Scalable Production of Mechanically Tunable Block Polymers from Sugar

Scalable production of mechanically tunable block polymers from sugar

Mingyong Xionga,1, Deborah K. Schneidermanb,1, Frank S. Batesa, Marc A. Hillmyerb,2, and Kechun Zhanga,2

Departments of aChemical Engineering and Materials Science and bChemistry, University of Minnesota, Minneapolis, MN 55455-0431

Edited by Malcolm H. Chisholm, The Ohio State University, Columbus, OH, and approved April 11, 2014 (received for review March 11, 2014) Development of sustainable and biodegradable materials is essen- physical and mechanical properties by controlling molecular ar- tial for future growth of the chemical industry. For a renewable chitecture, composition, and molar mass. product to be commercially competitive, it must be economically A specific example that illustrates the utility of this approach is viable on an industrial scale and possess properties akin or superior ABA triblock thermoplastic elastomers. Here, rigid (glassy or to existing petroleum-derived analogs. Few biobased polymers semicrystalline) “A” endblocks microphase separate to form nano- have met this formidable challenge. To address this challenge, scopic domains that tether together the soft, low glass transition we describe an efficient biobased route to the branched lactone, temperature (Tg) regions comprised of the center block “B” com- β-methyl-δ-valerolactone (βMδVL), which can be transformed into ponent (19–22). This design results in outstanding mechanical a rubbery (i.e., low glass transition temperature) polymer. We fur- properties and has been the basis for the commercially successful ther demonstrate that block copolymerization of βMδVL and lac- poly(styrene)–poly(diene) variants (23). Many PLA-containing tide leads to a new class of high-performance polyesters with block polymers have been reported; several have been shown to tunable mechanical properties. Key features of this work include be flexible, tough, elastic, and hydrolytically degradable (24–27). the creation of a total biosynthetic route to produce βMδVL, an Unfortunately the starting materials used to prepare these pol- efficient semisynthetic approach that employs high-yielding chem- ymers are either derived from fossil fuels or from natural prod- ical reactions to transform mevalonate to βMδVL, and the use of ucts that are prohibitively expensive. The development of an controlled polymerization techniques to produce well-defined economically viable rubbery polymer that can be used in com-

– β δ – CHEMISTRY PLA P M VL PLA triblock polymers, where PLA stands for poly bination with PLA to prepare mechanically superior block pol- (lactide). This comprehensive strategy offers an economically via- ymers therefore is the grand challenge that must be addressed ble approach to sustainable plastics and elastomers for a broad for these materials to be competitive with the analogous styrenic range of applications. block polymers. The need for an efficient, scalable, process to a methyl branched rubbery polyester | block copolymer | biobased production | lactone suitable for ring-opening transesterification polymerization mevalonate pathway (ROTEP) led us to examine candidates that could be produced by

biosynthesis with both high titer and high yield. After surveying SCIENCES olymeric materials account for nearly $400 billion in eco- the appropriate metabolic pathways, we identified β-methyl- Pnomic activity annually and represent the third largest δ-valerolactone (βMδVL) as an attractive target. Based on our APPLIED BIOLOGICAL manufacturing industry in the United States (1). Currently pe- experience with simple aliphatic polyesters we anticipated that troleum-derived polymers—for example polyethylene, polystyrene, the elastomeric polymer PβΜδVLcouldbecombinedwithhard and polyvinylchloride—dominate this market. Although these semicrystalline or glassy poly(lactide) (PLLA or PLA, respectively) materials are widely useful, their manufacture and disposal to produce P(L)LA–PβMδVL–P(L)LA triblock polymers that present inescapable environmental challenges that are costly to correct and simply unsustainable in the long term. To ensure the Significance continued vitality of the polymer enterprise, it is necessary to invent high-performance polymers that are both sustainable and In recent years there has been extensive research toward the cost-competitive. In recent years, ingenious advances in synthetic development of sustainable polymeric materials. However, biology have enabled the economical production of fuels (2–7), environmentally benign, bioderived polymers still represent chemicals (8–12), and complex natural products (13–16) from a woefully small fraction of plastics and elastomers on the renewable sugars. This elegant manipulation of existing organ- market today. To displace the widely useful oil-based polymers isms to efficiently produce valuable metabolites from inexpensive that currently dominate the industry, a bioderived synthetic feedstocks represents a triumph of modern science and engi- polymer must be both cost and performance competitive. In neering and offers society the promise of renewable, environ- this paper we address this challenge by combining the efficient mentally compatible next-generation materials. bioproduction of β-methyl-δ-valerolactone with controlled poly- Whereas there has been steady scientific progress toward the merization techniques to produce economically viable block production of synthetic polymers from renewable resources, the polymer materials with mechanical properties akin to com- fraction of high-performing, biobased, degradable polymers on mercially available thermoplastics and elastomers. the market is today minuscule (1). Arguably the most successful

