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PEER REVIEW TECHNOLOGY UPDATE

Development of a gas-phase bioprocess for isoprene-monomer production using metabolic pathway engineering
Gregory M. Whited,1 Frank J. Feher,2 David A. Benko,2 Marguerite A. Cervin,1 Gopal K. Chotani,1 Joseph C. McAuliffe,1 Richard J. LaDuca,1 Eliahu A. Ben-Shoshan,1 and Karl J. Sanford1
of industrial applications, ranging from the production of synthetic rubber for tires and coatings, to use in adhesives and development of specialty elastomers. Current production of isoprene is derived entirely from petrochemical sources. There is an increasing global need for more isoprene and a simultaneous environmental imperative to reduce greenhouse gases, both of which can be achieved by a highefficiency fermentation-based process for polymer-grade isoprene production. The key to such a process is engineering a microbial cell factory that is streamlined in physiology to drive sugar conversion through engineered biosynthetic pathways, to produce isoprene at near theoretical yields. Two biosynthetic isoprenoid pathways being optimized towards this end, and processes delivering >60g/L of isoprene are described. The product produced in the gas-phase is recovered from the fermentation off-gas in a continuous process. Current state-of-the-art technology has resulted in production, recovery, polymerization, and manufacture of tires with the isoprene component produced via fermentation. Continued improvements in both the cell factory and the production process are being actively pursued.

Genencor, division of Danisco USA Inc., 925 Page Mill Road, Palo Alto, California, USA 94304. Goodyear Innovation Center, The Goodyear Tire & Rubber Company, PO Box 3531, Akron, Ohio, USA 44309-3531.

Corresponding authors Karl J. Sanford, VP Technology Development, Genencor Phone: +001 (650) 846-7584; Email: karl. sanford@dansico.com David A. Benko, R&D Fellow, Goodyear Tire & Rubber Company Phone: +001 (330) 796-2592; Email: dbenko@goodyear.com Submitted: 1 February 2010; Revised: 12 March 2010; Accepted: 16 April 2010 KEYWORDS: biobased isoprene; microbial fermentation; gas phase; sugar conversion; biosynthetic isoprenoid pathway ACRONYMS: DMAPP, dimethylallyl pyrophosphate, DMAPP; IPP, isopentenyldiphosphate; MVA, mevalonic acid; 5-methyl erythritol phosphate; DOXP/ DXP, 1-deoxy-D-xylulose 5-phosphate; AcCoA, acetyl coenzyme A; IspS, isoprene synthases; PGL, phosphogluconolactonase

1. Introduction
soprene is a key chemical required to produce a diverse range of industrial products including a wide variety of specialty elastomers used in surgical gloves, motor mounts and fittings, rubber bands, golf balls, and shoes. Styrene-isoprene-styrene block copolymers form an essential part of most hot-melt pressure-sensitive adhesive formulations, and cis-polyisoprene or synthetic rubber is used in tire manufacture. All of the worlds isoprene is produced from petroleum-derived feedstocks and is subject to volatility in pricing and supply linked to oil. Nearly all major manufacturers of rubber goods depend on either imported natural rubber from Hevea brasiliensis (i.e., the Brazilian rubber tree) or petroleum-based synthetic rubber polymers. Unfortunately, neither material is obtainable from domestic US

Abstract
A sustainable production system for isoprene is being developed based on microbial fermentation of renewable sugars (BioIsoprene). Isoprene is an important commodity chemical used in a wide range

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sources in quantities sufficient to meet anticipated future demands. A method that is not limited either by petroleum refining capacity or by inefficient chemical synthesis is needed for the production of isoprene. In particular, a reliable, cost-competitive biobased process for isoprene production, which utilizes renewable feedstocks for an improved carbon footprint and decreased resource utilization, will be an industry-redefining development. Development of a biobased process to produce biochemicals typically requires a multi-step, iterative process. Establishing a production system that can deliver commercially relevant cost and capital investment targets involves the steps of: (1) engineering and building the core enzyme pathway(s) to produce the target molecule; (2) constricting and eliminating carbon flow through nonproductive or carbondepleting pathways; (3) balancing metabolic pathways to establish the required energy and cofactor balance; (4) optimizing the influx and efflux of key metabolites; (5) optimizing, engineering, and designing enzyme activities to mitigate key intermediate and/or end-product inhibition; and (6) integrating upstream fermentation-based processing with downstream recovery, purification, and/or chemical processing unit operations to deliver a product that meets specifications. To date, isoprene has not been included in the schema for significant biobased products.1 A biobased process for isoprene would require: (1) development of an aqueous process whereby the biological production system would remain viable during intracellular production of isoprene and the subsequent transport of isoprene in a gaseous phase through the cell membrane; and (2) development of a continuous gas-phase product recovery and purification strategy. This paper describes our approach at developing a scalable and commercially relevant bioprocess for isoprene production (BioIsoprene). Metabolic pathway engineering efforts outlined in Steps 1 through 4 in the previous paragraph are emphasized, and advantages that accrue due to the volatile nature (b.p. 34C) of isoprene (Steps 5 and 6, above) are discussed.

