USOO845.5236B2
(12) United States Patent (10) Patent No.: US 8,455,236 B2 Beck et al. (45) Date of Patent: Jun. 4, 2013
(54) COMPOSITIONS AND METHODS OF PGL WO WO 2009,076676 A2 6, 2009 FOR THE INCREASED PRODUCTION OF WO WO 2009,076676 A3 6, 2009 ISOPRENE WO WO 2009,132220 A9 10/2009 WO WO 2010/OO3OOT A2 1, 2010 WO WO 2010/OO3OOT A3 1, 2010 (75) Inventors: Zachary Q. Beck, Palo Alto, CA (US); WO WO 2010/O14825 A1 2, 2010 Marguerite A. Cervin, Redwood City, WO WO 2010/078457 A2 T 2010 WO WO 2010/078457 A3 T 2010 CA (US); Alex T. Nielsen, Rungsted WO WO 2010, 124146 A2 10/2010 Kyst (DK); Caroline M. Peres, Palo WO WO 2010, 124146 A3 10, 2010 Alto, CA (US) WO WO 2010, 148144 A1 12/2010 (73) Assignee: Danisco US Inc., Palo Alto, CA (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Thomason et al. (J Bacteriol. Dec. 2004; 186(24):8248-53.* Balbas et al. Gene. Jun. 12, 1996; 172(1):65-9.* patent is extended or adjusted under 35 Martin et al. Nat Biotechnol. Jul. 2003:21(7):796-802. Epub Jun. 1, U.S.C. 154(b) by 0 days. 2003.* Sasaki et al. FEBS Lett. Apr. 25, 2005:579(11):2514-8.* (21) Appl. No.: 12/978,324 Anderson, M.S. et al., (1989). “Isopentenyl Diphosphate: Dimethlal lyl Diphosphate Isomerare. An Improved Purification of the Enzyme (22) Filed: Dec. 23, 2010 and Isolation of the Gen From Saccharomyces cerevisia,” J. Biol. Chem. 264(32): 19169-19175. (65) Prior Publication Data Aon, J. et al., (2008) "Suppressing Posttranslational Gluconoylation US 2011/O1595.57 A1 Jun. 30, 2011 of Heterologous Proteins by Metabolic Engineering of Escherichia coli,' Appl. Environ. Microbiol. 74:950-958. Baba, T. et al., (2006) “Construction of Escherichia coli K-12 In Related U.S. Application Data Frame, Single-Gene Knockout Mutants: The Keio Collection.” Mol. Syst. Biol. 2: 2006.0008. (60) Provisional application No. 61/289.959, filed on Dec. Bouvier, F. et al., (2005) “Biogenesis, Moleculars Regulation and 23, 2009. Function of Plant Isoprenoids.” Progress in Lipid Res. 44:357-429. Campbell, E. et al., (1989) “Improved Transformation Efficiency of (51) Int. Cl. Aspregillus niger Using Homologous niaD Gene for Nitrate CI2P 7/42 (2006.01) Reductase. Curr. Genet. 16:53-56. CI2P 5/02 (2006.01) Cherepanov, P.P. et al., (1995) “Gene Disruption in Escherichia coli: CI2N 9/00 (2006.01) Tc' and Km' Cassettes with the Option of Flp-Catalyzed Excision of CI2N L/20 (2006.01) the Antibiotic-Resistance Determinant.” Gene 158(1):9-14. CI2N IS/00 (2006.01) Datsenko, K., et al., (2000) "One-Step Inactivation of Chromosomal C7H 2L/04 (2006.01) Genes in Escherichia coli K-12 Using PCR Products.” Proc. Nat. Acad. Sci. USA 97:6640-6645. (52) U.S. Cl. Dhe-Paganon, S. et al., (1994). “Mechanism of Mevalonate USPC ...... 435/252.3; 435/146; 435/167; 435/183; Pyrophosphate Decarboxylase: Evidence for a Carbocationic Tran 435/320.1; 536/23.2 sition State.” Biochemistry 33(45): 13355-13362. (58) Field of Classification Search EcoGene Accession No. EG13231, located at OTHER PUBLICATIONS Self, W.T. et al., (2001). “Molybdate Transport.” Res Microbiol. Grunden, A.M. et al., (1997). “Molybdate Transport and Regulation 152:311-321. in Bacteria, Arch Microbiol. 168:345-354. Sharkey, T. et al., (2005). “Evolution of the Isoprene Biosynthetic Hedl, M. et al., (2002). “Enterococcus faecalis Acetoacetyl-Coen Pathway in Kudza.” Plant Physiology 137:700-712. Zyme A Thiolase/3-Hydroxy-3-Methyglutary-Coenzyme A Silver, G. et al., (1995). “Characterization of Aspen Isoprene Reductase, A Dual-Function Protein of Isopentenyl Diphosphate Synthase. An Enzyme Responsible for Leaf Isoprene Emission to the Biosynthesis,” J. Bacteriol. 184(8):2116-2122. Atmosphere.” JBC 270(22): 13010-13016. Hoeffler, J-F. et al., (2002). “Isoprenoid Biosynthesis via the Sinha, A. et al., (1992). “Induction of Specific Enzymes of the Oxi Methylerythritol Phosphate Pathway. Mechanistic Investigations of the 1-Deoxy-D-Xylulose 5-Phosphate D Reductiosimerase.” Eur: J. dative Pentose Phosphate Pathway by Glucono-6-Lactone in Sac Biochen. 269:4446-4457. charomyces cerevisiae.” J. Gen. Microbiol. 138:1865-1873. International Search Report mailed on Jun. 15, 2011, for PCT Patent Sprenger, G.A. et al., (1997). “Identification of a Thiamin-Dependent Application No. PCT/US2010/062099, filed Dec. 23, 2010, 5 pages. Synthase in Escherichia coli Required for the Formation of the Koga, et al., (2007). "Biosyntheseis of Ester-Type Polar Lipids in 1-DeoxY-o-Xylulose 5-Phosphate Precursor to Isoprenoids, Archea and Evolutionary Consideration.” Microbiology and Mol. Thiamin, and Pyridoxol.” PNAS 94:12857-12862. Biology Reviews 71(1):97-120. Studier F.W. et al., (2009). “Understanding the Differences Between Kutsche, M. et al., (Apr. 1996). “Promoters Controlling Expression Genome Sequences of Escherichia coli B Strains REL606 and of the Alternative Nitrogenase and the Molybdenum Uptake System BL21 (DE3) and Comparison of the E. coli B and K-12 Genomes.”J. in Rhodobacter capsulatus Are Activated by NtrC, Independent of Mol. Biol. 394(4):653-680. o', and Repressed by Molybdenum.” Journal of Bacteriology Sutherlin, A. et al., (2002). “Enterococcus faecalis 3-Hydrozy-3- 178(7):2010-2017. Methylglutaryl Coenzyme A Synthase. An Enzyme of Isopentenyl Luttgen, H. etal. (2000). "Biosynthesis of Terpenoids:YchB Protein Diphosphate Biosynthesis,” J. Bacteriol. 184(15):4065-4070. Escherichia coli Phosphorylates the 2-Hydrozy Group of Thomason, L.C. et al., (Dec. 2004). “Identification of the Escherichia 4-Diphosphocytidyl-2C-Methyl-o-Erythritol.” PNAS 97(3):1062 coli K-12 ybhF Gene as pgil, Encoding 6-Phosphogluconolactonase.” 1067. J. Bact. 186(24):8248-8253. Miclet, E. et al., (Sep. 14, 2001). “NMR Spectroscopic Analysis of Thomason, L.C., (Jul. 2007). 'E. coli Genome Manipulation by Pl the First Two Steps of the Pentose-Phosphate Pathway Elucidates the Transduction.” Curr: Protocols Mol. Biol. Chapter 1, Unit 1.17. Role of 6-Phosphogluconolactonase.”.J. Biol. Chem. 276(37):34840 Tsay, Y.H. et al., (1991). “Cloning and Characterization of ERG8, An 34846. Essential Gene of Saccharomyces cerevisiae That Encodes Miller, B. etal. (2001). “First Isolation of an Isoprene Synthase Gene From Poplar and Successful Expression of the Gene in Escherichia Phosphomevalonate Kinase.” Mol Cell Biol. 11(2):620-631. coli. Planta 2 13:483-487. UniProtKB/Swiss-Prot Accession No. P38858 (PGL polypeptide), Oulmouden, A. et al., (1991). "Nucleotide Sequence of the ERG 12 located at U.S. Patent Jun. 4, 2013 Sheet 2 of 66 US 8,455,236 B2 Figure B. 2 CECO-S-CoA acetyl-CoA o o HC-C-Cig SCoA acetoacetyl-CoA Chico-S-CoA CO-S-CoA Chi HC-C-OH G-CoA E66HC 3 y CHOH CH2 HCCOH iseyasi acid 666HC * six CHO-P Chi Hscg3. 3-phosphatevatoric acid 666H 8 4CO s ATP CHO-P CHO-PP isopentenyi-P HaegCH HaegeChi sphomevalonic 2 - Af s 666H 9 • CO CHO-PP She isapeatenyi-P ÖH, f glo-PP dimethylaiyi-PP FiscC Chis U.S. Patent Jun. 4, 2013 Sheet 3 of 66 US 8,455,236 B2 Figure 2. T7 terminator f Origin P. alba HGS kan sequence Y. pET24 Paba HGS 69.57 bp T7 promoter A. lac operator --- N-CoIE1 pBR322 origin lac U.S. Patent Jun. 4, 2013 Sheet 6 of 66 US 8,455,236 B2 Figure 4. laco | trc promoter f /f RBS sy f /minicistron ORF NCO site lacq f s Y& Reinitiation RBS ...C \ Y. N.NcoI (412) 1...GGTAC M \\, NATGPst (437) WN 1 rrnB terminator PSt pTrcHis2B BSpH Site 4404 bp T. P.ensus alba HGS Amp resistance pBR322 ori U.S. Patent Jun. 4, 2013 Sheet 7 of 66 US 8,455,236 B2 Figure 5. lac operator net A. Ptrc promoter & P.alba HGS EWL230 (pTrc P.alba) 6068 bp rrn terminator U.S. Patent Jun. 4, 2013 Sheet 10 of 66 US 8,455,236 B2 Figure 7. -- lac operator - Ptric promoter P.alba HGS EWL230 (pTrCP.alba) PE, 6068 bp s y is site is: pBR322 Pst (2101) ACSFA ?tage vs. \ Pmei (2203) rrn terminator U.S. Patent Jun. 4, 2013 Sheet 11 of 66 US 8,455,236 B2 Figure 8. lac operator Ptrc promoter P.alba HGS / EWL244 (pTrc P.alba-mMVK) 6906 bp / \ bla \ rrn terminator U.S. Patent Jun. 4, 2013 Sheet 14 of 66 US 8,455,236 B2 uoedoKew.pediawonpºupleezau&q&& sºuseºaneserwoquebeq____\ lacuodzie U.S. Patent Jun. 4, 2013 Sheet 16 of 66 US 8,455,236 B2 Figure 10C. CCtgcacaactCtcCagtagtggtaactggttggacCCgCgaatacctggagcagcgCggttittaacgttaaggacctCtCCCtg CCgggcaacgctctgtaagcttcaacg.cgtctacaaataaaaaaggCacgtCagatgacgtgCCtttitttctgtctaga (SEQ ID NO:4) U.S. Patent Jun. 4, 2013 Sheet 17 of 66 US 8,455,236 B2 Figure 11A. if reverse primer T transcription terminator bla promoter Karr. p8R322 origin MCM376 - MMKfrom M. mazei archeat Lowerin pET2OOD U.S. Patent Jun. 4, 2013 Sheet 20 of 66 US 8,455,236 B2 Figure 12. ATG M13 Reverse pBBRCMPG|1.5pg| 7519 bp CDS 1 CMP-GI1.5-pg| lacz alpha M13 ForWard CDS2: Gentamycin resistance gene; CDS1: E. coli replication protein U.S. Patent Jun. 4, 2013 Sheet 23 of 66 US 8,455,236 B2 Figure 14A. 1 OOO -- - - 1 O O --- 10 1 O 5 1 O 15 20 25 30 35 4O 45 5O 55 6.O 65 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 24 of 66 US 8,455,236 B2 Figure 14B. 6O - 3O O 5 1 O 15 20 25 3O 35 4O 45 5O 55 6.O 65 Time (hr) Figure 14C. 40 2O . 1 O O 5 1 O 15 20 25 3O 35 4O 45 5O 55 6.O 65 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 25 of 66 US 8,455,236 B2 Figure 14D. 500 --- 450 400 - 350 - 3OO 250 - 2OO 150 - 1OO - O - -wn O 5 10 15 20 25 30 35 4O 45 5O 55 6O 65 Time (hr) Figure 14E. 3.0 - es u S 2.5 s O) Kane a 2.0 - 2 Average = 1.4 g/L/hr go 15 - between 19 and 59 hrs Ss 1.0 Average = 1.0 g/L/hr E between O and 40 hrs P O. 5 O D O. O | | O 5 10 15 20 25 30 35 4O 45 50 55 60 65 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 26 of 66 US 8,455,236 B2 Figure 14 F. 400 350 300 c 250 - E200 - 150 - OE. 100 - 50 - O O 5 10 15 20 25 30 35 40 45 50 55 60 65 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 27 of 66 US 8,455,236 B2 Figure 15. SEM : torr Faraday: torr HAL 7 RC RGA 201 12854 1.6e-08 14e–08 B : 1.2e-08 ...' ... 1e-08 ... 8e-09 Ge-09 | 4e–09 . . . 2e-09 | O -T-I-row... 