US 20140256009 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0256009 A1 Marliere (43) Pub. Date: Sep. 11, 2014 (54) METHOD FOR THE ENZYMATIC CI2N 9/88 (2006.01) PRODUCTION OF BUTADIENE CI2P 7/04 (2006.01) (52) U.S. Cl. (71) Applicant: SCIENTIST OF FORTUNE, S.A., CPC, C12P5/026 (2013.01); C12P 7/04 (2013.01); Luxembourg (LU) CI2N 9/1205 (2013.01); C12N 9/88 (2013.01); CI2N 9/1235 (2013.01) (72) Inventor: Philippe Marliere, Mouscron (BE) USPC 435/157:435/167; 435/252.33: 435/252.31; (73) Assignee: SCIENTIST OF FORTUNE, S.A., 435/252.3; 435/254.11: 435/254.21: 435/254.2: Luxembourg (LU) 435/254.3; 435/254.6:435/254.23; 435/257.2: 435/194 (21) Appl. No.: 14/352,825 (22) PCT Filed: Oct. 18, 2012 (57) ABSTRACT (86). PCT No.: PCT/EP2012/07O661 S371 (c)(1), Described is a method for the enzymatic production of buta (2), (4) Date: Apr. 18, 2014 diene which allows to produce butadiene from crotyl alcohol. Also described are enzyme combinations and compositions Related U.S. Application Data containing Such enzyme combinations which allow the enzy (60) Provisional application No. 61/549,149, filed on Oct. matic conversion of crotyl alcohol into butadiene. Further 19, 2011. more, the invention relates to microorganisms which have been genetically modified so as to be able to produce butadi (30) Foreign Application Priority Data ene from crotyl alcohol. Moreover, the invention relates to a method for the enzymatic Oct. 19, 2011 (EP) ................................... 111858,544 production of crotyl alcohol from crotonyl-Coenzyme A. The Publication Classification obtained crotyl alcohol can be further converted into butadi ene as described herein. Also described are enzyme combi (51) Int. Cl. nations which allow to convert crotonyl-Coenzyme A into CI2P 5/02 (2006.01) crotyl alcohol as well as (micro)organisms which express CI2N 9/12 (2006.01) Such enzyme combinations. Patent Application Publication Sep. 11, 2014 Sheet 1 of 10 US 2014/0256009 A1 1S-1)so Figure 1 Figure 2 -1s-1sO Figure 3 S-1s Figure 4 Patent Application Publication Sep. 11, 2014 Sheet 2 of 10 US 2014/0256009 A1 L-Lactate NAD" Lactate dehydrogenase 340 nm crotyl alcohol ATP Pyruvate NADH Studied enzyme C Pyruvate kinase Crotyl phosphate ADP Phosphoenolpyruvate Figure 5 s -S, 8-3.8rain 38-46 BS 15.2 €------ 8: f -------- 150 23. fi Figure 6 Patent Application Publication Sep. 11, 2014 Sheet 3 of 10 US 2014/0256009 A1 inters " " " " - ------- (S, ).5-9.7tain i{2-33) 43) Figure 7 O O O O -----S. o1 Yo-Yoh - 21- -Ho-1 DYo1- Yoh OH OH OH OH Dimethylallyl diphosphate isoprene Diphosphate O O O O 1S-1S-1 Yo1 Yoh 4N-1 * ...Ho1|yo1 -N-N YOH OH OH OH OH Croty diphosphate 1,3-Butadiene Diphosphate Figure 8 Patent Application Publication Sep. 11, 2014 Sheet 4 of 10 US 2014/0256009 A1 O ------H OH O O Figure 9 300 a Crotyl monophosphate Croty diphosphate 200 l 2.53.3 1513 Y 2335) OC 79.193.1 1203 O -Lld. ball 10 200 30. m/z Figure 10a Patent Application Publication Sep. 11, 2014 Sheet 5 of 10 US 2014/0256009 A1 Crotyl monophosphate 27 No croty diphosphate 4. 54 3. EC O Figure 10b 118 120 s 100 EO st D 60so 42 40 . it s 20 i 1 4.5 : O C. : Without (E)-beta Isoprene Monoterpene Monoterpene enzyme ocimene synthase P. synthase E. synthase Rt. synthase W. montana var. globulus alternifolia Winifera Iobata Figure 11 Patent Application Publication Sep. 11, 2014 Sheet 6 of 10 US 2014/0256009 A1 ------------------ saw Zjuu -- |||- || Patent Application Publication Sep. 11, 2014 Sheet 7 of 10 US 2014/0256009 A1 -se E s s s Patent Application Publication Sep. 11, 2014 Sheet 8 of 10 US 2014/0256009 A1 Z Patent Application Publication Sep. 11, 2014 Sheet 9 of 10 US 2014/0256009 A1 HejCTV/NWUU£’0 c ?7),aun61-I Patent Application Publication Sep. 11, 2014 Sheet 10 of 10 US 2014/0256009 A1 o'r G?eun61-I US 2014/0256009 A1 Sep. 11, 2014 METHOD FOR THE ENZYMATIC 0006 Thus, in a first aspect, the present invention relates PRODUCTION OF BUTADIENE to a process for the production of butadiene in which butadi ene is produced by the enzymatic conversion of crotyl alco 0001. The present invention relates to a method for the hol. Crotyl alcohol, also referred to as crotonyl alcohol or enzymatic production ofbutadiene which allows to produce crotonol, is an unsaturated alcohol of formula CHO (see butadiene from crotyl alcohol. The present invention also FIG. 1). Another name for crotyl alcohol is But-2-en-1-ol. It relates to microorganisms which have been genetically modi can be produced by reduction of crotonaldehyde (see FIG.3). fied so as to produce butadiene. According to the present invention crotyl alcohol can be 0002 The present invention also relates to a method for converted into butadiene by enzymatic reactions involving as the enzymatic production of crotyl alcohol from crotonyl intermediates crotyl phosphate and/or crotyl diphosphate. Coenzyme A. The obtained crotyl alcohol can be further Thus, the principle underlying the present invention is that converted into butadiene as described herein. The present crotyl alcohol is first enzymatically activated by the conver invention furthermore relates to enzyme combinations which sion into crotyl phosphate or crotyl diphosphate and is then allow to convert crotonyl-Coenzyme A into crotyl alcohol as further converted into butadiene by the use of appropriate well as to (micro)organisms which express such enzyme enzymes as described below. combinations. 