example to date is poly(lactide) (PLA), a compostable aliphatic Author contributions: M.X., D.K.S., M.A.H., and K.Z. designed research; M.X. and D.K.S. polyester derived from the fermentation product lactic acid. performed research; M.X., D.K.S., F.S.B., M.A.H., and K.Z. analyzed data; and M.X., D.K.S., However, the brittle nature of PLA and other commercial ali- F.S.B., M.A.H., and K.Z. wrote the paper. phatic polyesters such as poly(butylene succinate) and poly The authors declare no conflict of interest. (hydroxyalkanoate)s has thwarted their broad-based utility. Ex- This article is a PNAS Direct Submission. panded market penetration will pivot on the development of Freely available online through the PNAS open access option. products endowed with tunable combinations of properties, e.g., 1M.X. and D.K.S. contributed equally to this work. soft, ductile, and tough (17, 18). Among myriad strategies that 2To whom correspondence may be addressed. E-mail: [email protected] or hillmyer@ have been used to this end, PLA-containing block polymers umn.edu. represent a particularly attractive approach. These hybrid This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. materials offer opportunities to easily tune a wide range of 1073/pnas.1404596111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1404596111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 offer exceptional mechanical properties (26, 27). βMδVL can identify the optimum set of MvaS and MvaE for mevalonate pro- be generated by direct fermentation of glucose or through the duction (Fig. 2A and Table S2). high-performance liquid chro- production of the intermediate compound mevalonate. These matography (HPLC) analyses indicated that, with the exception two approaches to βMδVL, one entirely biochemical and the of MvaS from M. maripaludis and M. voltae,allheterologous other semisynthetic, represent facile chemical transformations enzymeswereactiveinE. coli. The strain expressing both mvaS – (Fig. 1). Because the theoretical yield of this lactone monomer is and mvaE genes from L. casei produced 14.6 g L 1 mevalonate – 0.42 g/g glucose, we estimate that this product can be produced from 40 g L 1 glucose, better than any other combination (V in for less than $2 a kilogram (Table S1), compatible with large- Fig. 2A). The strain expressing the L. casei enzymes produced 50% volume commodity applications. more mevalonate than other engineered E. coli strains described in With the bioderived βMδVL in hand, we demonstrated that previous work (30–32). bulk ROTEP using an organocatalyst proceeds at room temper- To biosynthesize anhydromevalonolactone from mevalonate, ature to high conversion, yielding an amorphous polyester with we explored the potential of siderophore pathway enzymes. In Tg = –51 °C (26, 28). Simple chain extension through reaction with fungi acyl-CoA ligase SidI converts mevalonate to mevalonyl- lactide (LLA or LA) leads to P(L)LA–PβMδVL–P(L)LA tri- CoA, and enoyl-CoA hydratase SidH transforms mevalonyl-CoA block polymers that can be tuned to have mechanical properties to anhydromevalonyl-CoA (33). We speculated that anhy- ranging from stiff tough plastics to soft and highly extendible dromevalonyl-CoA could spontaneously cyclize into anhy- elastomers (Fig. 1C). dromevalonolactone intracellularly. To test this hypothesis, we synthesized sidH and sidI genes based on E.