3. Historical overview of isoprene production


Isoprene was first synthetically produced in 1860 by thermal decomposition of natural rubber which contains thousands of isoprene monomers joined end-to-end to form long chains.5,6 Since that time, polymer chemists have sought to develop ways to reassemble isoprene back into a material that performs like natural rubber. These efforts, which intensified during and after World War II, led to two different methods for producing synthetic rubber from isoprene. Both methods were commercialized in the early 1960s, yielding limited commercial supplies of synthetic isoprene.7 Isoprene can be synthesized by many different reactions, but only a fraction of these have been attractive enough to warrant development into commercial processes. Today, fewer are proving to be commercially viable. The earliest methods for producing isoprene were based on classical industrial organic chemistry. Later methods were based on large-scale conversion of petrochemical feedstocks.

3.1 ISOPRENE PRODUCTION METHODS4,8,9


Reaction of acetone with acetylene10 (from calcium carbide) to produce isoprene was first used on a small scale by Germany during World War I. The process consists of three steps as shown in Figure 1, Scheme A. Although the process was used commercially until 1982, it could not compete with the more economical processes developed later, which were based on less complex, large-scale conversion of petrochemical feedstocks. Dimerization of propylene11 for isoprene production was jointly developed by The Goodyear Tire & Rubber Co. (Akron, Ohio, USA) and Scientific Design Company, Inc. (Little Ferry, New Jersey, USA. This three-step process, shown in Figure 1, Scheme B, involves production of a six-carbon intermediate, which when subjected to high-temperature hydrocarbon-cracking conditions yields isoprene and methane. Isoprene-selectivity in this process is about 50% based on propylene. This method, commercialized in 1962, was a viable source of isoprene when petroleum and energy were inexpensive and plentiful. It was abandoned by The Goodyear Tire & Rubber Co. in 1974 for more economical methods that were based on extraction of isoprene from hydrocarbon streams generated by petroleum refining operations near the Gulf of Mexico. Dehydrogenation of tertiary amylenes12 is a desirable route to produce isoprene when amylenes are readily available as a feedstock. The two tertiary amylene isomers (i.e., 2-methyl-1-butene and 2-methyl2-butene) can readily be obtained from the catalytic cracking operations at petroleum refineries. 2-Methyl-2-butene is also available via the olefin metathesis reaction of isobutylene and propylene. Tertiary amylenes, when heated to temperatures where unfavorable thermodynamics of the reaction can be overcome and C-H bonds easily broken, react to produce isoprene and hydrogen as major products, as shown in Figure 1, Scheme C. This process was commercialized by B.F. Goodrich and Shell Nederland Chemie in the early 1960s. Today, it is used commercially in the former Soviet Union. Isopentane dehydrogenation13 for isoprene production is similar to dehydrogenation of tertiary amylenes, except that the reaction is even

2. Isoprene background
Isoprene is a common synonym for the hydrocarbon chemical known as 2-methyl-1,3-butadiene (Figure 1). It is pervasive in the environment, produced naturally by plants, animals, humans, and bacteria. By far the largest amount of annual isoprene is produced biogenically by plants, which collectively release approximately 600 million tonnes per year into the atmosphere.2 It is important to note that this very large amount of isoprene is produced from carbon recycled in the atmosphere, rather than from petroleum sources. This amount of isoprene is enough to manufacture 60 billion car and truck tires, which is 50 times the current global production of 1.2 billion tires. Isoprene is also the most common hydrocarbon present in exhaled human breath; its etiology in the human body, however, is unknown.3 The collection of isoprene from plants and animals for commercial applications is economically unfeasible, so the current commercial routes to isoprene involve refinery-based conversion of petrochemical feedstocks. Global industrial production of synthetic isoprene from petrochemical feedstocks is less than 1 million tons per year.4