0007 Thus, the present invention relates, in a first aspect, 0003. Butadiene (1,3-butadiene) is a conjugated diene to a method for the production ofbutadiene comprising the with the formula CH (see FIG. 4). It is an important indus enzymatic conversion of crotyl alcohol into butadiene via trial chemical used as a monomer in the production of syn crotyl phosphate or crotyl diphosphate. thetic rubber. There exist different possibilities to produce 0008. The enzymatic conversion of crotyl alcohol into butadiene. Butadiene is, for example, produced as a by prod butadiene can occur via different alternative routes. In a first uct of the steam cracking process used to produce ethylene aspect (A), the present invention relates to a method for the and other olefins. In this process butadiene occurs in the C4 production of butadiene comprising the enzymatic conver stream and is normally isolated from other byproducts by sion of crotyl alcohol into butadiene via crotyl phosphate extraction into a polar aprotic solvent, such as acetonitrile, wherein said method comprises the steps of from which it is then stripped. Butadiene can also be pro duced by the catalytic dehydrogenation of normal butane or it (i) enzymatically converting crotyl alcohol into crotyl phos can be produced from ethanol. In the latter case, two different phate; and processes are in use. In a single-step process, ethanol is con (ii) enzymatically converting crotyl phosphate into butadi verted to butadiene, hydrogen and water at 400-450° C. over CC. a metal oxide catalyst (Kirshenbaum, I. (1978), Butadiene. In 0009. This alternative is in the following referred to as M. Grayson (Ed.), Encyclopedia of Chemical Technology, 3rd Alternative A and the different steps are referred to as A(i) and ed., vol. 4, pp. 313-337. New York: John Wiley & Sons). In a A(ii). two-step process, ethanol is oxidized to acetaldehyde which 0010. As regards step A(i), the enzymatic conversion of reacts with additional ethanol over a tantalum-promoted crotyl alcohol into crotyl phosphate is a phosphorylation step porous silica catalyst at 325-350° C. to yield butadiene (Kir and can be achieved by enzymes which catalyze the transfer shenbaum, I. (1978), loccit.). Butadiene can also be produced of a phospho group onto a molecule. Such as kinases. For by catalytic dehydrogenation of normal butenes. example, enzymes which can be employed in this reaction are 0004 For the past two decades, genetic engineering tech enzymes which are classified as E.C.2.7.1, i.e. phosphotrans nologies have made possible the modification of the metabo ferases with an alcohol group as acceptor, preferably lism of micro-organisms, and hence their use to produce key enzymes which are classified as 2.7.1.50 (hydroxyethylthiaz substances which they would otherwise produce at a low ole kinase) or which are classified as E.C. 2.7.1.89 (thiamine yield. By enhancing naturally occurring metabolic pathways, kinase). Preferably, ATP is the donor of the phospho group in these technologies open up new ways to bio-produce numer Such a reaction. Thus, in one embodiment the enzymatic ous compounds of industrial relevance. Several industrial conversion of crotyl alcohol into crotyl phosphate can, e.g., compounds Such as amino-acids for animal feed, biodegrad be achieved by the use of a hydroxyethylthiazole kinase (EC able plastics or textile fibres are now routinely produced using 2.7.1.50). Hydroxyethylthiazole kinase is an enzyme which genetically modified organisms. There are however no bio catalyzes the following reaction processes using micro-organisms in place for the production ATP+4-methyl-5-(2-hydroxyethyl)thiazole ADP+ of the major petrochemically derived molecules, in particular 4-methyl-5-(2-phosphoethyl)thiazole butadiene, since no micro-organisms are known as natural producers of butadiene even in Small quantities. Given the 0011. The occurrence of this enzyme has been described large amounts of rubber produced worldwide and the increas for several organisms, e.g.