'over- ---, -, or of to court. - -vir : O0:00 02:00 04:00 06:00 08:00 10:00 12:00 14:00 16:00 Time mm:ss * Hydrogen CO2 M Oxygen M. Isoprene U.S. Patent Jun. 4, 2013 Sheet 28 of 66 US 8,455,236 B2 Figure 16A. Primary Secondary carbon filter carbon filter Off-gas out t Dehumidifierunit condensateCooled V reservoir Off-gas in Sample point B U.S. Patent Jun. 4, 2013 Sheet 29 of 66 US 8,455,236 B2 Figure 16B. Steam in Water-cooled Condenser Glycol-cooled Cold trap Condenser (-78°C) (40°C) 1. (O'C) Carbon trap Gas out Carbon filter Steam lsoprene condenser collection U.S. Patent Jun. 4, 2013 Sheet 30 of 66 US 8,455,236 B2 || 3 | | LI3.InãIA Tesu.Odse>H EZO+eL \HOOOOOOG U.S. Patent Jun. 4, 2013 Sheet 31 of 66 US 8,455,236 B2 Ahridance Figure 18A -OOOO EO -OOOO OOOO 2OOOOO 18 E. -OOOO OOOOO 8OOOO EOOOO OOOO Time...: E.O 8.OO . . . E.O. 18.OO ... . .4. S. OOOOO Figure 18B. 8 EO OOOO Time-: E. E. . . 4. E.O. 18...... E. 8 Figure 18C. EO O Time-: E. 8. . . . . E. 18...... E. U.S. Patent Jun. 4, 2013 Sheet 32 of 66 US 8,455,236 B2 Figure 19A. Map of plasmid pDW34 lac operator ? Ptre promoter lacid | MCM65 N at . Ptrc, complete st gp10 RBS A N reinitiation RBS 47 W Truncated P.alba HGS (MEA) R pDW34a (MEA Truncation in pTrCP. alba-n MVK) 6858bp pBR322 ------RBS N \\ M. mazei MVK ORF / \ % \ bla \ rrn terminator U.S. Patent Jun. 4, 2013 Sheet 36 of 66 US 8,455,236 B2 U.S. Patent Jun. 4, 2013 Sheet 37 of 66 US 8,455,236 B2 Figure 21. 3.5 - 2.5 - 2 1.5 O5 - O A. i A A. A. -o- CMP312 OD 1CM -e-CMP323 OD 1CM A Sample Point U.S. Patent Jun. 4, 2013 Sheet 38 of 66 US 8,455,236 B2 Figure 22. Specific Productivity 2500 - 2000 - 1500 – 1 OOO – 5 O O Time (Hrs) -o-CMP312 S. Prod (ug/LIOD/h) -e-CMP323 S. Prod (ug/LIODIh) A Sample Point U.S. Patent Jun. 4, 2013 Sheet 39 of 66 US 8,455,236 B2 Figure 23. 1000 - - - 1OO – 10 - 1 O 5 1O 15 20 25 30 35 40 45 50 Time (hr) --DW 199 -o- CMP312 - A - CMP323 U.S. Patent Jun. 4, 2013 Sheet 40 of 66 US 8,455,236 B2 Figure 24. ------O 5 10 15 20 25 30 35 40 45 50 Time (hr) --DW 199 -o- CMP312 - A - CMP323 U.S. Patent Jun. 4, 2013 Sheet 41 of 66 US 8,455,236 B2 Figure 25. 700 - we —-.M 600 – 500 234 OOO OOO ---- 100 O 5 10 15 20 25 30 35 40 45 50 Time (hr) --DW 199 -O-CMP312 - A - CMP323 U.S. Patent Jun. 4, 2013 Sheet 42 of 66 US 8,455,236 B2 Figure 26. 12 5O 10 O 5 10 15 20 25 30 35 40 45 50 Time (hr) -- DW 199 -O- CMP312 - A - CMP323 U.S. Patent Jun. 4, 2013 Sheet 43 of 66 US 8,455,236 B2 Figure 27. 180 - - 1 6 O 2 O O | | O 5 10 15 20 25 Time (hr) -O- pg -O-pg U.S. Patent Jun. 4, 2013 Sheet 44 of 66 US 8,455,236 B2 Figure 28. 20 - - 18 16 - 14 - 12 - 10 - Time (hr) U.S. Patent Jun. 4, 2013 Sheet 45 of 66 US 8,455,236 B2 Figure 29. ------ | | | O 5 10 15 20 25 Time (hr) -O- pg -O- pg U.S. Patent Jun. 4, 2013 Sheet 46 of 66 US 8,455,236 B2 Figure 30. 1 OOO – 1OO - 10 - 1 O 5 1O 15 20 25 30 35 4O 45 50 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 47 of 66 US 8,455,236 B2 Figure 31. 35 - 3 O -- --- 122 5O5 10 O 5 10 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 48 of 66 US 8,455,236 B2 Figure 32. 250 - 200 - 1 5 O - 10 O O 5 10 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 49 of 66 US 8,455,236 B2 Figure 33. uu u u uu uu uu uu uu uu uu uu uu uuu uu uu uu uuu uu uu uu M MP uu uu vdvdd vdvd vdvd vdvd vdvdd vdvd vdvd vdvd vo MM uu uu MM 4 O | | O 5 10 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent Jun. 4, 2013 Sheet 50 of 66 US 8,455,236 B2 Figure 34A. Map of plasmid pPW15. mob AacC1 (Gent Resistance) M13 Reverse RBS? 1 ATG. Laczalph Ptrc mvaE M13 Forward U.S. Patent Jun. 4, 2013 Sheet 54 of 66 US 8,455,236 B2 Figure 35. Specific Productivity 0.4 O, 2 0. BL21 + pCL BL21 pg| + pCL BL21 + pBBR BL21 pg|+ pBBR BL21 + proUpper B 21 pg|+ proUpper Ptrcupper Ptrcuipper PtroUpper Ptre upper U.S. Patent Jun. 4, 2013 Sheet 55 of 66 US 8,455,236 B2 Figure 36. Sample 2 Sample 1 O. 1 O 1 OO 2OO 3OO 400 Time (min) --MCM872 BL21 ptro-upper --MCM876 BL21 pglpTrc-upper U.S. Patent Jun. 4, 2013 Sheet 56 of 66 US 8,455,236 B2 Figure 37. 0.06 - O.05 BL21 ptro-upper BL21 pglpTrc-upper U.S. Patent Jun. 4, 2013 Sheet 57 of 66 US 8,455,236 B2 Figure 38. 6.OE-06 5.OE-06 4.OE-06 O BL21 ptro-upper 3.OE-06 - BL21 pg pro-upper 2.OE-06 1.OE-06 - O.OE+OO Time point 1 Time point 2 U.S. Patent Jun. 4, 2013 Sheet 58 of 66 US 8,455,236 B2 Figure 39. 7.OE-06 6.OE-06 5.OE-06 4.OE-06 BL21 ptro-upper in BL21 pg. pirc-upper 3.OE-06 2.OE-06 1.OE-06 O.OE--OO -- Time point 1 Time point 2 U.S. Patent Jun. 4, 2013 Sheet 60 of 66 US 8,455,236 B2 Figure 40B. Amino acid sequence of PGL from P. aeruginosa (SEQ ID NO:12). MAISELKLPAGVGLQVWGSAAEQARGLAAEVAGRLRSALAEQGQALLVVSGGRSPVA FLEALSEEPLDWSRITVSLADERWVPESHADSNAGLVRRHLLRGEAAKARFIGLYQPAA SLEEAAELADHHLHELPLPIDVLVLGMGDDGHTASLFPNSPGLDLAMDPQGTRRCLPM WAPSVPHQRLTLPRAVLAAAKVQLLAIQGQSKLATLNAALAVEDERRMPVRAFLRAPL THWYP U.S. Patent Jun. 4, 2013 Sheet 62 of 66 US 8,455,236 B2 Figure 40D. l Ps aeruginosa pgll (1) -MAISEKLF Ecoli MC1655 pgll (1) MKQTVYIAS Consen SS () 51 PS aeruginoSa pg| (32) ------E. Ecoli MG 1655 pol (5) YVGVRP PGSPTHISTDHQGQFV CO. Sen SuS (51) I S 1 O1 Ps aeruginosa pol (62) LSFFPTDW Ecoli MG1655 poll (101) YNAGNVSVTR COn Sen SuS (101) L SR 51 Ps aeruginosa poli (108) FIGLYCPAASLE LGMGDDGHTASLFP Ecoli MG1655 pg| (151) ICLFTVSDDGH EOYAYCVNELN Consensus (151) AH 2O. Ps aeruginosa pgll (158) GLD) CLPMWAPSVP AAKVOLTAIOGOSKL Ecoli MC1655 pgl (201) DVW QTLDMMPENFSDTRWAADHITPDGRHLYACDRTA Consensus (201) L A 51 3 OO Ps aeruginosa pgl (208) ATLNAALAVEDERRMPVRAFLRAPLTIHWYP------Ecoli MG 1655 pol (251) SLTVFSVSEDGSWL SKEG Evolvini COI, Sen SuS (251) A I D I F 3 O1 331 Ps aeruginosa pg) (239) ------Ecoli MG1655 poll (301) WYEIVGEOGLLHEKGRYAVGOGPMWVVVNAH Consensus (301) Identity 9.7%; Similarity 17.8% Vector Nti Align X E. coli MG 1655 pgl Pseudomonas aeruginosa pg| U.S. Patent Jun. 4, 2013 Sheet 63 of 66 US 8,455,236 B2 Figure 41A. U.S. Patent Jun. 4, 2013 Sheet 64 of 66 US 8,455,236 B2 Figure 41B. O.8 Around 15% increase in Spec. Crowth rate O.7 O6 O5 S. O3 O2 O1 EFT (h) --BL21 pg| U.S. Patent Jun. 4, 2013 Sheet 65 of 66 US 8,455,236 B2 Figure 42A. i DW202 --CMP234 20.0 1.O.O co-wroto O.O 10.0 50.0 600 U.S. Patent Jun. 4, 2013 Sheet 66 of 66 US 8,455,236 B2 Figure 42B. 18O 1. 4. O 1. 2. O 1. O. O W 8. O W202 --CMP234 6. O US 8,455,236 B2 1. 2 COMPOSITIONS AND METHODS OF PGL improvements to reduce the operational costs associated with FOR THE INCREASED PRODUCTION OF the production of isoprene derived from biological sources ISOPRENE and to increase yields of isoprene are needed. Such as the improved compositions and methods for the increased pro CROSS-REFERENCE TO RELATED duction of isoprene and other heterologous polypeptides APPLICATIONS capable of biological activity provided herein. All patents, patent applications, documents, nucleotide and This application claims priority to U.S. Provisional Patent protein sequence database accession numbers and articles Application No. 61/289.959, filed on Dec. 23, 2009, the dis cited herein are incorporated herein by reference in their closure of which is hereby incorporated herein by reference in 10 entirety. its entirety. SUMMARY FIELD OF THE INVENTION Disclosed herein are improved compositions and methods This disclosure relates to improved compositions and 15 for the increased production of isoprene. Also provided methods for the increased production of biochemicals in E. herein are improved compositions and methods for the coli, as well as improved compositions and methods for the increased production of heterologous polypeptides capable of increased production ofisoprene in E. coli. biological activity. The invention is based in part on the obser Vation that chromosomal integration of 6-phosphoglucono BACKGROUND lactonase (PGL) into E. coli strains which lack nucleic acids encoding for PGL polypeptide improves the production of Isoprene (2-methyl-1,3-butadiene) is an important organic different types of products, for example, isoprene or meva compound used in a wide array of applications. For instance, lonate. isoprene is employed as an intermediate or a starting material Accordingly, in one aspect, the invention provides for in the synthesis of numerous chemical compositions and 25 recombinant cell(s) of an Escherichia coli (E. coli) strain, or polymers. Isoprene is also an important biological material progeny thereof, capable of producing isoprene, the cellcom that is synthesized naturally by many plants and animals. prising: (a) one or more copies of a heterologous nucleic Isoprene became an important monomer for utilization in acid(s) encoding a PGL polypeptide wherein the nucleic acid the synthesis of cis-1,4-polybutadiene when its stereo-regu is integrated in the E. coli chromosome; and (b) one or more lated polymerization became commercially possible in the 30 heterologous nucleic acid(s) encoding isoprene synthase; early 1960s. cis-1,4-Polyisoprene made by such stereo-regu wherein prior to the integration, the E. coli cell does not lated polymerizations is similar instructure and properties to contain nucleic acid(s) encoding a PGL polypeptide, and natural rubber. Even though it is not identical to natural rub wherein the resulting recombinant cell produces isoprene at a ber it can be used as a substitute for natural rubber in many greater titer than that of the same cells that do not comprise (a) applications. For instance, synthetic cis-1,4-polyisoprene 35 and (b). rubber is widely used in manufacturing vehicle tires and other In any of the aspects herein, one or more copies of a rubber products. This demand for synthetic cis-1,4-polyiso heterologous nucleic acid encoding a molybdenum uptake prene rubber consumes a majority of the isoprene available in polypeptide is additionally integrated in the E. coli chromo the worldwide market. The remaining isoprene is used in some. In any of the aspects herein, the molybdenum uptake making other synthetic rubbers, block copolymers, and other 40 polypeptide is selected from the group consisting of modF. chemical products. For instance, isoprene is used in making modE, modA, modB and modC. In any of the aspects herein, butadiene-isoprene rubbers, styrene-isoprene copolymer rub one or more copies of a heterologous nucleic acid encoding a bers, styrene-isoprene-butadiene rubbers, styrene-isoprene galactose metabolism polypeptide is additionally integrated styrene block copolymers, and styrene-isoprene block in the E. coli chromosome. In any of the aspects herein, the copolymers. 