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A) ACETONE / ACETLYENE ROUTE B) PROPYLENE DIMER ROUTE more thermodynamically unfavorable and O higher temperatures are required to break the + C2H2 2 C-H bonds, as shown in Figure 1, Scheme D. This process, which was used in the former 10 40C Soviet Union, can be used to make isoprene (nPr)3A1 Liquid NH3 2 MPa when energy is cheap and isopentane readily available from petroleum refineries. OH Isobutylene-formaldehyde condensation14 to make isoprene was first developed in the early 1960s and is practiced today by chemicals manufacturer Kuraray (Osaka, SiO2 / A12O3 100C H2 Pd/C Japan) and companies in the former Soviet Union. Although the early process was very OH energy-intensive, recent improvements have resulted in increased energy efficiency to the point where the process today remains comA12O3 mercially viable.15 The process itself occurs in > 600C 250 300C CH4 two steps, as shown in Figure 1, Scheme E. H2O In the first step, isobutylene reacts with two molecules of formaldehyde (which can be produced via oxidation of methanol) to produce Fe2O3 / K2CO3 / Cr2O3 > 600C an intermediate containing two oxygen atoms. This intermediate decomposes in the presence 2H2 600C ISOPRENE of acid at higher temperatures to produce iso-H2 (2-methyl-1,3-butadiene) prene, water, and a molecule of formaldehyde. Extractive-distillation of isoprene16,17 from C) ISOAMYLENE ROUTE D) ISOPENTANE ROUTE petroleum cracking streams, first commercialized in 1975 by the company now known as Nippon Zeon (Tokyo, Japan), has become a H+ CH2O, H2O 240 - 400C major source of petroleum-based isoprene. The process itself is based on the ability of certain polar solvents (e.g., acetonitrile, dimethylformamide, N-methylpyrrolidone) O H+ to selectively extract isoprene from complex + 2 CH2O mixtures of hydrocarbons generated during O 95C steam-cracking of petroleum. These mixtures typically contain 1020% isoprene mixed E) ISOBUTYLENE / FORMALDEHYDE ROUTE with tertiary amylenes, more than 15 other C5 hydrocarbons and dozens of other lowFigure 1. Synthetic routes for isoprene production molecular-weight hydrocarbons in amounts less than 1%. Extractive-distillation is an effective method for obtaining isoprene but is energy-intensive and synthetic isoprene in the US, produces and consumes approximately has a utility limited by the availability of isoprene-containing hydro13% (120,000 tonnes)4 of the worlds supply at its chemical operations carbon streams. When petroleum refineries use heavy hydrocarbons in Beaumont, Texas, USA. as feedstocks for hydrocarbon-cracking, the waste streams are plentiful in, and contain greater amounts of, crude isoprene; when light 3.2 CURRENT PETROCHEMICAL SOURCES OF ISOPRENE hydrocarbons or natural gas are used as feedstocks, the amount of Worldwide, high-purity isoprene is produced at a number of proisoprene produced via cracking is much less. The industry trend is duction sites (Figure 2), at volumes in excess of 1.7 billion pounds per towards the use of lighter hydrocarbon feedstock streams for cracking, year.4 Nonetheless, the current supply and demand balance within the and thus the supplies of crude C5 streams (and high purity petroleumglobal isoprene industry is at risk in the coming years. Downstream demand for isoprene-based derivatives (driven primarily by tires in derived isoprene derived from these crude C5 streams) are lessening. the automotive sector and adhesives) has been hindered significantly The Goodyear Tire & Rubber Co., which is the leading producer of

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in the wake of the current global economic crisis; but as demand regenerates, questions regarding sufficient supply of isoprene arise. Supply of isoprene is further threatened as availability of natural rubber tightens and its price increases. The shutdown of Royal Dutch Shells Pernis isoprene unit at the end of 2009 reduced the global supply of isoprene several percent. In addition, the trend towards declining availability of crude C5 feed streams from olefin crackers for isoprene extraction units further limits isoprene supply, by requiring the units to operate at lower utilization rates. Still, lighter feed slates would lead to reduced amounts of butadiene and reduced supply of alternate synthetic rubber. Consumers of isoprene, anticipating these limitations in supply, are looking for additional sources for isoprene. Biological sources of isoprene and the potential for biotechnology: By far, plants produce the largest volume of global biogenic isoprene, but a commercial process to produce and recover isoprene from plants is not practical. Isoprene production by bacteria has also been shown to be widespread in nature, although its global contribution is small.18,19 The common soil bacterium, Bacillus subtilis, is found to be the best natural producer of isoprene. Genetic approaches to identify the gene responsible for encoding the catalytic activity for isoprene synthesis have been pursued, but without success.20,21 Cell-free studies with B. subtilis extracts revealed that isoprene was produced from dimethylallyl pyrophosphate (DMAPP), a natural intermediate in biological isoprenoid biosynthesis, but the enzyme

responsible for catalyzing the conversion of DMAPP to isoprene was not identified in these studies.22 With this foundation in mind, the commercial production of isoprene via a microbial fermentation process, using metabolically engineered microorganisms, is an attractive proposition. Pioneering work by Zimmer and colleagues demonstrated that recombinant Escherichia coli, expressing isoprene synthase derived from a plant, produces isoprene in culture.23 These studies have led the way to consideration of applying the tools of metabolic pathway engineering to microorganisms, for conversion of sugars to isoprene in large-scale bioreactors as a commercial process. Subsequently, other groups have reported the production of low amounts of isoprene in recombinant organisms by expressing isoprene synthase derived from a plant in photosynthetic bacteria.24,25 The isoprene concentrations reported in these studies have not yet been shown to be viable at a commercial scale.

