxococcus xanthus  (NC_008095) optimized for  Escherichia coli : MXAN_3757mxO1ec contains the following sequence (SEQ ID No37): 
     
       
         
           
               
            
               
                 ATGCCTGAGTTTAAAGTTGATGCACGTGGTCCGATTGAAATTTGGAC 
               
               
                   
               
               
                 CATTGATGGTGAAAGCCGTCGTAATGCAATTAGCCGTGCAATGCTGA 
               
               
                   
               
               
                 AAGAACTGGGTGAACTGGTTACCCGTGTTAGCAGCAGCCGTGATGTT 
               
               
                   
               
               
                 CGTGCAGTTGTTATTACCGGTGCCGGTGATAAAGCATTTTGTGCCGG 
               
               
                   
               
               
                 TGCCGATCTGAAAGAACGTGCAACAATGGCCGAAGATGAAGTTCGTG 
               
               
                   
               
               
                 CATTTCTGGATGGTCTGCGTCGTACCTTTCGTGCAATTGAAAAAAGC 
               
               
                   
               
               
                 GATTGCGTTTTTATTGCCGCAATTAATGGTGCAGCACTGGGTGGTGG 
               
               
                   
               
               
                 CACCGAACTGGCACTGGCATGTGATCTGCGTGTTGCAGCACCGGCAG 
               
               
                   
               
               
                 CGGAACTGGGTCTGACCGAAGTTAAACTGGGCATTATTCCGGGTGGT 
               
               
                   
               
               
                 GGTGGTACACAGCGTCTGGCACGTCTGGTTGGTCCGGGTCGTGCAAA 
               
               
                   
               
               
                 AGATCTGATTCTGACCGCACGTCGTATTAATGCAGCAGAAGCATTTA 
               
               
                   
               
               
                 GCGTTGGTCTGGCAAATCGCCTGGCACCGGAAGGTCATCTGCTGGCA 
               
               
                   
               
               
                 GTTGCCTATGGTCTGGCCGAAAGCGTTGTTGAAAATGCACCGATTGC 
               
               
                   
               
               
                 AGTTGCAACCGCCAAACATGCAATTGATGAAGGCACCGGTCTGGAAC 
               
               
                   
               
               
                 TGGATGATGCACTGGCCCTGGAACTGCGTAAATATGAAGAAATTCTG 
               
               
                   
               
               
                 AAAACCGAAGATCGCCTGGAAGGCCTGCGTGCATTTGCAGAAAAACG 
               
               
                   
               
               
                 TGCACCGGTGTATAAAGGTCGTTAA 
               
            
           
         
       
     
     The MXAN_3757mxO1ec synthetic gene is amplified by PCR with primers Ptrc01/RBS01*2-MXAN_3757mxO1ec-SacI F (SEQ ID No29) and MXAN_3757 mxO1ec-KpnI R (SEQ ID No30) using the pM vector harbouring the MXAN_3757mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the Sad and KpnI sites of the pCL1920. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec. 
     Ptrc01/RBS01*2-MXAN_3757mxO1ec-SacI F (SEQ ID No29) 
     with 
     
       
         
           
               
            
               
                 Ccgagctc gagctgttgacaattaatcatccggctcgtataatg   
               
               
                   
               
               
                   tgtggaa GTCGACGTTAACACGCGT taaggaggttataa   ATGCC   
               
               
                   
               
               
                 
                   TGAGTTTAAAGTTG 
                 
               
            
           
         
       
         
         
           
             sequence (lower case) for SacI restriction site and extrabases, 
             sequence (underlined lower case) for the trc promoter sequence (Amman et al., 1983), 
             sequence (upper case) for SalI, HpaI and MluI restriction sites, 
             sequence (bold lower case) corresponding to RBS consensus sequence with a PsiI restriction site, 
             sequence (italic upper case) homologous to the beginning of MXAN_3757mxO1ec synthetic gene sequence, 
           
         
       
    
     MXAN_3757mxO1ec-KpnI R(SEQ ID No30) 
     with 
     
       
         
           
               
            
               
                   CGGGGTACC TTAACGACCTTTATACACCG 
               
            
           
         
       
         
         
           
             sequence (underlined upper case) for KpnI restriction site and extrabases, 
             sequence (upper case) homologous to the end of the MXAN_3757mxO1ec synthetic gene sequence. 
           