-coli-optimized codons Results for protein expression. Using BlastP analysis, homologous SidI Construction of a Nonnatural Metabolic Pathway for Biosynthesis of and SidH from Aspergillus fumigatus, Neurospora crassa, Phaeos- βMδVL. Because βMδVL is not a natural metabolite, we con- phaeria nodorum,andSclerotinia sclerotiorum were chosen for structed an artificial biosynthetic pathway to create an engineered fermentation experiments (34). Results revealed that the strain – Escherichia coli strain (Fig. 1A). Our biosynthetic strategy builds expressing SidI and SidH from A. fumigatus produced 730 mg L 1 on the mevalonate pathway that previously has been engineered anhydromevalonolactone; the strain carrying enzymes from – for the synthesis of artemisinin and terpenoids in Saccharomyces N. crassa also generated 540 mg L 1 anhydromevalonolactone cerevisiae and E. coli, respectively (29, 30). In this work we im- (Fig. 2B and Table S3). The other two enzyme pairs did not proved the production of mevalonate in E. coli and also ex- function properly in E. coli. panded the pathway to synthesize βMδVL from this precursor. To explore the reduction of anhydromevalonolactone to βMδVL The overall nonnatural pathway has three components: (i) by enoate reductases, we cloned Oye2 and Oye3 from S. cerevisiae overexpression of the mevalonate-producing enzymes; (ii)in- (35), wild-type and mutant YqjM (C26D and I69T) from Bacillus troduction of the fungal siderophore proteins to synthesize subtilis (36). The strains carrying the anhydromevalonolactone anhydromevalonolactone (AML); (iii) reduction of AML to βMδVL pathway and Oye2 or mutant YqjM successfully produced 180 or –1 by enoate reductases. 270 mg L βMδVL, respectively, directly from glucose (Fig. 2C To generate an acetoaceyl-CoA pool we used the E. coli and Table S4). Thus, we successfully constructed an artificial endogenous enzyme acetyl-CoA acetyltransferase AtoB. First biosynthetic pathway to βMδVL from glucose. HMG-CoA synthase (MvaS) and HMG-CoA reductase (MvaE) were cloned to provide a route for the production of mevalonate Semisynthesis of βMδVL from Mevalonate. Whereas directed evo- from this pool. To maximize mevalonate flux, the Protein-Protein Basic lution approaches undoubtedly will improve the aforementioned β δ Local Alignment Search Tool (BlastP) was used to identify MvaS M VL biosynthetic pathway (5), we also developed a semi- and MvaE from various organisms: Enterococcus faecalis, Staph- synthetic approach for the immediate large-scale production of β δ ylococcus aureus, Lactobacillus casei, Methanococcus maripaludis, M VL. In this route the fermented mevalonate is first dehy- β δ and Methanococcus voltae. Combinatorial tests were used to drated to anhydromevalonolactone, then reduced to M VL (Fig. 1B). To scale up the production of mevalonate, the E. coli strain carrying genes from L. casei was tested for fermentation in a 1.3-L bioreactor. During the fermentation, the strain achieved a produc- – – – tivity of 2 g L 1h 1 mevalonate with the final titer reaching 88 g L 1 (Fig. 2D and Table S5). The yield for this semibatch fermentation was 0.26 g/g glucose. To prepare anhydromevalonolactone we added sulfuric acid directly to the fermentation broth and heated to reflux to catalyze the dehydration of mevalonate (Fig. 2E and Fig. S1). At a catalyst loading of 10% by volume, 98% of the mevalonate was converted to anhydromevalonolactone with a se- lectivity of 89% (Table S6). The resulting anhydromevalonolactone was isolated by solvent extraction using chloroform and hydroge- nated to βMδVL using Pd/C as the catalyst (bulk, room temperature, 350 psi H2,12h)to>99% conversion. The crude product was subsequently purified by distillationtoobtainpolymerization-grade monomer (Fig. 2F).