4. BioIsoprene platform
The development of an integrated process for producing, recovering, and purifying isoprene monomer from a recombinant microorganism (BioIsoprene) presents many challenges requiring the integration of biotechnology and chemical engineering at a skill level not usually resident in a single company. A research collaboration between Genencor (a division of Danisco US Inc.) and The Goodyear Tire & Rubber Company has provided a forum in which a major C5

Figure 2. Current global production of isoprene

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chemical can be developed using a bioCollaborative research initiative based manufacturing method (Figure 3). The production of BioIsoprene by engineered bacteria from renewable feedstocks, offers the opportunity to deliver a sustainable manufacturing process to supply isoprene for its many current uses. Through use of renewable feedstocks, the production of BioIsoprene is expected to be advantageous compared to petroleum-based proBioIsoprene cesses for producing isoprene, with respect Renewable Microbial strain Large-scale Polymer-grade recovery & feedstock(s) development fermentation BioIsoprene purification to reductions in nonrenewable-energy know-how demand and global warming potential based on greenhouse gas emissions. Such a process would also provide for the potential Integrated biobased process for production of BioIsoprene unmet needs of isoprene not currently met due to volatility in cost and limited avail- Figure 3. Collaborative research initiative between Genencor and The Goodyear Tire & Rubber ability of petroleum-derived isoprene. For Company to develop an integrated biobased process to produce BioIsoprene the United States, a domestic, reliable, and renewable source of isoprene is imperative to national security and an issue of economic competitiveness. A sustainable Glucose manufacturing process for isoprene would C6 3C reduce US dependence on foreign oil and MEP pathway (C5) offer jobs to feedstock suppliers. Sucrose/ biomass Design considerations for a cell factory Isoprene Isoprene Cells synthase recovery 3C 2C producing chemicals: Genencor has a long Glycerol history in the design and operation of cell MVA pathway 16C 2C factories. Over the years, the company has produced industrial enzymes, commodPlant oils ity chemicals, and biochemicals including 1,3-propanediol,26 indigo,27 tryptophan,28 and ascorbic acid.29 The production of iso- Figure 4. Conceptual view of BioIsoprene cell factory prene by fermentation is similar to previous metabolic engineering efforts with respect to the development of the production organism, but encomTable 1. Catalytic parameters of characterized plant passes some unique differences, such as the extremely flammable isoprene synthases nature of the product and the recovery of the molecule as a gas rather Specific Activity KmDMAPP than a liquid. Design of the production strain involves engineering Enzyme (U/mg) (M) of endogenous and heterologous pathways using molecular biology, Kudzu (recombinant)27 0.075 7,700 biochemistry, metabolomics, and fermentation techniques combined 20 Aspen (native) 0.5 8,000 with special safety considerations to ensure proper handling and con28 tainment of the isoprene. The general Scheme for designing a flexible Poplar (recombinant) 0.16 9,000 microbial cell factory for isoprene production is shown in Figure 4. The use of many different sustainable industrial carbon sources are shown, with their direct links into central metabolism. Further details The key enzyme isoprene synthase: Since a microbial isoprene of design concepts are discussed below. The key components for isosynthase has yet to be identified, the enzyme used for BioIsoprene prene production are the enzyme isoprene synthase and the isoprenoid synthesis has been derived from plants. Many plants naturally proprecursor pathway(s) that are engineered into a cell to maximize carduce isoprene by the catalytic elimination of pyrophosphate from bon flux, while maintaining the energy and redox balance. Isoprene DMAPP. A number of biochemical studies of plant isoprene synthase produced in the fermentor is recovered continuously from the off-gas have been reported.23,30,31 However, sequence data for isoprene synoutside the production vessel. thase exists for only two plant families, namely, kudzu (the Asian