45 galactose metabolism polypeptide is selected from the group The isoprene used in industrial applications is typically consisting of galM, galK, galT and galE. In any of the aspects produced as a by-product of the thermal cracking of petro herein, one or more copies of a heterologous nucleic acid leum or naphtha or is otherwise extracted from petrochemical encoding a galactose metabolism polypeptide and one or streams. This is a relatively expensive, energy-intensive pro more copies of a heterologous nucleic acid encoding a molyb cess. With the worldwide demand for petrochemical based 50 denum uptake polypeptide are additionally integrated in the products constantly increasing, the cost of isoprene is E. coli chromosome. In any of the aspects herein, (a) the PGL expected to rise to much higher levels in the long-term and its polypeptide is an E. coli PGL polypeptide; (b) the molybde availability is limited in any case. There is concern that future num uptake polypeptide is selected from the group consisting Supplies of isoprene from petrochemical-based sources will of modF, modE, modA, modB and modC.; and (c) the galac be inadequate to meet projected needs and that prices will rise 55 tose metabolism polypeptide is selected from the group con to unprecedented levels. Accordingly, there is a need to pro sisting of galM, galK, galT and galE. In any of the aspects cure a source ofisoprene from a low cost, renewable source herein, nucleic acids encoding the PGL polypeptide, galac which is environmentally friendly. The improved methods tose metabolism polypeptide, and molybdenum uptake and compositions described herein provide Such a source of polypeptide are part a 17.257 base pair piece as shown in FIG. isoprene, capable of being derived at low cost and from 60 20. In any of the aspects herein, the recombinant cell produces renewable sources. isoprene at a higher specific productivity than that of the same Several recent advancements have been made in the pro cells that do not contain (a) and (b). duction of isoprene from renewable sources (see, for In any of the aspects herein, the recombinant cell has a example, International Patent Application Publication No. specific productivity of at least 15 mg/OD/hr. In any of the WO 2009/076676 A2). Such methods produce isoprene at 65 aspects herein, the nucleic acids encoding PGL polypeptide, rates, titers, and purity that may be sufficient to meet the molybdenum uptake polypeptide, and/or galactose metabo demands of a robust commercial process, however process lism polypeptide are from E. coli strain K12 MG 1655 or a US 8,455,236 B2 3 4 derivative of E. coli strain K12 MG 1655. In any of the aspects encoding a PGL polypeptide with one or more associated herein, the cell is of E. coli strain B. In any of the aspects expression control sequences is/are chromosomal copies herein, the cell is of E. coli strain BL21. In any of the aspects (e.g., integrated into the E. coli chromosome). In some herein, the cell is of E. coli strain BL21 (DE3). aspects, the E. coli cells are in culture. In some aspects, the In any of the aspects herein, the recombinant E. coli cell 5 cells are of E. coli strain B. In some aspects, the cells are of E. further comprises (c) a heterologous nucleic acid encoding an coli strain BL21. In some aspects, the cells are of E. coli strain upper mevalonate (MVA) pathway polypeptide and/or a BL21 (DE3). In some aspects, the minimal medium is supple lower MVA pathway polypeptide. mented with 0.1% (w/v) yeast extract or less. In some aspects, In any of the aspects herein, the recombinant E. coli cell the minimal medium is Supplemented with 1% (w/v) glucose further comprises (d) a heterologous nucleic acid encoding an 10 or less. In some aspects, the minimal medium is Supple upper mevalonate (MVA) pathway polypeptide and/or a mented with 0.1% (w/v) yeast extract or less and 1% (w/v) lower MVA pathway polypeptide. In any of the aspects glucose or less. In some aspects, the heterologous gene herein, the upper MVA pathway polypeptide is selected from encoding a PGL polypeptide is from E. coli strain K12 the group consisting of: (i) an acetoacetyl-Coenzyme A Syn MG1655 or a derivative of E. coli Strain K12 MG 1655. In thase (thiolase) polypeptide; (ii) a 3-hydroxy-3-methylglu 15 Some aspects, the heterologous gene encoding a PGL taryl-Coenzyme A synthase polypeptide; and (iii) a 3-hy polypeptide is from the genus Pseudomonas. In some aspects, droxy-3-methylglutaryl-Coenzyme A reductase polypeptide. the Pseudomonas is Pseudomonas aeruginosa. In any of the aspects herein, the lower MVA pathway In some aspects, the heterologous polypeptide capable of polypeptide is selected from the group consisting of: (i) biological activity comprises one or more polypeptides mevalonate kinase (MVK); (ii) phosphomevalonate kinase involved in the biosynthesis ofterpenoid (isoprenoid) or caro (PMK); (iii) diphosphomevalonate decarboxylase (MVD); tenoid compounds, and the cells produce a terpenoid or caro and (iv) isopentenyl diphosphate isomerase (IDI). In any of tenoid at a higher specific productivity than that of the same the aspects herein, (a) the PGL polypeptide is an E. coli PGL cells lacking one or more copies of a heterologous gene polypeptide; (b) the molybdenum uptake polypeptide is encoding a PGL polypeptide with one or more associated selected from the group consisting of modF, modE, modA, 25 expression control sequences, when the cells are cultured in modB and modC.; and (c) the galactose metabolism polypep minimal medium. In some aspects, the terpenoid is selected tide is selected from the group consisting of galM, galK, galT from the group consisting of hemiterpenoids, monoterpe and galE. In any of the aspects herein, the isoprene synthase noids, sesquiterpenoids, diterpenoids, Sesterterpenoids, trit polypeptide is from Populus alba. erpenoids, tetraterpenoids, and higher polyterpenoids. In The invention also provides for methods of producing iso 30 Some aspects, the hemiterpenoid is prenol (i.e., 3-methyl-2- prene, the method comprising: (a) culturing a composition buten-1-ol), isoprenol (i.e., 3-methyl-3-buten-1-ol), 2-me comprising any of the recombinant cell described herein thyl-3-buten-2-ol, or isovaleric acid. In some aspects, the under suitable culture conditions for the production of iso monoterpenoid is geranyl pyrophosphate, eucalyptol, prene and (b) producing isoprene. In some aspects, the limonene, or pinene. In some aspects, the sesquiterpenoid is method comprises further recovering the isoprene. In other 35 farnesyl pyrophosphate, artemisinin, or bisabolol. In some aspects, the recombinant cell has a specific productivity aspects, the diterpenoid is geranylgeranyl pyrophosphate, ret greater than about 15 mg/OD/hr of isoprene. inol, retinal, phytol, taxol, forskolin, or aphidicolin. In some The invention also provides for methods of producing aspects, the triterpenoid is squalene or lanosterol. In some mevalonate, the method comprising: (a) culturing a compo aspects, the tetraterpenoid is lycopene or carotene. In some sition comprising the recombinant cell of claim 15 under 40 aspects, the carotenoid is selected from the group consisting suitable culture conditions for the production of mevalonate of Xanthophylls and carotenes. In some aspects, the Xantho and (b) producing mevalonate. In some aspects, the method phyll is lutein or zeaxanthin. In some aspects, the carotene is comprises further recovering the mevalonate. C-carotene, B-carotene, Y-carotene, B-cryptoxanthin or lyco The invention also provides for methods of making any of pene. the recombinant cell described herein comprising: (a) trans 45 In another aspect, provided herein are cells of an Escheri ducing a heterologous nucleic acid encoding a PGL polypep chia coli strain that does not encode a PGL polypeptide, tide into an E. coli cell, wherein prior to the integration, the E. wherein the E. coli cells comprise one or more copies of a coli cell does not contain nucleic acid(s) encoding a PGL heterologous gene encoding a PGL polypeptide with one or polypeptide; (b) allowing the nucleic acid encoding a PGL more associated expression control sequences and a heterolo polypeptide to integrate in the E. coli chromosome; and (c) 50 gous nucleic acid encoding an isoprene synthase polypeptide, introducing one or more heterologous nucleic acid(s) encod and wherein the cells have a specific productivity of isoprene ing isoprene synthase into E. coli cell. greater than that of the same cells lacking one or more copies The invention also provides for compositions comprising of a heterologous gene encoding a PGL polypeptide with one any of the recombinant cell described herein. or more associated expression control sequences, when the In other aspects, provided herein are cells of an Escherichia 55 cells are cultured in minimal medium. In some aspects, the coli strain that does not encode a PGL polypeptide, wherein one or more copies of the heterologous gene encoding a PGL the E. coli cells comprise one or more copies of a heterolo polypeptide with one or more associated expression control gous gene encoding a PGL polypeptide with one or more sequences is/are chromosomal copies (e.g., integrated into associated expression control sequences and a nucleic acid the E. coli chromosome). In some aspects, the E. coli cells are encoding a heterologous polypeptide capable of biological 60 in culture. In some aspects, the cells further comprise a het activity, and wherein the cells produce the heterologous erologous nucleic acid encoding an MVA pathway polypep polypeptide capable of biological activity at a specific pro tide. In some aspects, the MVA pathway polypeptide is an ductivity greater than that of the same cells lacking one or upper MVA pathway polypeptide. In