         
       
    
     4.3.2—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) Plasmid 
     pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) plasmid is derived from pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec described above and MXAN_4264mxO1ec synthetic gene described below. 
     MXAN_4264mxO1ec Synthetic Gene 
     A synthetic gene of the  Myxococcus xanthus  MXAN_4264 gene coding for a 3-methylglutaconyl-CoA decarboxylase subunit A is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to  Escherichia coli  according to the supplier&#39;s matrix. The construct is cloned into supplier&#39;s pM vectors and verified by sequencing. 
     MXAN_4264 gene sequence from  Myxococcus xanthus  (NC_008095) optimized for  Escherichia coli : MXAN_4264mxO1ec contains the following sequence (SEQ ID No38): 
     
       
         
           
               
            
               
                 ATGAAAACCGCACGTTGGTGTAGCCTGGAAGAAGCAGTTGCAAGCAT 
               
               
                   
               
               
                 TCCGGATGGTGCAAGCCTGGCAACCGGTGGTTTTATGCTGGGTCGTG 
               
               
                   
               
               
                 CACCGATGGCACTGGTTATGGAACTGATTGCACAGGGTAAACGTGAT 
               
               
                   
               
               
                 CTGGGTCTGATTAGCCTGCCGAATCCGCTGCCAGCAGAATTTCTGGT 
               
               
                   
               
               
                 TGCCGGTGGTTGTCTGGCACGTCTGGAAATTGCATTTGGTGCACTGA 
               
               
                   
               
               
                 GCCTGCAAGGTCGTGTTCGTCCGATGCCGTGTCTGAAACGTGCAATG 
               
               
                   
               
               
                 GAACAGGGCACCCTGGCATGGCGTGAACATGATGGTTATCGTGTTGT 
               
               
                   
               
               
                 TCAGCGTCTGCGTGCAGCAAGCATGGGTCTGCCGTTTATTCCGGCAC 
               
               
                   
               
               
                 CGGATGCAGATGTTAGTGGCCTGGCACGTACCGAACCGCCTCCGACC 
               
               
                   
               
               
                 GTTGAAGATCCGTTTACAGGTCTGCGTGTTGCAGTTGAACCGGCATT 
               
               
                   
               
               
                 TTATCCGGATGTTGCCCTGCTGCATGCACGTGCCGCAGATGAACGTG 
               
               
                   
               
               
                 GTAATCTGTATATGGAAGATCCGACCACCGATCTGCTGGTTGCGGGT 
               
               
                   
               
               
                 GCAGCAAAACGTGTTATTGCAACCGTGGAAGAACGTGTTGCAAAACT 
               
               
                   
               
               
                 GCCTCGTGCAACCCTGCCTGGTTTTCAGGTTGATCGTATTGTTCTGG 
               
               
                   
               
               
                 CACCGGGTGGTGCCCTGCCGACCGGTTGTGCAGGTCTGTATCCGCAT 
               
               
                   
               
               
                 GATGATGAAATGCTGGCACGTTATCTGAGCCTGGCAGAAACCGGTCG 
               
               
                   
               
               
                 TGAAGCCGAGTTTCTGGAAACCCTGCTGACCCGTCGTGCAGCATAA 
               
            
           
         
       
     
     The MXAN_4264mxO1ec synthetic gene is amplified by PCR with primers RBS01*2-MXAN_4264mxO1ec-SmaI F (SEQ ID No31) and MXAN_4264mxO1ec-BamHI R(SEQ ID No32) using the pM vector harbouring the MXAN_4264mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the SmaI and BamHI sites of the pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264). 
     RBS01*2-MXAN_4264mxO1ec-SmaI F (SEQ ID No31) 
     with 
     
       
         
           
               