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GLUCOSE

G6P F6P F1,6BP Triose-3P

6PG

P5P

MEP Pathway
S7P IPP +CO2 E4P Thiamine

Isoprene
Idi DMAPP

MEP

IspS

IspH FldA - Fpr

-NADPH

HDMAPP IspG FldA - Fpr -NADPH

DXS DXP

DXR

IspD

IspE DCMEP -ATP

IspF MECP

MEP DCM -NADPH -CTP Pyridoxal MvaE

MvaS AcAc-CoA HMG-CoA

MvaE Mevalonate -2 NADPH MVK -ATP PMev

Pyr Ac-CoA AcCoA

Ac-CoA

TCA

MVA Pathway

-ATP PPMev -ATP +CO 2 IPP

PMK

MVD DMAPP Idi IspS

Isoprene

MVA

Figure 5. Connection of the two isoprenoid pathways to central metabolism. The central pathways for carbon metabolism in E. coli; glycolysis (light blue arrows), pentose phosphate (dark blue arrows), Entner-Doudoroff (green arrows), and the TCA cycle (purple arrows) are shown with the branch points for the MVA (yellow arrows) and MEP (orange arrows) pathways for isoprenoid production (adapted from Ref. 32). Abbreviations and pathway components: ATP, adenosine triphosphate; CTP, cytosine triphosphate; G6P, glucose-6-phophate; F6P, fructose6-phosphate; F1,6BP, fructose 1,6-bisphosphate; pyruvate; AcCoA, acetyl-coenzyme A; NAD+/NADH, nicotinamide adenine dinucleotide/ reduced form; NADP+/NADPH, nicotinamide adenine dinucleotide phosphate/reduced form. CO2, carbon dioxide; 6PG, 6-phosphogluconate; P5P, pyridoxal-5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reductase; MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol; IspD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; DCM, 4-diphosphocytidyl-2-C-methylerythritol; IspE, 4-diphosphocytidyl-2-C-methyl-Derythritol kinase; DCMEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MECP, 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate; IspG, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate synthase; HDMAPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; IspH, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate reductase; FldA, flavodoxin I; Fpr, ferredoxin NADP+ reductase; DMAPP, dimethylallyl pyrophosphate; IPP, isopentenyl diphosphate; IDI, IPP isomerase; IspS, isoprene synthase; MvaE, acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase; MvaS, mevalonate synthase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, diphosphomevalonate decarboxylase; AcAcCoA, acetoacetyl coenzyme A; HMG-CoA, 3-hydroxy3-methyl-glutaryl-CoA; PMev, phosphomevalonate; PPMev, diphosphomevalonate; IsopreneMEP/IsopreneMVA, isoprene produced via the MEP or the MVA pathway, respectively. vine, Pueraria montana) and poplar (Populus). Due to the poor kinetics of this class of enzyme (Table 1), protein engineering is required to obtain an improved enzyme capable of providing the rates of production of isoprene needed for a commercially viable process. MVA and MEP pathways for production of DMAPP: DMAPP, the substrate for isoprene synthase and its isomer isopentenyl diphosphate (IPP), together known as isoprene units, are the building blocks of terpenes and higher isoprenoids (C10 to >C55). Isoprene, a hemiterpene, is formed by catalytic elimination of pyrophosphate from DMAPP by isoprene synthase. DMAPP and IPP are essential compounds in all living organisms and are produced via one of two known pathways, the mevalonic acid (MVA) pathway present in eukaryotes and

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some prokaryotes, and the 5-methyl erythritol phosphate (MEP), also known as DOXP or DXP pathway present in prokaryotes and plants (Figure 5).32 Carbon flux into both pathways stems from intermediates derived from sugar metabolism via central metabolism (acetylCoA for MVA, pyruvate and glyceraldehyde 3-phosphate for MEP). Thus, isoprene is a suitable chemical to be produced by engineered microorganisms through either of the isoprenoid pathways, as sugars derived from biomass hydrolysis represent a sustainable feedstock for isoprene production at industrial scale. While DMAPP can be produced by both isoprenoid pathways, the MVA pathway is by far the best characterized as it has been exploited industrially for production of isoprenoids in both yeast and bacteria. This pathway is amenable to molecular biology manipulations and metabolic pathway engineering in both prokaryotes and eukaryotes (yeast), and high titers of isoprenoids from this pathway have been reported.33 The MEP pathway, on the other hand, was discovered relatively recently and has not been studied to the same extent.34 The pathway is linked to essential cell functions and contains iron-sulfur cluster enzymes which are poorly characterized on a biochemical level.35,36 There are a few examples of isoprenoid production via the MEP pathway by overexpression of one or more enzymes in the pathway; however, levels of product for commercial production were not achieved.37-40 Optimization of the carbon flux through the MEP pathway sufficient for commercial production of isoprene or isoprenoids is challenging. Despite the challenge, the optimization of the MEP pathway is worth pursuing, as it provides an increased yield of product from feedstock compared to that by the MVA pathway (30.2%, compared to 25.2% yield on glucose), as shown by the theoretical/ calculated yield equations below. Production of isoprene from glucose in presence of oxygen via engineered MVA pathway: E. coli engineered with MVA pathway to isoprene requires three AcCoA, three ATP and two NAD(P)H per isoprene produced. Therefore: (1) 1.5 Glc 3 AcCoA + 3 ATP + 3 CO2 + 6 NAD(P)H (glycolysis) (2) 3 AcCoA + 2 NAD(P)H MVA (3) MVA + 3 ATP Isoprene + CO2 (1) to (3) 1.5 Glc Isoprene + 4 CO2 + 4 NAD(P)H Overall 1.5 Glc + 2 O2 Isoprene + 4 CO2 + 5 H2O Isoprene mass yield on glucose is 25.2% Production of isoprene from glucose in presence of oxygen via engineered MEP pathway: E. coli engineered with MEP pathway to isoprene requires pyruvate (Pyr) and glyceraldehyde 3-phosphate (Gap), three ATP and three NAD(P)H per isoprene produced. Therefore: (1) Glc Pyruvate + Glyceraldehyde 3-phosphate + NAD(P)H (glycolysis) (2) Pyruvate + Glyceraldehyde 3-phosphate + 3 ATP + 3 NAD(P)H IPP (3) IPP DMAPP Isoprene (1) to (3) Glc + 3ATP + 2 NAD(P)H Isoprene + CO2 Overall 1.25 Glc + 0.5 O2 Isoprene + 2.5 CO2 + 3.5 H2O Isoprene mass yield on glucose is 30.2%