some aspects, the MVA more copies of a heterologous gene encoding a PGL polypep pathway polypeptide is a lower MVA pathway polypeptide. tide with one or more associated expression control 65 In some aspects, the upper MVA pathway polypeptide is sequences, when the cells are cultured in minimal medium. In selected from the group consisting of: (i) an acetoacetyl Some aspects, the one or more copies of the heterologous gene Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hy US 8,455,236 B2 5 6 droxy-3-methylglutaryl-Coenzyme A synthase polypeptide; diphosphomevalonate decarboxylase (MVD); and a nucleic and (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reduc acid encoding isopentenyl diphosphate isomerase (IDI); (ii) a tase polypeptide. In some aspects, the upper MVA pathway nucleic acid encoding P alba isoprene synthase; (iii) a polypeptide is from the genus Enterococcus. In some aspects, nucleic acid encoding M. mazeimevalonate kinase; and (iv) a the upper MVA pathway polypeptide is from Enterococcus nucleic acid encoding the upper MVA pathway from Entero faecalis. In some aspects, the lower MVA pathway polypep COccus faecalis, comprising a nucleic acid encoding an tide is selected from the group consisting of: (i) mevalonate acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; a kinase (MVK); (ii) phosphomevalonate kinase (PMK); (iii) nucleic acid encoding a 3-hydroxy-3-methylglutaryl-Coen diphosphomevalonate decarboxylase (MVD); and (iv) iso Zyme A synthase polypeptide; and a nucleic acid encoding a pentenyl diphosphate isomerase (IDI). In some aspects, the 10 3-hydroxy-3-methylglutaryl-Coenzyme A reductase lower MVA pathway polypeptide is an MVK polypeptide. In polypeptide. some aspects, the MVK polypeptide is from the genus Metha In another aspect, provided herein are improved methods nosarcina. In some aspects, the MVK polypeptide is from of producing a heterologous polypeptide capable of biologi Methanosarcina mazei. cal activity, the method comprising: (a) culturing cells of an In some aspects, the cells are of E. coli Strain B. In some 15 E. coli strain that does not encode a PGL polypeptide, aspects, the cells are of E. coli strain BL21. In some aspects, wherein the E. coli cells comprise one or more copies of a the cells are of E. coli strain BL21 (DE3). In some aspects, the heterologous gene encoding a PGL polypeptide with one or minimal medium is supplemented with 0.1% (w/v) yeast more associated expression control sequences and a nucleic extract or less. In some aspects, the minimal medium is acid encoding a heterologous polypeptide capable of biologi Supplemented with 1% (w/v) glucose or less. In some aspects, cal activity; and (b) producing the heterologous polypeptide the minimal medium is supplemented with 0.1% (w/v) yeast capable of biological activity, wherein the cells produce the extract or less and 1% (w/v) glucose or less. In some aspects, heterologous polypeptide at a specific productivity greater the heterologous gene encoding a PGL polypeptide is from E. than that of the same cells lacking one or more copies of a coli strain K12 MG 1655 or a derivative of E. coli strain K12 heterologous gene encoding a PGL polypeptide with one or MG 1655. In some aspects, the heterologous gene encoding a 25 more associated expression control sequences, when the cells PGL polypeptide is from the genus Pseudomonas. In some are cultured in minimal medium. In some aspects, the one or aspects, the Pseudomonas is Pseudomonas aeruginosa. more copies of the heterologous gene encoding a PGL In some aspects, the cells have a specific productivity polypeptide with one or more associated expression control greater than about 20 mg/OD/hr ofisoprene. In some aspects, sequences are chromosomal copies (e.g., integrated into the the cells have a specific productivity greater than about 25 30 E. coli chromosome). In some aspects, the method further mg/OD/hr of isoprene. In some aspects, the heterologous comprises the step of recovering the heterologous polypep nucleic acid encoding an isoprene synthase polypeptide is tide capable of biological activity. operably linked to a promoter and the cells have a specific In some aspects, the cells are of E. coli Strain B. In some productivity greater than about 20 mg/OD/hr of isoprene. In aspects, the cells are of E. coli strain BL21. In some aspects, Some aspects, the heterologous nucleic acid encoding an iso 35 the cells are of E. coli strain BL21 (DE3). In some aspects, the prene synthase polypeptide is operably linked to a promoter minimal medium is supplemented with 0.1% (w/v) yeast and the cells have a specific productivity greater than about 25 extract or less. In some aspects, the minimal medium is mg/OD/hr of isoprene. Supplemented with 1% (w/v)glucose or less. In some aspects, In some aspects, the isoprene synthase polypeptide is a the minimal medium is supplemented with 0.1% (w/v) yeast plant isoprene synthase polypeptide. In some aspects, the 40 extract or less and 1% (w/v) glucose or less. In some aspects, cells further comprise a heterologous nucleic acid encoding the heterologous polypeptide having PGL activity is from E. an IDI polypeptide. In some aspects, the cells further com coli strain K12 MG 1655 or a derivative of E. coli strain K12 prise a chromosomal copy of an endogenous nucleic acid MG 1655. In some aspects, the heterologous polypeptidehav encoding an IDI polypeptide. In some aspects, the cells fur ing PGL activity is from the genus Pseudomonas. In some ther comprise a heterologous nucleic acid encoding a DXS 45 aspects, the Pseudomonas is Pseudomonas aeruginosa. polypeptide. In some aspects, the cells further comprise a In some aspects, the heterologous polypeptide capable of chromosomal copy of an endogenous nucleic acid encoding a biological activity comprises one or more polypeptides DXS polypeptide. In some aspects, the cells further comprise involved in the biosynthesis ofterpenoid (isoprenoid) or caro one or more nucleic acids encoding an IDI polypeptide and a tenoid compounds, and the cells produce a terpenoid or caro DXS polypeptide. In some aspects, one nucleic acid encodes 50 tenoid at a higher specific productivity than that of the same the isoprene synthase polypeptide, IDI polypeptide, and DXS cells lacking one or more copies of a heterologous gene polypeptide. In some aspects, one plasmid encodes the iso encoding a PGL polypeptide with one or more associated prene synthase polypeptide, IDI polypeptide, and DXS expression control sequences, when the cells are cultured in polypeptide. minimal medium. In some aspects, the terpenoid is selected In some aspects, the isoprene synthase polypeptide is a 55 from the group consisting of hemiterpenoids, monoterpe naturally-occurring polypeptide from the genus Pueraria. In noids, sesquiterpenoids, diterpenoids, Sesterterpenoids, trit Some aspects, the isoprene synthase polypeptide is a natu erpenoids, tetraterpenoids, and higher polyterpenoids. In rally-occurring polypeptide from Pueraria montana. In some Some aspects, the hemiterpenoid is prenol (i.e., 3-methyl-2- aspects, the isoprene synthase polypeptide is a naturally buten-1-ol), isoprenol (i.e., 3-methyl-3-buten-1-ol), 2-me occurring polypeptide from the genus Populus. In some 60 thyl-3-buten-2-ol, or isovaleric acid. In some aspects, the aspects, the isoprene synthase polypeptide is a naturally monoterpenoid is geranyl pyrophosphate, eucalyptol, occurring polypeptide from Populus alba. limonene, or pinene. In some aspects, the sesquiterpenoid is In some aspects, the cells comprise (i) an integrated nucleic farnesyl pyrophosphate, artemisinin, or bisabolol. In some acid encoding the lower MVA pathway from S. cerevisiae aspects, the diterpenoid is geranylgeranyl pyrophosphate, ret comprising a glucose isomerase promoter and a nucleic acid 65 inol, retinal, phytol, taxol, forskolin, or aphidicolin. In some encoding mevalonate kinase (MVK); a nucleic acid encoding aspects, the triterpenoid is squalene or lanosterol. In some phosphomevalonate kinase (PMK); a nucleic acid encoding aspects, the tetraterpenoid is lycopene or carotene. In some US 8,455,236 B2 7 8 aspects, the carotenoid is selected from the group consisting In some aspects, the isoprene synthase polypeptide is a of Xanthophylls and carotenes. In some aspects, the Xantho plant isoprene synthase polypeptide. In some aspects, the phyll is lutein or zeaxanthin. cells further comprise a heterologous nucleic acid encoding In another aspect, provided herein are improved methods an IDI polypeptide. In some aspects, the cells further com of producing isoprene, the method comprising: (a) culturing 5 prise a chromosomal copy of an endogenous nucleic acid cells of an E. coli strain that does not encode a PGL polypep encoding an IDI polypeptide. In some aspects, the cells fur tide, wherein the E. coli cells comprise one or more copies of ther comprise a heterologous nucleic acid encoding a DXS a heterologous gene encoding a PGL polypeptide with one or polypeptide. In some aspects, the cells further comprise a more associated expression control sequences and a heterolo chromosomal copy of an endogenous nucleic acid encoding a gous nucleic acid encoding an isoprene synthase polypeptide; 10 DXS polypeptide. In some aspects, the cells further comprise and (b) producing isoprene, wherein the cells have a specific one or more nucleic acids encoding an IDI polypeptide and a productivity of isoprene greater than that of the same cells DXS polypeptide. In some aspects, one nucleic acid encodes lacking one or more copies of a heterologous gene encoding the isoprene synthase polypeptide, IDI polypeptide, and DXS a PGL polypeptide with one or more associated expression polypeptide. In some aspects, one plasmid encodes the iso control sequences, when the cells are cultured in minimal 15 prene synthase polypeptide, IDI polypeptide, and DXS medium. In some aspects, the one or more copies of the polypeptide. In some aspects, the isoprene synthase polypep heterologous gene encoding a