            
               
                 cggggggtacccccggg TAAGGAGGTTATAA ATGAAAACCGCACGTTGGTG 
               
            
           
         
       
         
         
           
             sequence (lower case) for KpnI and SmaI restriction sites and extrabases, 
             sequence (bold upper case) for RBS consensus sequence with a PsiI restriction site, 
             sequence (upper case) homologous to the beginning of MXAN_4264mxO1ec synthetic gene sequence, 
           
         
       
    
     MXAN_4264mxO1ec-BamHI R(SEQ ID No32) 
     with 
     
       
         
           
               
               
            
               
                   
                   GCTCTAGAGGATCC TTATGCTGCACGACGGGTCAG 
               
            
           
         
       
         
         
           
             sequence (underlined upper case) for BamHI and XbaI restriction sites and extrabases, 
             sequence (upper case) homologous to the end of the MXAN_4264mxO1ec synthetic gene sequence. 
           
         
       
    
     4.3.3—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265) Plasmid 
     pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265) plasmid is derived from pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) described above and MXAN_4265mxO1ec synthetic gene described below and a transcriptional terminator. 
     Synthetic Gene MXAN_4265mxO1ec 
     A synthetic gene of the  Myxococcus xanthus  MXAN_4265 gene coding for a 3-methylglutaconyl-CoA decarboxylase subunit B is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to  Escherichia coli  according to the supplier&#39;s matrix. The construct is cloned into supplier&#39;s pM vectors and verified by sequencing. 
     MXAN_4265 gene sequence from  Myxococcus xanthus  (NC_008095) optimized for  Escherichia coli : MXAN_4265mxO1ec contains the following sequence (SEQ ID No39): 
     
       
         
           
               
            
               
                 ATGAGCGCAACCCTGGATATCACACCGGCAGAAACCGTTGTTAGCCT 
               
               
                   
               
               
                 GCTGGCACGTCAGATTGATGATGGTGGTGTTGTTGCAACCGGTGTTG 
               
               
                   
               
               
                 CAAGTCCGCTGGCAATTCTGGCCATTGCAGTTGCACGTGCCACCCAT 
               
               
                   
               
               
                 GCACCGGATCTGACCTATCTGGCATGTGTTGGTAGCCTGGACCCGGA 
               
               
                   
               
               
                 AATTCCGACCCTGCTGCCGAGCAGCGAAGATCTGGGTTATCTGGATG 
               
               
                   
               
               
                 GTCGTAGCGCAGAAATTACCATTCCGGACCTGTTTGATCATGCACGT 
               
               
                   
               
               
                 CGTGGTCGTGTTGATACCGTTTTTTTTGGTGCAGCCGAAGTTGATGC 
               
               
                   
               
               
                 CGAAGGTCGTACCAATATGACCGCAAGCGGTAGTCTGGATAAACCGC 
               
               
                   
               
               
                 GTACCAAATTTCCTGGTGTTGCCGGTGCAGCAACCCTGCGTCAGTGG 
               
               
                   
               
               
                 GTTCGTCGTCCGGTTCTGCTGGTTCCGCGTCAGAGCCGTCGTAATCT 
               
               
                   
               
               
                 GGTTCCGGAAGTTCAGGTTGCCACCACCCGTGATCCGCGTCGTCCTG 
               
               
                   
               
               
                 TTACCCTGATTAGCGATCTGGGTGTTTTTGAACTGGGTGCAAGCGGT 
               
               
                   
               
               
                 GCACGTCTGCTGGCTCGCCATCCGTGGGCAAGCGAAGAACATATTGC 
               
               
                   
               
               
                 AGAACGTACCGGTTTTGCATTTCAGGTTAGCGAAGCACTGAGCGTTA 
               
               
                   
               
               
                 CCAGCCTGCCGGATGCACGTACCGTTGCAGCAATTCGTGCAATTGAT 
               
               
                   
               
               
                 CCGCATGGTTATCGTGATGCACTGGTTGGTGCATAA 
               
            
           
         
       