Isoprene is derived from intermediates in central metabolism: As described above, the first enzymes in both the MEP and MVA pathways use intermediates in central metabolism as substrates, namely, glyceraldehyde-3-phosphate + pyruvate or acetyl-CoA, respectively. These pathways have differing requirements for reducing power and energy. Thus, in addition to optimizing carbon flux through the MEP or MVA pathways, the levels of the central metabolism intermediates must also be maximized, and the reducing power/energy balance optimized by metabolic engineering. There are several examples of metabolic engineering of the glycolytic pathway to enhance the production of small molecules, and many of the approaches are applicable to isoprene production. Production of isoprene at high titer: We have exploited the MVA pathway in combination with plant (Populus alba and kudzu) isoprene synthases (IspS) combined with central metabolism engineering to successfully demonstrate isoprene production at high titer in E. coli. The MVA pathway was cloned into E. coli (which already has its native MEP pathway) as two synthetic operons the upper pathway converting AcCoA to MVA, and the lower pathway producing DMAPP from MVA, consisting of a combination of bacterial and yeast enzymes. The lower pathway operon is integrated into the chromosome while the upper pathway operon and the isoprene synthase gene plus an additional copy of mvk (from Methanosarcina mazei) are expressed on two different plasmids. Both plasmid-encoded gene sets are driven from the inducible Ptrc promoter, while the lower MVA pathway is driven from a constitutive promoter (Figure 6). The first synthetic operon, the upper pathway, from Enterococcus faecalis consists of two genes (mvaE and mvaS) encoding three enzymatic activities. The mvaE gene is a single gene encoding a protein that is a fusion of the thiolase and HMG-CoA reductase activities (the first and third enzymatic steps respectively; Figures 5 and 6). The mvaS gene encodes HMG-CoA synthase, the protein for the second enzymatic step. These enzymes together produce one molecule of mevalonic acid from three molecules of AcCoA. Mevalonic acid is converted to DMAPP through the second synthetic operon the lower MVA pathway which consists of MVK, PMK MVD, and IDI enzymes from Saccharomyces cerevisiae and Methanosarcina mazei (Figure 5). This engineered pathway plus the cloned isoprene synthase gene from kudzu (Figure 6, plasmid 2a) in E. coli together are capable of producing isoprene from glucose at a high titer of approximately 20 g/L in 40 h of fermentation. The optical density (OD550) of the three strains described in Figure 6 following fermentation did not change as a result of the increased isoprene production (20 g/L to 60g/L), indicating that the modifications resulted in a more efficient isoprene-producing cell (Figure 7A). By changing the isoprene synthase gene to that from P. alba IspS (Figure 6, plasmid 2), the titer further increased to approximately 30 g/L in 40 h of fermentation and to greater than 40 g/L after 55 h of fermentation (Figure 7B). Additional increases in isoprene titer were achieved by improving the carbon flux through the pentose phosphate pathway, but E. coli

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mvaE

mvaS

Plasmid 1: Upper pathway


Ptrc RBS RBS Terminator

Plasmid 2: a/b) Kudzu/P. alba isoprene synthase + MVK

Kudzu or P. alba ISPS

mvk (archaeal)

Ptrc RBS

RBS

Terminator

P. alba ISPS

mvk (archaeal) pgl

c) P. alba isoprene synthase + MVK + PGL

Ptrc RBS

RBS

RBS

Terminator

pstS

MVK

PMK

MVD

IDI

glmS

PGI1.2

RBS

RBS

Terminator

Lower pathway integrated on the chromosome

Figure 6. Construction of the high titer BioIsoprene-producing E. coli strain. The upper pathway (Plasmid1) consists of mvaE and mvaS cloned behind the Ptrc in the plasmid backbone pCL1920. Plasmid 2 consists of either Kudzu (a) or P. alba (b) ISPS and mvk (archaeal) from Methanosarcina mazei or P. alba ISPS, mvk (archaeal) plus pgl (c) cloned behind Ptrc in the plasmid backbone PtrcHis2. The lower pathway consists of MVK, PMK, MVD, and IDI from S. cerevisiae cloned behind the PGI1.2 promoter and integrated at the att7 site in the E. coli chromosome. Ptrc: trc promoter; RBS: ribosome binding site; ISPS: gene encoding isoprene synthase; MVK: gene encoding mevalonate kinase; pgl: E. coli gene encoding phosphonogluconolactonase; pstS: gene upstream of the att7 integration site; PGI1.2: GI1.2 promoter19; MVK: mevalonate kinase gene from S. cerevisiae; PMK; phosphomevalonate kinase gene from S. cerevisiae; MVD: phosphomevalonate decarboxylase gene from S. cerevisiae; and IDI: IPP isomerase gene from S. cerevisiae; glmS: gene downstream of the att7 integration site.

strain BL21 has low phosphogluconolactonase (PGL) activity resulting in low carbon flux through the pentose phosphate pathway.41 The MVA pathway requires NADPH as a cofactor (3-hydroxy-3-methyl-glutarylCoA reductase, or HMG-CoA reductase), and it has been shown that expression of secreted proteins can be improved by overexpression of phosphogluconolactonase (PGL) in E. coli strain BL21. By constitutively expressing the pgl gene (ybhE ) from E. coli strain MG1655 on the same plasmid as the mvk and ispS genes (Figure 6, plasmid 2c), we have demonstrated that the newly engineered E. coli strain is able to produce isoprene from glucose or biomass at titers greater than 60 g/L in a fed batch 14-liter fermentation process (Figure 7B). The BioIsoprene released in the off-gas is of high purity and can be recovered from the gas-phase with methods discussed further below.