PGL polypeptide with one or tide is a naturally-occurring polypeptide from the genus more associated expression control sequences are chromo Pueraria. In some aspects, the isoprene synthase polypeptide Somal copies (e.g., integrated into the E. coli chromosome). is a naturally-occurring polypeptide from Pueraria montana. In some aspects, the improved method further comprises a In some aspects, the isoprene synthase polypeptide is a natu step of recovering the isoprene. In some aspects, the cells rally-occurring polypeptide from the genus Populus. In some further comprise a heterologous nucleic acid encoding an aspects, the isoprene synthase polypeptide is a naturally MVA pathway polypeptide. In some aspects, the MVA path occurring polypeptide from Populus alba. way polypeptide is an upper MVA pathway polypeptide. In In Some aspects, the cells comprise (i) an integrated nucleic some aspects, the MVA pathway polypeptide is a lower MVA 25 acid encoding the lower MVA pathway from S. cerevisiae pathway polypeptide. comprising a glucose isomerase promoter and a nucleic acid In some aspects, the upper MVA pathway polypeptide is encoding mevalonate kinase (MVK); a nucleic acid encoding selected from the group consisting of: (i) an acetoacetyl phosphomevalonate kinase (PMK); a nucleic acid encoding Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hy diphosphomevalonate decarboxylase (MVD); and a nucleic droxy-3-methylglutaryl-Coenzyme A synthase polypeptide; 30 acid encoding isopentenyl diphosphate isomerase (IDI); (ii) a and (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reduc nucleic acid encoding P alba isoprene synthase; (iii) a tase polypeptide. In some aspects, the upper MVA pathway nucleic acid encoding M. mazeimevalonate kinase; and (iv) a polypeptide is from the genus Enterococcus. In some aspects, nucleic acid encoding the upper MVA pathway from Entero the upper MVA pathway polypeptide is from Enterococcus COccus faecalis, comprising a nucleic acid encoding an faecalis. In some aspects, the lower MVA pathway polypep 35 acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; a tide is selected from the group consisting of: (i) mevalonate nucleic acid encoding a 3-hydroxy-3-methylglutaryl-Coen kinase (MVK); (ii) phosphomevalonate kinase (PMK); (iii) Zyme A synthase polypeptide; and a nucleic acid encoding a diphosphomevalonate decarboxylase (MVD); and (iv) iso 3-hydroxy-3-methylglutaryl-Coenzyme A reductase pentenyl diphosphate isomerase (IDI). In some aspects, the polypeptide. lower MVA pathway polypeptide is an MVK polypeptide. In 40 some aspects, the MVK polypeptide is from the genus Metha BRIEF DESCRIPTION OF THE DRAWINGS nosarcina. In some aspects, the MVK polypeptide is from Methanosarcina mazei. In some aspects, the cells are of E. FIG. 1A shows the MVA and DXP metabolic pathways for coli strain B. In some aspects, the cells are of E. coli strain isoprene (based on F. Bouvier et al., Progress in Lipid Res.44: BL21. In some aspects, the cells are of E. coli strain BL21 45 357-429, 2005). The following description includes alterna (DE3). In some aspects, the minimal medium is Supple tive names for each polypeptide in the pathways and a refer mented with 0.1% (w/v) yeast extractor less. In some aspects, ence that discloses an assay for measuring the activity of the the minimal medium is Supplemented with 1% (w/v) glucose indicated polypeptide. Mevalonate Pathway: AACT; Acetyl or less. In some aspects, the minimal medium is Supple CoA acetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacte mented with 0.1% (w/v) yeast extract or less and 1% (w/v) 50 riol. 184:2116-2122, 2002; HMGS: Hydroxymethylglutaryl glucose or less. In some aspects, the heterologous gene CoA synthase, MvaS, EC 2.3.3.10. Assay: J. Bacteriol. 184: encoding a PGL polypeptide is from E. coli strain K12 4065-4070, 2002; HMGR: 3-Hydroxy-3-methylglutaryl MG1655 or a derivative of E. coli Strain K12 MG 1655. In CoA reductase, MvaE, EC 1.1.1.34. Assay: J. Bacteriol. 184: Some aspects, the heterologous gene encoding a PGL 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC polypeptide is from the genus Pseudomonas. In some aspects, 55 2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phospho the Pseudomonas is Pseudomonas aeruginosa. mevalonate kinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol. In some aspects, the cells have a specific productivity 11:620-631, 1991; DPMDC; Diphosphomevalonate decar greater than about 20 mg/OD/hr ofisoprene. In some aspects, boxylase, MVD1, EC 4.1.1.33. Assay: Biochemistry the cells have a specific productivity greater than about 25 33: 13355-13362, 1994; IDI: Isopentenyl-diphosphate delta mg/OD/hr of isoprene. In some aspects, the heterologous 60 isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem. 264: nucleic acid encoding an isoprene synthase polypeptide is 19169-19175, 1989. DXP Pathway: DXS: 1-Deoxyxylulose operably linked to a promoter, and wherein the cells have a 5-phosphate synthase, dxs, EC 2.2.1.7. Assay: PNAS specific productivity greater than about 20 mg/OD/hr of iso 94:12857-62, 1997: DXR: 1-Deoxy-D-xylulose 5-phosphate prene. In some aspects, the heterologous nucleic acid encod reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem. ing an isoprene synthase polypeptide is operably linked to a 65 269:4446-4457, 2002: MCT: 4-Diphosphocytidyl-2C-me promoter, and wherein the cells have a specific productivity thyl-D-erythritol synthase, IspD, EC 2.7.7.60. Assay: PNAS greater than about 25 mg/L/hr ofisoprene. 97:6451-6456, 2000; CMK: 4-Diphosphocytidyl-2-C-me US 8,455,236 B2 9 10 thyl-D-erythritol kinase, IspE, EC 2.7.1.148. Assay: PNAS FIG. 14D shows the time course of total isoprene produced 97: 1062-1067, 2000; MCS: 2C-Methyl-D-erythritol 2,4-cy from the 15-L bioreactor fed with glucose. FIG. 14E shows clodiphosphate synthase, Isp, EC 4.6.1.12. Assay: PNAS volumetric productivity within the 15-L bioreactor fed with 96:11758-11763, 1999: HDS: 1-Hydroxy-2-methyl-2-(E)- glucose. FIG. 14F shows carbon dioxide evolution rate butenyl 4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. (CER), or metabolic activity profile, within the 15-L biore Org. Chem. 70:9168-9174, 2005; HDR: 1-Hydroxy-2-me actor fed with glucose. thyl-2-(E)-butenyl 4-diphosphate reductase, IspH. EC FIGS. 15A-B are graphs showing analysis of off-gas from 1.17.1.2. Assay: JACS, 126:12847-12855, 2004. fermentation in 15 L. bioreactors. Sample A is strain FIG. 1B illustrates the classical and modified MVA path RM111608-2 sampled at 64.8 hours. Sample B is strain ways. 1, acetyl-CoA acetyltransferase (AACT); 2. HMG 10 EWL256 was E. coli BL21 (DE3), pCL upper, cmR-gi1.2- CoA synthase (HMGS);3, HMG-CoA reductase (HMGR);4, yKKDyI, pTrcAlba-mMVK sampled at 34.5 hours. Hydro mevalonate kinase (MVK); 5, phosphomevalonate kinase gen is detected above the baseline (0.95x10 torr) for both (PMK); 6, diphosphomevalonate decarboxylase (MVD or samples. DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8. FIG. 16A shows an exemplary isoprene recovery unit. phosphomevalonate decarboxylase (PMDC); 9, isopentenyl 15 FIG. 16B shows an exemplary isoprene desorption/con phosphate kinase (IPK). The classical MVA pathway pro densation setup. ceeds from reaction 1 through reaction 7 via reactions 5 and 6. FIG. 17 shows a GC/FID chromatogram of an isoprene while a modified MVA pathway goes through reactions 8 and product. The material was determined to be 99.7% pure. 9. P and PP in the structural formula are phosphate and FIGS. 18A-C show the GC/FID chromatograms of an iso pyrophosphate, respectively. This figure was taken from prene sample before (A) and after treatment with alumina (B) Koga and Morii, Microbiology and Mol. Biology Reviews or silica (C). The isoprene peak is not shown in these chro 71.97-120, 2007. The modified MVA pathway is present, for matograms. example, in some archaeal organisms, such as Methanosa FIG. 19A shows a map of plasmid p)W34, encoding a rcina mazei. truncated version of P alba isoprene synthase (MEA variant) FIG. 2 is a map of plasmid plT24 P. alba HGS. 25 under the control of the PTrc promoter and M. mazei MVK. FIG. 3A-B are the nucleotide sequence of plasmid pET24 FIG. 19B-D shows the complete nucleotide sequence of plas P alba HGS (SEQID NO:1). mid plDW34 (SEQID NO:7). FIG. 4 is a schematic diagram showing restriction sites FIG. 20 shows the chromosomal organization of E. coli used for endonuclease digestion to construct plasmid K12 strain MG 1655 around the pgl locus (Graph imported EWL230 and compatible cohesive ends between BspHI and 30 from www.ecocyc.com). The region deleted in E. coli BL21 NcoI sites. (DE3) compared to E. coli K12 MG655 and restored instrains FIG. 5 is a map of plasmid EWL230. CMP215 and CMP258 is shown in brackets. The predicted FIGS. 6A-B are the nucleotide sequence of plasmid ORF of the ybgS gene is circled. A forward arrow (->) indi EWL230 (SEQ ID NO:2). cates the annealing site of the galMF primer (SEQID NO:8). FIG. 7 is a schematic diagram showing restriction sites 35 A reverse arrow (e-) indicates the annealing site of the galMR used for endonuclease digestion to construct plasmid primer (SEQ ID NO:9). EWL244 and compatible cohesive ends between NsiI and FIG. 21 shows optical density (OD) plots from microfer Pst sites. mentation experiments conducted with PGL+ (CMP312) and FIG. 8 is a map of plasmid EWL244. PGL- (CMP323) cultures. Black triangles along the X-axis FIGS. 9A-B are the nucleotide sequence of plasmid 40 indicate when offline samples were taken. Other OD values EWL244 (SEQ ID NO:3). are interpolated. FIG.10A is a map of the M. mazei archaeal Lower Pathway FIG. 22 shows isoprene specific productivity plots from operon. microfermentation experiments conducted with PGL+ FIGS. 10B-C are the nucleotide sequence of the M. mazei (CMP312) and PGL- (CMP323) cultures. Black triangles archaeal Lower Pathway operon (SEQID NO:4). 