Polymerization of βMδVL. Based on previous work with alkyl- substituted δ-valerolactones, we suspected the ceiling temperature for the polymerization of βMδVL might be low (28, 37, 38). To favor high conversion we therefore conducted the polymer- Fig. 1. (A) Total biosynthetic pathway for the production of βMδVL. (B)A izations in bulk monomer at room temperature using the highly semisynthetic route to produce βMδVL from mevalonate. (C) Conversion of active organocatalyst triazabicyclodecene (TBD) (Fig. 3) (39). The βMδVL to an elastomeric triblock polymer that can be repeatedly stretched addition of TBD to neat βMδVL in the presence of benzyl alcohol to 18 times its original length without breaking. (BnOH) as the initiator ([βMδVL]0/[TBD]0/[BnOH]0 = 492/1/1.7)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1404596111 Xiong et al. Downloaded by guest on September 26, 2021 CHEMISTRY

Fig. 2. Total biobased production of βMδVL and semisynthetic route to this monomer. (A) Fermentation of mevalonate from different combinations of MvaS and MvaE. MvaE from E. faecalis plus MvaS from: I, E. faecalis; II, Staphylococcus aureus; III, L. casei. MvaS from L. casei plus MvaE from: IV, S. aureus;V,L. casei; SCIENCES VI, M. maripaludis; VII, M. voltae.(B) Anhydromevalonolactone fermentation with siderophore enzymes SidI and SidH from: A, A. fumigatus;B,N. crassa;C,P. nodorum;D,S. sclerotiorum.(C) Production of βMδVL through fermentation with enoate-reductase: 1, Oye2 from S. cerevisiae; 2, Oye3 from S. cerevisiae;3, APPLIED BIOLOGICAL wild-type YqjM from B. subtilis; 4, Mutant YqjM (C26D and I69T) from B. subtilis.(D) Production of mevalonate by fermentation of glucose in a 1.3-L bioreactor. (E) Acid catalyzed dehydration of mevalonate to anhydromevalonolactone monitored by refractive index (RI). (F) NMR spectrum of purified βMδVL prepared via hydrogenation of anhydromevalonolactone. Data shown in A–C include error bars that identify the range of the results obtained from three experiments (n = 3).

resulted in the rapid production of poly(βMδVL)—within 1 h at of lactide, and the polymerization initiated by addition of TBD.] room temperature (T = 18 °C) 75% of the monomer was con- Although depolymerization can be problematic for poly(βMδVL) sumed, within 3 h the reaction approached equilibrium (Fig. S2). when diluted in the presence of TBD, the conversion of βMδVL Determination of the residual monomer concentration over a range was virtually unchanged before and after the chain extension with of temperatures allowed us to calculate the thermodynamic param- lactide, suggesting that the addition of lactide to the end of the poly Δ o = − : ± : À1 eters for this polymerization ( Hp 13 8 0 3 kJ mol and (βMδVL) prevents significant depolymerization. Compared with Δ o = − ± −1 −1 Sp 46 1 J mol K ); these values correspond to a ceiling the poly(βMδVL) macroinitiators, polymers prepared using either β δ temperature (Tc) of 227 °C for the polymerization of neat M VL. extension strategy exhibited expected increases in mass average Practically, the limiting conversion for the bulk polymerization molarmass(M ) as determined by multiangle laser light scattering- is about 91% and 60% at 18 °C and 120 °C, respectively (Fig. m size exclusion chromatography (Fig. 4A). In addition, the 13C NMR S3). At 18 °C with as little as 0.05 wt% TBD, the bulk polymerization is well controlled with the conversion and ratio of monomer to added initiator dictating the molar mass of the polymer (Table S7). Using the diol initiator 1,4-phenylenedimethanol we obtained dihydroxy terminated poly(βMδVL).

Synthesis and Mechanical Properties of Block Copolymers. Poly (βMδVL) is an amorphous aliphatic polyester with a low glass transition temperature (Tg = –51 °C). To explore the potential of this rubbery polymer as the soft segment in thermoplastic elastomers we used dihydroxy terminated poly(βMδVL) to prepare triblock polymers with PLA endblocks. This was easily accom- plished by adding a solution of (±)or(–)-lactide (LA or LLA) β δ β δ β δ Fig. 3. Polymerization of M VL leading to P M VL and chain extension directly to a polymerization of M VL that was near equilibrium with (±)- or (-)-lactide (LA or LLA) yielding P(L)LA–PβMδVL–P(L)LA triblock (Fig. 3). [Alternatively, purfied telechelic poly(βMδVL) (free of polymer. TBD and HO-R-OH represent triazabicyclodecene and 1,4-phenyl- residual monomer and catalyst) could be dissolved in a solution enedimethanol, respectively.

