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Microorganisms and Methods for the Biosynthesis of Aromatics, 2,4-Pentadienoate and 1,3- Butadiene

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(19) TZZ Z¥Z_T (11) EP 2 607 340 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: C07C 11/167 (2006.01) C12P 5/02 (2006.01) 26.06.2013 Bulletin 2013/26 C12N 15/52 (2006.01) C12P 7/16 (2006.01) C12N 1/15 (2006.01) C12N 1/19 (2006.01) (2006.01) (21) Application number: 13154607.9 C12N 1/21 (22) Date of filing: 26.07.2011 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Osterhout, Robin E. GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO San Diego, CA 92121 (US) PL PT RO RS SE SI SK SM TR • Burgard, Anthony P. Designated Extension States: San Diego, CA 92121 (US) BA ME •Pharkya,Priti San Diego, CA 92121 (US) (30) Priority: 26.07.2010 US 367792 P •Burk,Mark J. 27.07.2010 US 368223 P San Diego, CA 92121 (US) 09.09.2010 US 381407 P (74) Representative: Jones Day (62) Document number(s) of the earlier application(s) in Rechtsanwälte,Attorneys- at-Law,Patentanwälte accordance with Art. 76 EPC: Prinzregentenstrasse 11 11740777.5 80538 München (DE) (71) Applicant: Genomatica, Inc. Remarks: San Diego, CA 92121 (US) This application was filed on 08-02-2013 as a divisional application to the application mentioned under INID code 62. (54) Microorganisms and methods for the biosynthesis of aromatics, 2,4-pentadienoate and 1,3- butadiene (57) The invention provides non-naturally occurring The invention additionally provides methods of using microbial organisms having a 1,3-butadiene pathway. such organisms to produce 1,3-butadiene. EP 2 607 340 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 607 340 A1 Description BACKGROUND OF THE INVENTION 5 [0001] The present invention relates generally to biosynthetic processes, and more specifically to organisms having toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy) phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene biosynthetic capability. [0002] Toluene is a common solvent that has replaced benzene due to benzene’s greater carcinogenicity and is an industrial feedstock and used in the manufacture of TNT, polyurethane foam, benzaldehyde and benzoic acid. Toluene 10 is a byproduct in the manufacture of gasoline and exists in small concentrations in crude oil. [0003] Benzene is often used as an intermediate to make other chemicals. Its most widely-produced derivatives include styrene, which is used to make polymers and plastics, phenol for resins and adhesives, via cumene, and cyclohexane, which is used in the manufacture of Nylon. Benzene is also used to make some types of rubbers, lubricants, dyes, detergents, drugs, explosives, napalm and pesticides. Benzene production in the petroleum industry is made by various 15 energy intensive processes including, catalytic reforming, toluene hydrodealkylation, toluene disproportionation, and steam cracking. [0004] Styrene is the precursor to polystyrene and numerous copolymers. Styrene based products include, acrylonitrite 1,3-butadiene styrene (ABS), styrene-1,3-butadiene (SBR) rubber, styrene-1,3-butadiene latex, SIS (styrene- isoprene- styrene), S-EB-S (styrene-ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), and unsaturated polyesters. 20 These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing. [0005] Styrene is most commonly produced by the catalytic dehydrogenation of ethylbenzene. Ethylbenzene is mixed in the gas phase with 10-15 times its volume in high-temperature steam, and passed over a solid catalyst bed. Most ethylbenzene dehydrogenation catalysts are based on iron(III) oxide, promoted by several percent potassium oxide or 25 potassium carbonate. Steam serves several roles in this reaction. It is the source of heat for powering the endothermic reaction, and it removes coke that tends to form on the iron oxide catalyst through the water gas shift reaction. The potassium promoter enhances this decoking reaction. The steam also dilutes the reactant and products, shifting the position of chemical equilibrium towards products. A typical styrene plant consists of two or three reactors in series, which operate under vacuum to enhance the conversion and selectivity. Typical per-pass conversions are ca. 65% for 30 two reactors and 70-75% for three reactors. [0006] Over 25 billion pounds of 1,3-butadiene (or just butadiene or BD) are produced annually and is applied in the manufacture of polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1,4-butanediol. 1,3-butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. 35 The ability to manufacture 1,3- butadiene from alternative and/or renewable feedstocks would represent a major advance in the quest for more sustainable chemical production processes [0007] One possible way to produce 1,3-butadiene renewably involves fermentation of sugars or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to 1,3- butadiene in a second step involving metal-based catalysis. Direct fermentative production of 1,3-butadiene from re- 40 newable feedstocks would obviate the need for dehydration steps and 1,3-butadiene gas (bp -4.4°C) would be contin- uously emitted from the fermenter and readily condensed and collected. Developing a fermentative production process would eliminate the need for fossil- based 1,3-butadiene and would allow substantial savings in cost, energy, and harmful waste and emissions relative to petrochemically-derived 1,3-butadiene. [0008] 2,4-Pentadienoate is a useful substituted butadiene derivative in its own right and a valuable intermediate en 45 route to other substituted 1,3-butadiene derivatives, including, for example, 1- carbamoyl-1,3-butadienes which are ac- cessible via Curtius rearrangement. The resultant N-protected-1,3-butadiene derivatives can be used in Diels alder reactions for the preparation of substituted anilines. 