45 along the X-axis indicate when offline samples were taken. FIG. 11A is a map of MCM376-MVK from M. mazei Other OD values are interpolated. archaeal Lower in pET200D. FIG. 23 shows a time course of optical density in a 15-L FIGS. 11B-C are the nucleotide sequence of MCM376 bioreactor fed with glucose. MVK from M. mazei archaeal Lower in pET200D (SEQ ID FIG. 24 shows a time course of isoprene titer in a 15-L NO:5). 50 bioreactor fed with glucose. Isoprene titer is defined as the FIG. 12 is a map of plasmid pBBRCMPGI1.5-pgl. amount of isoprene produced per liter of fermentation broth. FIGS. 13 A-B are the nucleotide sequence of plasmid The equation for calculating isoprenetiter is: (Instantaneous pBBRCMPGI1.5-pgl (SEQID NO:6). isoprene production rate, g/L/hr)dt from t=0 to thrs = g/L FIGS. 14A-F are graphs of isoprene production by E. coli broth. strain expressing M. mazei mevalonate kinase, P alba iso 55 FIG. 25 shows the time course of total isoprene produced prene synthase, and pgl (RHM1 11608-2), and grown in fed from the 15-L bioreactors fed with glucose. batch culture at the 15-L scale. FIG. 14A shows the time FIG. 26 shows isoprene specific productivity within the course of optical density within the 15-L bioreactor fed with 15-L bioreactors fed with glucose. Equation for calculating glucose. FIG. 14B shows the time course of isoprene titer Specific Productivity levels: (mg isoprene-mg isoprene)/ within the 15-L bioreactor fed with glucose. The titer is 60 (OD550, L broth-OD550*L broth)/(2.7 OD*L/g cell)/ defined as the amount of isoprene produced per liter offer (t-to) – mg isoprene/g cell/hr. mentation broth. Method for calculating isoprene: cumulative FIG. 27 shows a time course of optical density within the isoprene produced in 59 hrs, g/Fermentor volume at 59 hrs, 15-L bioreactor fed with glucose. The pgl+ sample was a L = g/L broth. FIG. 14C also shows the time course of culture of strain CMP312. The pgl-sample was a culture of isoprene titer within the 15-L bioreactor fed with glucose. 65 Strain CMP323. Method for calculating isoprene: f(Instantaneous isoprene FIG. 28 shows a time course of isoprene titer within the production rate, g/L/hr)dt from t=0 to 59 hours = g/L broth. 15-L bioreactor fed with glucose. The titer is defined as the US 8,455,236 B2 11 12 amount of isoprene produced per liter of fermentation broth. FIG. 41A-B shows the growth rate of BL21 (Novagen) and The pgl+ sample was a culture of strain CMP312. The pgl strain CMP258 (example 6), labeled as BL21 pgl. Growth sample was a culture of strain CMP323. Equation for calcu was assessed in M9 minimal medium (6g/L NaHPO, 3 g/L lating Isoprene Titer: (Instantaneous isoprene production KHPO, 0.5g/L NaCl, 0.5 g/L NHCl, 0.1 mM CaCl2 mM rate, g/L/hr)dt from t=0 to 20 hrs = g/L broth. MgSO) containing 0.4% (w/v) glucose. Growth was mea FIG. 29 shows isoprene specific productivity within the sured at ODoo. FIG. 41A shows the growth of BL21 and 15-L bioreactor fed with glucose. The pgl+ sample was a strain CMP258 (labeled as BL21 pgl). FIG. 41B shows spe culture of strain CMP312. The pgl-sample was a culture of cific growth rate (LL) of BL21 with and without pgl. Restoring strain CMP323. Equation for calculating Specific Productiv the 17,257 bp deletion comprising pgl in BL21 results in a ity levels: (mg isoprene-mg isoprene)/OD550,L broth 10 strain with around 15% increase in specific growth rate. OD550*L broth)/(2.7 OD*L/g cell)/(t-to) = mg iso FIG. 42A shows a time course of isoprene titer in a 15-L prenelg cell/hr bioreactor fed with glucose. Isoprene titer is defined as the FIG. 30 shows a time course of optical density within a amount of isoprene produced per liter of fermentation broth. 15-L bioreactor containing E. coli K12 strain MG 1655 fed The equation for calculating isoprenetiter is: (Instantaneous with glucose. 15 isoprene production rate, g/L/hr)dt from t=0 to thrs = g/L FIG. 31 shows a time course of isoprene titer within the broth. FIG. 42B shows isoprene specific productivity within 15-L bioreactor containing E. coli K-12 strain MG 1655 fed the 15-L bioreactors fed with glucose. Equation for calculat with glucose. The titer is defined as the amount of isoprene ing Specific Productivity levels: (mg isoprene-mg iso produced per liter of fermentation broth. Equation for calcu prene)/OD550, L broth-OD550*L broth)/(2.7 lating Isoprene Titer: (Instantaneous isoprene production OD*L/g cell)/(t-to) = mg isoprene/g cell/hr. rate, g/L/hr)dt from t=0 to thrs = g/L broth. FIG. 32 shows a time course of total isoprene produced DETAILED DESCRIPTION from the 15-L bioreactor containing E. coli K-12 strain MG1655 fed with glucose. E. coli BL21 and BL21 (DE3) are widely used hosts for the FIG.33 shows a time course of isoprene specific produc 25 production of recombinant proteins. They can also be used to tivity in a 15-L bioreactor containing E. coli strain K12 produce other products, such as isoprene. Yields of recombi MG 1655 fed with glucose. Equation for calculating specific nant proteins, biochemicals, and other products in Such E. coli productivity: (mg isoprene-mg isoprene)/OD550,L strains can be improved by increasing activity of the pentose broth-OD550*L broth)/(2.7 OD*L/g cell)/(t-to) = mg phosphate pathway, a metabolic pathway important for cell isoprene/g cell/hr. 30 growth. Comparison of the genomic sequence of E. coli BL21 FIG. 34A shows a map of plasmid pW15, expressing the prepared by Codon Genomics (St. Louis, Mo.) using an Illu upper MVA pathway polypeptides mvaE and mvaS from mina Genome Analyzer II (GAII) Sequencing System to that Enterobacter faecalis. FIGS. 34B-D shows the complete of E. coli MG1655 (GenBank Accession No. U00096) nucleotide sequence of plasmid pPW15 (SEQID NO:10). revealed that the E. coli BL21 genome carried a deletion of FIG. 35 shows mevalonate specific productivity of bacte 35 17,257 bp in the region encoding genes involved in the utili rial strains in TM3 minimal medium containing 0.1% yeast Zation of galactose as well as other genes that are described in extract and 1% glucose. Experiments were run in triplicate greater detail herein. Unexpectedly, that deletion also encom from unique colonies. Strains are described in more detail in passed the ybhE gene (Thomason, L., Court, D., Datta, A., Table 29. BL21+pCL pTrcUpper strain MCM870; BL21 Khanna, R. and Rosner, J., “Identification of the Escherichia pgl+pCL pTrcUpper strain MCM874; BL21+pBBR 40 coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolac pTrcUpper-strain MCM871; BL21 pgl+pBBR tonase” J. Bact. 186:8248-8253 (2004)), which encodes the pTrcUpper strain MCM875; BL21+pTrcUpper-MCM872: enzyme 6-phosphogluconolactonase (PGL), the second BL21 pgl+pTrcUpper-MCM876. enzyme in the pentose phosphate pathway. The deletion was FIG. 36 shows growth of E. coli strains MCM872 and made by UV irradiation of a parent strain of E. coli BL21 and MCM876 in TM3 minimal medium containing 0.02% yeast 45 passed via P1 transduction (Studier F. Daegelen, P., Lenski, extract and 1% glucose. R., Maslov, S. Kim, J. F., “Understanding the differences FIG.37 shows mevalonate production rate of E. coli strains between genome sequences of Escherichia coli B strains MCM872 (BL21 pTrc-Upper) and MCM876 (BL21 pgl REL606 and BL21 (DE3) and comparison of the E. coli Band pTrc-Upper) in TM3 minimal medium containing 0.02% K-12 genomes. J. Mol. Biol. published ahead of print Sep. yeast extract and 1% glucose. 50 15, 2009). Consequently, E. coli BL21 and BL21 (DE3) lack FIG.38 shows the concentration of MvaS protein per OD both PGL activity and the ability to utilize galactose as a in E. coli strains MCM872 (BL21 pTrc-Upper) and MCM876 carbon Source. (Aon et al., “Suppressing posttranslational (BL21 pglpTrc-Upper) in TM3 minimal medium with 0.02% gluconoylation of heterologous proteins by metabolic engi yeast extract, taken at two different timepoints. neering of Escherichia coli,' Appl. Environ. Microbiol. FIG.39 shows the concentration of MvaE per OD in E. coli 55 74:950-958 (2008)). strains MCM872 (BL21 pTrc-Upper) and MCM876 (BL21 Additionally, the deletion also included genes required for pglpTrc-Upper) grown in TM3 minimal medium with 0.02% high affinity transport of molybdate. While required in only yeast extract. trace amounts, molybdenum plays an important role in sev FIG. 40A shows the amino acid sequence of 6-phospho eral metabolic pathways in all organisms. Molybdate is used gluconolactonase (PGL) from E. coli K12 MG1655 (SEQID 60 as an enzymatic cofactor by bacteria in a number of oxidation/ NO:11). FIG. 40B shows the amino acid sequence of PGL reduction reactions, plays a critical role in nitrogen metabo from P. aeruginosa (SEQ ID NO:12). FIG. 40C shows the lism, and, particularly in the case of anaerobic respiration, amino acid sequence of PGL from S. cerevisiae (SEQ ID contributes to energy production. (See, e.g., Self et al., Res NO:13). FIG. 40D shows an alignment of the amino acid Microbiol. 152:311-321 (2001); Grunden & Shanmugam, sequences of E. coli PGL and P. aeruginosa PGL. Identical 65 Arch Microbiol. 