Xiong et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 spectra of the resulting triblocks revealed no evidence of significant Conclusion tranesterification between the poly(βMδVL) and poly(lactide) blocks, We have developed a semisynthetic approach to βMδVL from consistent with clean formation of the desired ABA triblock archi- glucose that relies upon the fermentation of mevalonate and tecture (Fig. 4B). subsequent transformation of mevalonate to βMδVL. The high − Despite the structural similarity of poly(lactide) and poly titer of the fermentation (88 g L 1) and the efficiency of the β δ – β δ – ( M VL), P(L)LA P( M VL) P(L)LA triblock polymers readily chemical reactions used to produce the final product make the microphase separate at only moderate molar masses as evidenced overall process scalable and commercially promising. We also by differential scanning calorimetry (DSC) and small-angle X-ray have described a nonnatural total biosynthetic pathway for the scattering (SAXS). Both PLA–P(βMδVL)–PLA [from (±)-lactide, production of βMδVL , which obviates the need for additional LA] and PLLA–P(βMδVL)–PLLA [from (-)-lactide, LLA] exhibit chemical transformations. Optimization of this all-biosynthetic separate glass transitions for the midblock and endblock segments process (i.e., titer and yield comparable to the semisynthetic route) by DSC (Fig. 4C). In addition, the SAXS data from these tri- would further reduce the production cost of βMδVL. This bio- blocks showed well-defined scattering peaks that correspond to derived monomer can be readily converted from the neat state to self-assembled nanostructures with principal spacings ranging from 20 to 50 nm (Fig. 4D and Table S8). a rubbery hydroxytelechelic polymer using controlled polymeri- ± Predictably, the mechanical and thermal properties of these zation techniques at ambient temperature; addition of either ( ) β δ ordered block polymers are influenced by molar mass, tacticity or (-)-lactide to a poly( M VL) midblocks leads to well-defined of the poly(lactide) segments, and composition. By changing the ABA triblock polymers. We have shown that the thermal and endblock from the minority component (fLA = 0.29) to the majority mechanical properties of these materials can be tuned by con- component (fLA = 0.59) it is possible to access either elastomers or trolling molar mass, architecture, and endblock tacticity and have tough plastics. Moreover, at a fixed composition and molar mass, specifically demonstrated thermoplastic elastomers with proper- the use of semicrystalline PLLA endblocks leads to remarkably ties similar to commercially available styrenic block polymers. strong elastomers that rival commercially available petroleum based This work lays the foundation for the production of new biobased block copolymers in terms of recoverability, tensile strength, and polymeric materials with a wide range of potential properties ultimate elongation (Fig. 5 and Table S9) (23). and applications.

− Fig. 4. (A) Overlay of size exclusion chromatography traces obtained from PβMδVL (20.0 kg mol 1) and a corresponding PLLA–PβMδVL–PLLA (9.1–20.0–9.1 − − kg mol 1) triblock polymer. (B) 13C NMR spectra obtained from (Bottom)PβMδVL, (Middle) PLLA (10.0 kg mol 1), and (Top) PLLA–PβMδVL–PLLA (9.1–20.0–9.1 − − kg mol 1). (C) DSC thermograms recorded for (Bottom)PβMδVL and (Middle)PLA–PβMδVL–PLA (16.2–20.0–16.2 kg mol 1), and (Top) PLLA–PβMδVL–PLLA − − (9.1–20.0–9.1 kg mol 1). Data were taken while heating at a rate of 5 °C min 1 after cooling from 200 °C at the same rate. (D) SAXS pattern recorded at room temperature from PLA–PβMδVL–PLA (16.2–20.0–16.2 kg mol−1). Diffraction peaks at q*=0.185 nm−1, 2q*, 3q*, and 4q* are consistent with a periodic (d = 33 nm) lamellar morphology.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1404596111 Xiong et al. Downloaded by guest on September 26, 2021 Fig. 5. (A) Representative stress (σ) versus strain (e) results obtained in uniaxial extension for triblock polymers containing different volume fractions of

semicrystalline (fLLA) and glassy (fLA) blocks. Incorporation of relatively small amounts of the hard block (fLA = 0.29 and fLLA = 0.32) results in a soft (elastic modulus E = 1.9 and 5.9 MPa, respectively), highly extendable elastic material. Increasing the hard block content (fLA = 0.59) leads to a stiff (E = 229 MPA) and −1 ductile plastic. (B) Stress versus strain response of PLLA–PβMδVL–PLLA (18.6–70.0–18.6 kg mol )(fLLA = 0.32) during cyclic loading (1–20 cycles) to 67% strain − at a rate of 5 mm min 1. These results demonstrate nearly ideal elastic behavior with nearly complete recovery of the applied strain.

Materials and Methods basic alumina using cyclohexane as an eluent. The cyclohexane was removed β δ For details, see SI Appendix. under vacuum to obtain purified M VL.