Further progress in optimizing the process described above with E. coli as a model and potential first-generation production organism has been made. Deep-tank culture conditions have been used to produce isoprene and recover the off-gas of the fermentation. Using this type of system, we have produced isoprene at greater than 60 g/L culture broth titers, with a volumetric productivity of 2 g/L/hr, a yield of 11% isoprene from glucose, and cell productivity of 0.85 g isoprene/g dry cell weight.42 The potential yield of the MEP pathway is significantly higher than that of the MVA pathway. Metabolic engineering of the MEP pathway has been demonstrated in other systems to increase carbon flux to isoprenoids.37-39 An optimization strategy, similar to that described above for the MVA pathway, is being pursued in utilizing the MEP pathway for isoprene production.42

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ant of high levels of CO2. Carbon loadings of 10% to 15% w/w are possible before significant breakthrough of BioIsoprene product occurs. Desorption can be achieved by steam or nitrogen, 100 followed by condensation of the BioIsoprene vapor to the concentrated liquid product. The purity of the liquid product is very high (usually over 99.5% w/w); however, the nature of the impurities present is of key importance. 10 The catalysts used to polymerize isoprene to cis Kudzu IspS + MVK polyisoprene are very sensitive to certain com Poplar IspS + MVK Poplar IspS + MVK + PGL pound classes, such as organosulfur compounds, ketones, and acidic hydrocarbons like acetylenes 1 and 1,3-cyclopentadiene. Analysis of BioIsoprene 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Time (hr) lots by gas chromatography revealed that most impurities are polar, oxygenated compounds such 70 as alcohols, esters, and ketones, with little to no Kudzu IspS + MVK detectable C5 hydrocarbons other than isoprene. Poplar IspS + MVK 60 This greatly simplifies purification of BioIsoprene Poplar IspS + MVK + PGL relative to conventional processes that are used to 50 recover petroleum-derived isoprene. The BioIsoprene production system is a unique 40 case of in situ product removal which has substantial advantages from the standpoint of end30 product toxicity and final product recovery and 20 purification. Generally, biobased production systems are challenged by toxicity and recovery 10 issues associated with a product that accumulates either in the extracellular aqueous phase or in 0 an oil phase in the fermentation broth. Isoprene, 0 5 10 15 20 25 30 35 40 45 50 55 60 65 being a high-vapor-pressure compound (400 Time (hr) mm Hg at 20C) that is a gas at fermentation temperatures, renders the intracellular environFigure 7. BioIsoprene titers from 14 L fed batch fermentation. E. coli strains containing the constructs described in Figure 6 were grown in glucose fed batch, 14-liter fermentations ment and culture medium substantially free of for the indicated time. (A) Cell density of the culture was determined by monitoring optical the product, and thus does not accumulate at density (OD) at a wavelength of 550 nm. (B) BioIsoprene titer was measured from the offtoxic concentrations. As inexpensive sugar feedgas using a mass spectrometer and is presented as g/L fermentation broth. Strains contain stocks move to more crude forms, the advanthe integrated lower pathway and plasmid-borne upper pathway plus either kudzu isoprene tage of the gas-phase recovery becomes more synthase + MVK (archaeal; blue diamonds); P. alba isoprene synthase + MVK (archaeal; yellow squares); or P. alba isoprene synthase + MVK (archaeal) + pgl (red triangles). obvious and important for the economics of the overall process. Many of the future feedstocks, e.g., cellulosic hydrolysates, contain a variety of small- and large-molecular-weight compounds and color bodies. BioIsoprene recovery: BioIsoprene vapor can be recovered from Such feedstocks will be useful to produce products that are volatile fermentation off-gas and concentrated into the liquid form by variat ambient fermentation temperature and recovered in the gas phase, ous means, including adsorption/desorption from activated carbon, thus reducing the purification requirements. absorption/stripping from a solvent, compression/condensation, and membrane permeation.43-45 The choice of method is dictated by the BioIsoprene-containing tires: A biobased process can be deemed commercially relevant only when the performance of the product percent isoprene in the off-gas, and the levels of other gases such as derived from it matches or exceeds that of its chemically derived CO2 and water vapor, in addition to the scale of the recovery operation. counterpart. Samples of high-purity monomer (Figs. 8A, B) were On smaller scales the most convenient method employs carbonpolymerized into the synthetic rubber, cis-1,4-polyisoprene followbased adsorbents which can remove BioIsoprene vapor from fering well-known procedures.46 The catalyst is a multi-component, mentation off-gas with high efficiency. The method is applicable over a wide range of BioIsoprene vapor concentrations and is tolerheterogeneous mixture made up of titanium tetrachloride, anhydrous
Isoprene titer (g/L broth)
Optical density (OD550)