     
     The MXAN_4265mxO1ec synthetic gene is amplified by PCR with primers RBS01*2-MXAN_4265mxO1ec-XbaI F (SEQ ID No33) and MXAN_4265mxO1ec-PstI R (SEQ ID No34) using the pM vector harbouring the MXAN_4265mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the XbaI and PstI sites of the pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264). The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265). 
     RBS01*2-MXAN_4265mxO1ec-XbaI F (SEQ ID No33) 
     with 
     
       
         
           
               
               
            
               
                   
                 gctctaga TAAGGAGGTTATAA ATGAGCGCAACCCTGGATATC 
               
            
           
         
       
         
         
           
             sequence (lower case) for XbaI restriction site and extrabases, 
             sequence (bold upper case) for RBS consensus sequence with a PsiI restriction site, 
             sequence (upper case) homologous to the beginning of MXAN 13  4265mxO1ec synthetic gene sequence, 
           
         
       
    
     MXAN_4265mxO1ec-PstI R (SEQ ID No34) 
     with 
     
       
         
           
               
            
               
                    GCCAAGCTTCTGCAG   GCAGAAAGGCCCACCCGAAGGTGAGCCAG gtat 
               
               
                   
               
               
                 acTTATGCACCAACCAGTGCATC 
               
            
           
         
       
         
         
           
             sequence (underlined upper case) for HindIII and PstI restriction sites and extrabases, 
             sequence (bold upper case) for T7Te transcriptional terminator sequence from T7 phage (Harrington et al., 2011), 
             sequence (lower case) for BstZ17I restriction site, 
             sequence (upper case) homologous to the end of the MXAN_4265mxO1ec synthetic gene sequence. 
           
         
       
    
     4.4—Construction of Strain 4: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07) 
     Construction of a strain with increased prenol pathway flux expressing the atoB gene to produce acetoacetyl-CoA, the optimized mvaS gene from  Enterococcus faecalis  to produce 3-hydroxy-3-methylglutaryl-CoA, the optimized MXAN(3757-4264-4265) operon from  Myxococcus xanthus  to produce 3-methylcrotonyl-CoA and the adhE2ca gene from  Clostridium acetobutylicum  to produce prenol. 
     The pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 and pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07 plasmids are introduced by electroporation into the strain 3 (Table 1). The presence of the two plasmids is verified and the resulting strain MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07) is called strain 4 (Table 1). 
     Example 5 
     Culture of the Above Described Prenol Production Strains on Glucose 
     Production strains 1, 2, 3 and 4 are evaluated in small Erlenmeyer flasks using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32:120-128) that is supplemented with 10 g·L −1  MOPS and 10 g·L −1  glucose and adjusted to pH 6.8. 
     A 5 mL preculture is grown at 37° C. for 6.5 hours in a mixed medium (10% LB medium (Sigma 25%) with 2.5 g·L −1  glucose and 90% minimal medium described above). It is used to inoculate a 50 mL culture to an OD 600  of 0.1 in minimal medium. When necessary, antibiotics are added at concentrations of 50 mg·L −1  for ampicillin and spectinomycin and 10 mg·L −1  for gentamycin. The temperature of the cultures is 37° C. When the culture reaches an OD 600  of 7 to 9, extracellular metabolites are analyzed using HPLC with refractometric detection (organic acids and glucose). Production of prenol is determined by GC/MS. 
     The strains 2 and 4 produce prenol with a concentration ranging between 0.001 and 100 mM whereas strains 1 and 3 do not produce it. 
     PATENT REFERENCES 
     
         
         WO 2009/006429 
         WO 2009/076676 
         WO 2010/031076 
         US 2010/0216958 
         WO 2010/031076 
         WO 2008/052973 
         WO 2008/052595 
         WO 2008/040387 
         WO 2007/144346 
         WO 2007/141316 
         WO 2007/077041 
         WO 2007/017710 
         WO 2006/082254 
         WO 2006/082252 
         WO 2005/111202 
         WO 2005/073364 
         WO 2005/047498 
         WO 2004/076659 
       
    
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