168:345-354 (1997)). amino acids are shown highlighted in grey. Conservative The pentose phosphate pathway (PPP) is used during amino acid Substitutions are shown highlighted in black. growth to provide NADPH and pentoses (5-carbon sugars) US 8,455,236 B2 13 14 (Neidhart, F., Ingraham, J., and Schaechter, M., 1990, Physi the recombinant E. coli cell can use promoter(s) and/or other ology of the bacterial cell: a molecular approach (Sinauer regulatory systems that have been introduced to the E. coli Associates, Inc. Sunderland, Mass.)). The PPP has two dis cell to regulate the transcription and Subsequent expression of tinct phases: (1) the oxidative phase, in which NADPH is the integrated PGL nucleic acid. generated; and (2) the non-oxidative synthesis of 5-carbon The invention also encompasses cells of an Escherichia sugars. The PPP is an alternative to glycolysis, and while it coli strain that does not encode a 6-phosphogluconolactonase does involve oxidation of glucose, its primary role is anabolic (PGL) polypeptide, wherein the E. coli cells comprise one or rather than catabolic. The primary results of the pathway are: more copies of a heterologous gene encoding a PGL polypep (1) the generation of reducing equivalents in the form of tide with one or more associated expression control NADPH, for use in reductive biosynthesis reactions within 10 sequences and a nucleic acid encoding a heterologous cells. Such as fatty acid synthesis; (2) production of ribose-5- polypeptide capable of biological activity. In one aspect, the phosphate (R5P), used in the synthesis of nucleotides and PGL polypeptide is not encoded by nucleic acids on a plas nucleic acids; and (3) production of erythrose-4-phosphate mid. In some aspects, the E. coli cells produce the polypeptide (E4P), used in the synthesis of aromatic amino acids. Aro capable of biological activity at a specific productivity greater matic amino acids, in turn, are precursors for many biosyn 15 than that of the same cells lacking one or more copies of a thetic pathways. Dietary pentose Sugars derived from the heterologous gene encoding a PGL polypeptide with one or digestion of nucleic acids may be metabolized through the more associated expression control sequences, when the cells pentose phosphate pathway, and the carbon skeletons of are cultured in minimal medium. Also provided herein are dietary carbohydrates may be converted into glycolytic or improved methods of producing heterologous polypeptides gluconeogenic intermediates. In mammals, the PPP occurs capable of biological activity, comprising the steps of cultur exclusively in the cytoplasm, and is one of the three main ing the E. coli cells that do not encode a PGL polypeptide in ways the body creates molecules with reducing power, minimal medium, wherein the cells comprise one or more accounting for approximately 60% of NADPH production in copies of a heterologous gene encoding a PGL polypeptide humans. with one or more associated expression control sequences Restoring the PGL gene and its associated expression con 25 and a nucleic acid encoding a heterologous polypeptide trol sequences in E. coli BL21 and BL21 (DE3) strains con capable of biological activity, and producing the heterologous veys a Substantial growth benefit, as the pentose phosphate polypeptide. In some aspects, the cells produce the heterolo pathway provides reducing equivalents for use in reductive gous polypeptide at a specific productivity greater than that of biosynthesis reactions within cells, such as fatty acid synthe the same cells lacking one or more copies of a heterologous sis, ribose-5-phosphate (R5P) for use in the synthesis of 30 gene encoding a PGL polypeptide with one or more associ nucleotides and nucleic acids, and (3) erythrose-4-phosphate ated expression control sequences, when the cells are cultured (E4P) for use in the synthesis of aromatic amino acids. In in minimal medium. addition, it will be useful for industrial purposes to have a In another aspect, provided herein are cells of an Escheri homologous strain (e.g., an E. coli BL21 or BL21 (DE3) chia coli strain that does not encode a PGL polypeptide, strain) able to utilize galactose, in order to extend the range of 35 wherein the E. coli cells comprise one or more copies of a available carbon Sources. heterologous gene encoding a PGL polypeptide with one or Furthermore, restoring genes that encode high affinity more associated expression control sequences and a heterolo molybdate transport proteins will provide an additional gous nucleic acid encoding an upper mevalonate (MVA) growth benefit, as the cell will be able to utilize molybdate pathway polypeptide, a lower MVA pathway polypeptide, more efficiently in those metabolic reactions that require 40 and/oran isoprene synthase polypeptide. In some aspects, the molybdenum as a cofactor. The invention encompasses E. coli cells have a specific productivity of isoprene greater improved methods and compositions for recombinant bacte than that of the same cells lacking one or more copies of a rial cells expressing a heterologous nucleic acid encoding a heterologous gene encoding a PGL polypeptide with one or PGL polypeptide integrated into the bacterial chromosome. more associated expression control sequences, when the cells The PGL integration alone or in combination with one or 45 are cultured in minimal medium. Also provided herein are more other heterologous nucleic acids encoding polypeptides improved methods of producing isoprene, comprising the for galactose metabolism and/or molybdenum transport can steps of culturing the E. coli cells that do not encode a PGL improve a recombinant bacterial cells ability for the produc polypeptide in minimal medium, wherein the cells comprise tion of isoprene. one or more copies of a heterologous gene encoding a PGL Accordingly in one aspect, the invention encompasses 50 polypeptide with one or more associated expression control recombinant cell(s) of an Escherichia coli (E. coli) strain sequences and a heterologous nucleic acid encoding an upper capable of producing isoprene, wherein the cell(s) comprise: mevalonate (MVA) pathway polypeptide, a lower MVA path (a) one or more copies of a heterologous nucleic acid(s) way polypeptide, or an isoprene synthase polypeptide, and encoding a PGL polypeptide wherein the nucleic acid(s) producing isoprene. In some aspects, the cells have a specific is/are integrated in the E. coli chromosome; and (b) one or 55 productivity of isoprene greater than that of the same cells more heterologous nucleic acid(s) encoding isoprene Syn lacking one or more copies of a heterologous gene encoding thase; wherein prior to the integration, the E. coli cell does not a PGL polypeptide with one or more associated expression contain (a) nucleic acid(s) that encodes a encoding a PGL control sequences, when the cells are cultured in minimal polypeptide, and wherein the resulting recombinant cell pro medium. duces isoprene at a greater titer than that of the same cell(s) 60 General Techniques that do not comprise (a) and (b). In some cases, the recombi The practice of the present invention will employ, unless nant E. coli cell can use its own endogenous promoter(s) otherwise indicated, conventional techniques of molecular and/or its other regulatory systems to regulate the transcrip biology (including recombinant techniques), microbiology, tion and Subsequent expression of the integrated PGL nucleic cell biology, biochemistry, and immunology, which are acid. In Such cases, the expression of the heterologous nucleic 65 within the skill of the art. Such techniques are explained fully acids (e.g., PGL or isoprene) is not constitutive expression in the literature, “Molecular Cloning: A Laboratory Manual', driven by a plasmid or elements on a plasmid. In other cases, second edition (Sambrook et al., 1989); “Oligonucleotide US 8,455,236 B2 15 16 Synthesis” (M. J. Gait, ed., 1984): “Animal Cell Culture' (R. acid. In some cases, a heterologous nucleic acid can be a I. Freshney, ed., 1987); "Methods in Enzymology” (Aca heterologous gene. One of skill in the art would appreciate the demic Press, Inc.); "Current Protocols in Molecular Biology’ differences and also be able to use the context of the teaching (F.M. Ausubel et al., eds., 1987, and periodic updates); "PCR: accordingly. The Polymerase Chain Reaction', (Mullis et al., eds., 1994). As used herein, an “expression control sequence” means a Singleton et al., Dictionary of Microbiology and Molecular nucleic acid sequence that directs transcription of a nucleic Biology 2nded. J. Wiley & Sons (New York, N.Y. 1994), and acid of interest. An expression control sequence can be a March, Advanced Organic Chemistry Reactions, Mecha promoter. Such as a constitutive or an inducible promoter, or nisms and Structure 4th ed., John Wiley & Sons (New York, an enhancer. An expression control sequence can be "native' N.Y. 1992), provide one skilled in the art with a general guide 10 or heterologous. A native expression control sequence is to many of the terms used in the present application. derived from the same organism, species, or strain as the gene Definitions being expressed. A heterologous expression control sequence The term "isoprene’ refers to 2-methyl-1,3-butadiene is derived from a different organism, species, or strain as the (CASH78-79-5). It can be the direct and final volatile C5 gene being expressed. An "inducible promoter” is a promoter hydrocarbon product from the elimination of pyrophosphate 15 that is active under environmental or developmental regula from 3.3-dimethylallyl pyrophosphate (DMAPP). It may not tion. The expression control sequence is operably linked to involve the linking or polymerization of IPP molecules to the nucleic acid segment to be transcribed. DMAPP molecules. The term "isoprene' is not generally As used herein, the terms “minimal medium' or “minimal intended to be limited to its method of production unless media' refer to growth medium containing the minimum indicated otherwise herein. nutrients possible for cell growth, generally without the pres As used herein, the term “6-phosphogluconolactone' ence of amino acids. Minimal medium typically contains: (1) refers to 6-phospho-D-glucono-1,5-lactone (CASH2641-81 - a carbon Source for bacterial growth; (2) various salts, which 8). As used herein, the term “6-phosphogluconate” refers to may vary among bacterial species and growing conditions; 6-phospho-D-gluconate (CASH921-62-0). and (3) water. The carbon Source can vary significantly, from As used herein, the term “polypeptides includes polypep 25 simple Sugars like glucose to more complex hydrolysates of tides, proteins, peptides, fragments of polypeptides, and other biomass. Such as yeast extract, as discussed in more fusion polypeptides. detail below. The salts generally provide essential elements As used herein, an "isolated polypeptide' is not part of a Such as magnesium, nitrogen, phosphorus, and Sulfur to allow library of polypeptides, such as a library of 2, 5, 10, 20, 50 or the cells to synthesize proteins and nucleic acids. Minimal more different polypeptides and is separated from at least one 30 medium can also be supplemented with selective agents. Such component with which it occurs in nature. An isolated as antibiotics, to select for the maintenance of certain plas polypeptide can be obtained, for example, by expression of a mids and the like. For example, if a microorganism is resistant recombinant nucleic acid encoding the polypeptide. An iso to a certain antibiotic, such as amplicillin or tetracycline, then lated polypeptide can be a non-naturally occurring polypep that antibiotic can be added to the medium in order to prevent tide. 35 cells lacking the resistance from growing. Medium can be By "heterologous polypeptide' is meant a polypeptide Supplemented with other compounds as necessary to select encoded by a nucleic acid sequence derived from a different for desired physiological or biochemical characteristics. Such organism, species, or strain than the host cell. In some as particular amino acids and the like. aspects, a heterologous polypeptide is not identical to a wild As used herein, the term “terpenoid' or "isoprenoid refers type polypeptide that is found in the same host cell in nature. 