2,4- Pentadienoate can be used in the preparation of various polymers and co-polymers. [0009] Terephtalate (also known as terephthalic acid and PTA) is the immediate precursor of polyethylene terephthalate 50 (PET), used to make clothing, resins, plastic bottles and even as a poultry feed additive. Nearly all PTA is produced from para-xylene by oxidation in air in a process known as the Mid Century Process. This oxidation is conducted at high temperature in an acetic acid solvent with a catalyst composed of cobalt and/or manganese salts. Para- xylene is derived from petrochemical sources and is formed by high severity catalytic reforming of naphtha. Xylene is also obtained from the pyrolysis gasoline stream in a naphtha steam cracker and by toluene disproportion. 55 [0010] Cost-effective methods for generating renewable PTA have not yet been developed to date. PTA, toluene and other aromatic precursors are naturally degraded by some bacteria. However, these degradation pathways typically involve monooxygenases that operate irreversibly in the degradative direction. Hence, biosynthetic pathways for PTA are severely limited by the properties of known enzymes to date. 2 EP 2 607 340 A1 [0011] A promising precursor for PTA is p-toluate, also known as p-methylbenzoate. P-Toluate is an intermediate in some industrial processes for the oxidation of p-xylene to PTA. It is also an intermediate for polymer stabilizers, pesticides, light sensitive compounds, animal feed supplements and other organic chemicals. Only slightly soluble in aqueous solution, p-toluate is a solid at physiological temperatures, with a melting point of 275°C. Microbial catalysts for synthe- 5 sizing this compound from sugar feedstocks have not been described to date. [0012] Thus, there exists a need for alternative methods for effectively producing commercial quantities of compounds such as styrene, 2,4-pentadienoate, 1,3- butadiene, p-toluate, terephthalate, benzene and toluene. The present invention satisfies this need and provides related advantages as well. 10 SUMMARY OF THE INVENTION [0013] The invention provides non-naturally occurring microbial organisms having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, sty- rene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway. The invention additionally provides methods of using 15 such organisms to produce toluene, benzene, p- toluate, terephthalate, (2- hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene. [0014] The invention also provides non-naturally occurring microbial Organisms having a (2-hydroxy-3-methyl-4-ox- obutoxy)phosphonate (2H3M4OP) pathway, a p- toluate pathway, a terephthalate pathway, a (2 -hydroxy- 4-oxobutoxy) phosphonate (2H4OP) pathway, and/or a benzoate pathway. The invention additionally provides methods of using such 20 organisms to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, p- toluate, terephthalate, (2-hydroxy-4-oxobu- toxy)phosphonate, or benzoate. BRIEF DESCRIPTION OF THE DRAWINGS 25 [0015] Figure 1 shows the conversion of phenylalanine to toluene via phenylacetate. Enzymes are A. phenylalanine ami- notransferase and/or phenylalanine oxidoreductase (deaminating), B. phenylpyruvate decarboxylase, C. phenyla- cetaldehyde dehydrogenase and/or oxidase, D. phenylacetate decarboxylase, E. phenylacetaldehyde decarbony- 30 lase, and F. phenylpyruvate oxidase. Figure 2 shows the conversion of phenylalalanine to benzene by phenylalanine benzene-lyase. Figure 3 shows pathways to styrene from benzoyl-CoA. Enzymes are: A. benzoyl-CoA acetyltransferase, B. 3-oxo- 35 3-phenylpropionyl-CoA synthetase, transferase and/or hydrolase, C. benzoyl- acetate decarboxylase, D. acetophe- none reductase and E. 1-phenylethanol dehydratase, F. phosphotrans-3-oxo-3-phenylpropionylase, G. benzoyl- acetate kinase. Figure 4 shows the conversion of muconate stereoisomers to 1,3-butadiene.
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  • Microbial Biochemistry, 2Nd Edition

    Microbial Biochemistry, 2Nd Edition

    Microbial Biochemistry . G.N. Cohen Microbial Biochemistry Second Edition Prof. G.N. Cohen Institut Pasteur rue du Docteur Roux 28 75724 Paris France [email protected] ISBN 978-90-481-9436-0 e-ISBN 978-90-481-9437-7 DOI 10.1007/978-90-481-9437-7 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010938472 # Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Foreword This book originates from almost 60 years of living in the company of micro- organisms, mainly with Escherichia coli. My scientific life has taken place almost exclusively at the Institut Pasteur in Paris, where many concepts of modern molecular biology were born or developed. The present work emphasizes the interest of microbial physiology, biochemistry and genetics. It takes into account the considerable advances which have been made in the field in the last 30 years by the introduction of gene cloning and sequencing and by the exponential development of physical methods such as X-ray crystallog- raphy of proteins. The younger generation of biochemists is legitimately interested in the problems raised by differentiation and development in higher organisms, and also in neuros- ciences.