1000

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hexane, diphenyl ether, and triisobutyl aluminum. Polymerization is conducted at 40C, and triisopropanol amine is used as the shortstop. Butylated hydroxytoluene is added as an antioxidant to stabilize the polymer during transportation and storage. As expected, polymerization of the samples provided by Genencor produced a polymer that was equivalent to commercially produced Natsyn (Goodyear polymerized cis-1,4 polyisoprene elastomers) within experimental limits. The BioNatsyn polymer itself, shown in Figure 8C, had an average Mooney viscosity of 71 (5), Tg of 61.7C (1), cis-1,4 content of 98.5%, and trans-1,4-content of 1.5%. The Mooney viscosity is a measure of the polymers molecular weight and distribution. In addition, tan . versus frequency curves as obtained on an RPA2000 Rubber Process Analyzer (Alpha Technologies, Akron, Ohio, USA), for polymers derived from the BioIsoprene monomer and petroleumbased isoprene, were superimposable within experimental limits over the frequency range of 120 to 120,000 Hz. The tire tread compound was prepared according to the recipe shown in Table 2. As expected, the tread composition containing BioNatsyn behaved similar to a composition containing petroleumderived polymer. Measured properties included cure time at 150C (19.6 min), 300% moduls (9.2 MPa), tensile strength (16.5 MPa), elongation at break (542%), Mooney viscosity (45), and Shore A hardness (71). These compared well to properties obtained from commercially prepared Natsyn polyisoprene (petroleum based): cure time (21.8 min), 300% modulus (7.2 MPa), tensile strength (16.1 MPa), elongation break (598%), Mooney viscosity (45), and Shore A hardness (62). This tread compound was used to produce the prototype tires, one of which is shown in Figure 8D. These results convincingly demonstrate the commercial reality for the production of BioIsoprene for tires and other applications. The image shown in Figure 8D is a photograph of an actual tire made with BioIsoprene monomer.

quantities of a basic C5 hydrocarbon that, in principle, can be used as a feedstock for a large number of value-added products. One of these products is a synthetic cis-polyisoprene (synthetic rubber), which for decades has been recognized as a suitable replacement for natural rubber in many applications. Additionally, there is potential for creating polyisoprenes with improved material properties.47 The BioIsoprene process offers a real possibility for obtaining the quantities of low-cost isoprene needed to produce a meaningful large-volume alternative to Hevea natural rubber and petroleum-derived isoprene. BioIsoprene will have broader commercial applications beyond the biochemical uses of isoprene in synthetic rubber, adhesives, and specialty elastomers. As a C5 hydrocarbon, BioIsoprene has inherent

TABLE 2. Tire tread recipe


Ingredient
BioNatsyn Carbon black Silica Oils/waxes Antidegradants Curatives

Amount (parts per hundred rubber)


100 60 12.5 13 8.25 10.8

5. Summary
A first-of-its-kind biobased process to produce isoprene (BioIsoprene) from renewable raw materials is in development by Genencor and The Goodyear Tire & Rubber Company. Early-stage efforts have resulted in the development of an E. coli-based production system capable of channeling carbon through the MVA biosynthetic pathway to deliver isoprene at titers exceeding 60 g/L. Unlike other biobased systems to produce biochemicals, BioIsoprene is produced as a gas-phase product that is released as soon as it is produced into the vapor phase of the reactor without any noticeable negative physical or inhibitory impacts on the biological host. Polymer-grade BioIsoprene is recovered from the integrated process. Potential benefits of the gas-phase nature of the product include: (1) reduction and/or elimination of feedback inhibition by the isoprene product on further synthesis; (2) efficient recovery and purification of polymer-grade BioIsoprene from the fermentation broth; and (3) possibility to use crude, low-cost feedstocks. The development of BioIsoprene represents a major achievement for industrial biotechnology because it has the potential to enable production of isoprene from renewable raw materials to deliver commercial

Figure 8. Production and purification of BioIsoprene-containing concept tires. (A) Small scale sample of liquid BioIsoprene monomer from fermentation off-gas; (B) Several kilogram samples of liquid BioIsoprene; (C) Synthetic rubber sample polymerized from BioIsoprene monomer; (D) Graphic representation of the concept tires produced by Goodyear from BioIsoprene-containing synthetic rubber.

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fuel properties and represents a key biobased intermediate that can be converted to a drop-in transportation fuel additive (using chemical catalysis) to C10 and C15 biobased hydrocarbon fuels, thus addressing performance gasoline, jet fuel, and biodiesel markets. As the technology is expanded into commercial development, the model used will be based on the strategy of building a BioIsoprene biorefinery that has, as its center point, the low-cost manufacture of high-purity isoprene that can then be sold both into the chemical and biofuel markets. This route to biochemicals and to biobased hydrocarbon fuels for the gasoline, diesel, and aviation markets has many attractive features.

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Acknowledgments
The authors thank Roopa Ghirnikar for expert editorial assistance.

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