Plasmids and Strains. All cloning procedures were carried out in the E. coli Polymer Synthesis. To synthesize poly(βMδVL) a monofunctional (benzyl al-

strain XL10-gold (Stratagene). Genes for mevalonate and βMδVL pathway cohol) or difunctional (1,4 benzene dimethanol) alcohol was added to CHEMISTRY were either PCR amplified from genomic DNA templates or codon-opti- monomer in a pressure vessel or glass vial and stirred with a magnetic stir mized and synthesized by GenScript. Three kinds of plasmids have been bar until completely dissolved. The ratio of monomer to alcohol was varied

constructed for the biosynthesis of βMδVL (Fig. S4). BW25113 (rrnBT14 to target polymers of different molecular weights. When the initiator was Δ Δ Δ lacZWJ16 hsdR514 araBADAH33 rhaBADLD78) transformed with the rele- dissolved, an appropriate amount of catalyst (∼0.05–0.2 mol% TBD or ∼0.5 vant plasmids was used to produce mevalonate or βMδVL. mol% diphenyl phosphate) relative to monomer was added to initiate the polymerization. The reaction was stirred and the conversion monitored us- Culture Condition and Analysis. E. coli strains were growth at 37 °C in 2XYT ing 1H NMR of crude quenched aliquots. Poly((L)LA)-b-poly(βMδVL)-b-poly rich medium (16 g/L Bacto-tryptone, 10 g/L yeast extract, and 5 g/L NaCl) sup- β δ ((L)LA) was prepared from a previously isolated and purified poly( M VL). SCIENCES μ plemented with appropriate antibiotics (ampicillin 100 g/mL and kanamycin The poly(βMδVL) prepolymer was first dissolved in a 1 M solution of (±)- μ 50 g/mL). Shake flask fermentations were performed with 125-mL conical flasks lactide or (–)-lactide in dichloromethane. After dissolution of the prepol- APPLIED BIOLOGICAL β containing M9 medium. After adding 0.1 mM isopropyl- -D-thiogalactoside, the ymer, 0.1 mol% TBD (relative to lactide) was added. The solution was stirred fermentation was performed for 48 h at 30 °C. Scale-up fermentation was for 10 min and then quenched by adding 5 equivalents of benzoic acid performed in 1.3 L Bioflo 115 Fermentor (NBS). The culturing condition was relative to TBD. The crude polymer samples were purified by precipitation in set at 34 °C, dissolved oxygen level 20%, and pH 7.0. The concentrations of methanol from dichloromethane/chloroform and subsequently dried. metabolites were measured by HPLC. ACKNOWLEDGMENTS. D.K.S. thanks Byeongdu Lee for assistance with Dehydration, Hydrogenation, and Purification. The fermentation supernatant acquisition of SAXS data, and Justin Bolton for photography assistance. was treated with concentrated H2SO4 to dehydrate mevalonate into anhy- Funding for this work was provided by the Center for Sustainable Polymers dromevalonolactone. The reaction temperature was 121 °C. Chloroform was at the University of Minnesota, a National Science Foundation (NSF)-supported used to extract anhydromevalonolactone from the reaction mixture. The Center for Chemical Innovation (CHE-1136607). The BioTechnology Institute results of dehydration are shown in Fig. S1 and Table S6. For hydrogenation, at the University of Minnesota is also acknowledged for support. Portions unreduced palladium on activated carbon [10% (wt/wt) Acros Organics] was of this work were performed at Sector 12-IDB of the Advanced Photon used as the catalyst. In a typical hydrogenation procedure, 10 g of catalyst were Source (APS). Use of the APS, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne added to a 300 mL high-pressure reactor with 50 mL anhydromevalonolactone. ∼ National Laboratory, was supported by the US DOE under Contract DE- The reaction was then allowed to stir under H2 ( 350 pounds per square inch AC02-06CH11357. Additional parts of this work were carried out in the gauge) at room temperature overnight to ensure quantitative conversion. University of Minnesota College of Science and Engineering Characteriza- The crude βMδVL was dried over calcium hydride for 12 h and then distilled tion Facility, which receives partial support from NSF through the National under vacuum (50 mTorr, 30 °C); the distilled product was passed through dry Nanotechnology Infrastructure Network program.

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