40 to a large and diverse class of naturally-occurring organic As used herein, a “nucleic acid refers to two or more chemicals similar to terpenes. Terpenoids are derived from deoxyribonucleotides and/or ribonucleotides covalently five-carbon isoprene units assembled and modified in a vari joined together in either single or double-stranded form. ety of ways, and are classified in groups based on the number By “recombinant nucleic acid' is meant a nucleic acid of of isoprene units used in group members. Hemiterpenoids interest that is free of one or more nucleic acids (e.g., genes) 45 have one isoprene unit. Monoterpenoids have two isoprene which, in the genome occurring in nature of the organism units. Sesquiterpenoids have three isoprene units. Diterpe from which the nucleic acid of interest is derived, flank the noids have four isoprene units. Sesterterpenoids have five nucleic acid of interest. The term therefore includes, for isoprene units. Triterpenoids have six isoprene units. Tetrater example, a recombinant DNA which is incorporated into a penoids have eight isoprene units. Polyterpenoids have more vector, into an autonomously replicating plasmid or virus, or 50 than eight isoprene units. into the genomic DNA of a prokaryote or eukaryote, or which As used herein, the term “carotenoid refers to a group of exists as a separate molecule (e.g., a cDNA, a genomic DNA naturally occurring organic pigments produced in the chloro fragment, or a cDNA fragment produced by PCR or restric plasts and chromoplasts of plants, of some other photosyn tion endonuclease digestion) independent of other sequences. thetic organisms, such as algae, in some types of fungus, and In some aspects, a recombinant nucleic acid is a nucleic acid 55 in Some bacteria. Carotenoids include the oxygen-containing that encodes a non-naturally occurring polypeptide. Xanthophylls and the non-oxygen-containing carotenes. By "heterologous nucleic acid' is meant a nucleic acid Unless defined otherwise herein, all technical and scien sequence derived from a different organism, species or strain tific terms used herein have the same meaning as commonly than the host cell. In some aspects, the heterologous nucleic understood by one of ordinary skill in the art to which this acid is not identical to a wild-type nucleic acid that is found in 60 invention pertains. the same host cell in nature. For example, a nucleic acid As used herein, the singular terms “a,” “an, and “the encoding a PGL polypeptide isolated from E. coli K12 strain include the plural reference unless the context clearly indi MG 1655 or a derivative thereof, integrated into the chromo cates otherwise. some of E. coli BL21 (DE3) by P1 transduction and expressed It is intended that every maximum numerical limitation in the cell is a heterologous nucleic acid. In one aspect, a 65 given throughout this specification includes every lower "heterologous nucleic acid can mean the introduction of a numerical limitation, as if Such lower numerical limitations nucleic acid into a host cell that does not have that nucleic were expressly written herein. Every minimum numerical US 8,455,236 B2 17 18 limitation given throughout this specification will include restored to the BL21 or BL21 (DE3) genome are PGL and every higher numerical limitation, as if such higher numerical modE. In some aspects, the genes restored to the BL21 or limitations were expressly written herein. Every numerical BL21 (DE3) genome are PGL and modA, modB, and modC. range given throughout this specification will include every In some aspects, the genes restored to the BL21 or BL21 narrower numerical range that falls within such broader 5 (DE3) genome are PGL, modF, and modE. In some aspects, numerical range, as if such narrower numerical ranges were the genes restored to the BL21 or BL21 (DE3) genome are all expressly written herein. PGL, modF, modA, modB, and modC. In some aspects, the Genes Encoding Polypeptides Restored to E. coli B121 or genes restored to the BL21 or BL21 (DE3) genome are PGL, BL21(DE3) modE, modA, modB, and modC. In some aspects, the genes The 17,257 bp deletion in the E. coli BL21 and BL21 (DE3) 10 genomes includes the yghE gene (PGL), genes encoding restored to the BL21 or BL21 (DE3) genome are PGL, modF. proteins involved in the utilization of galactose as a carbon modE, modA, modB, and modC. Source, genes encoding proteins involved in molybdenum In some aspects, the genes restored to the BL21 or BL21 transport, as well as several other genes of unknown function (DE3) genome are PGL, galM, and modF. In some aspects, ality. See, for example, FIG. 20. The genes involved in the 15 the genes restored to the BL21 or BL21 (DE3) genome are utilization of galactose are galM which encodes galactose-1- PGL, galM, and modE. In some aspects, genes restored to the epimerase, galK, which encodes galactokinase, galT, which BL21 or BL21 (DE3) genome are PGL, galM, and modA, encodes galactose-1-phosphate uridylyltransferase, and modB, and modC. In some aspects, the genes restored to the galE, which encodes UDP-glucose 4-epimerase. The genes BL21 or BL21 (DE3) genome are PGL, galK, and modF. In encoding proteins involved in molybdenum transport are some aspects, the genes restored to the BL21 or BL21 (DE3) modF, which encodes the fused molybdate transporter sub genome are PGL, galK, and modE. In some aspects, the genes units of the ABC Superfamily, modE, which encodes the restored to the BL21 or BL21 (DE3) genome are PGL, galK, repressor of the modABC operon for molybdenum transport, modA, modB, and modC. In some aspects, the genes restored and modA, modB, and modC, which each encode a molyb to the BL21 or BL21 (DE3) genome are PGL, galT, and modF. date transporter subunit protein. 25 In some aspects, the genes restored to the BL21 or BL21 Accordingly, bacterial (e.g., E. coli) cells can be engi (DE3) genome are PGL, galT, and modE. In some aspects, the neered to integrate nucleic acids encoding a PGL polypeptide genes restored to the BL21 or BL21 (DE3) genome are PGL, in the E. coli chromosome. Introduction of heterologous galT, modA, modB, and modC. In some aspects, the genes nucleic acids encoding for isoprene synthase (e.g., P alba restored to the BL21 or BL21 (DE3) genome are PGL, galK, isoprene synthase) can increase the total titer and/or specific 30 and modF. In some aspects, the genes restored to the BL21 or activity for isoprene production. Furthermore, in addition to BL21 (DE3) genome are PGL, galE, and modE. In some the PGL integration, one or more genes encoding proteins aspects, the genes restored to the BL21 or BL21 (DE3) involved in the utilization of galactose as a carbon source or genome are PGL, galE, modA, modB, and modC. proteins involved in molybdenum transport can also be intro In some aspects, the genes restored to the BL21 or BL21 duced into the E. coli cell to increase the overall fitness of the 35 (DE3) genome are PGL, galM, galK, and modF. In some recombinant cell, which, in turn, can lead to increased pro aspects, the genes restored to the BL21 or BL21 (DE3) duction of isoprene. genome are PGL, galM, galK, and modE. In some aspects, the Various options of integrated PGL alone or integrated PGL genes restored to the BL21 or BL21 (DE3) genome are PGL, in combination with one or more genes encoding proteins galM, galK, and modA, modB, and modC. In some aspects, involved in the utilization of galactose as a carbon source or 40 the genes restored to the BL21 or BL21 (DE3) genome are proteins involved in molybdenum transport are contemplated PGL, galM, galT, and modF. In some aspects, the genes within the scope of the invention. Thus, in Some aspects, the restored to the BL21 or BL21 (DE3) genome are PGL, galM, gene restored to the BL21 or BL21 (DE3) genome is PGL. In galT, and modE. In some aspects, the genes restored to the some aspects, the genes restored to the BL21 or BL21 (DE3) BL21 or BL21 (DE3) genome are PGL, galM, galT, and genome are PGL and galM. In some aspects, genes restored to 45 modA, modB, and modC. In some aspects, the genes restored the BL21 or BL21 (DE3) genome are PGL and galK. In some to the BL21 or BL21 (DE3) genome are PGL, galM, galE, and aspects, the genes restored to the BL21 or BL21 (DE3) modF. In some aspects, the genes restored to the BL21 or genome are PGL and galT. In some aspects, the genes BL21 (DE3) genome are PGL, galM, galE, and modE. In restored to the BL21 or BL21 (DE3) genome are PGL and some aspects, the genes restored to the BL21 or BL21 (DE3) galE. In some aspects, the genes restored to the BL21 or 50 genome are PGL, galM, galE, and modA, modB, and modC. BL21 (DE3) genome are PGL, galM, and galK. In some In some aspects, the genes restored to the BL21 or BL21 aspects, the genes restored to the BL21 or BL21 (DE3) (DE3) genome are PGL, galK, galT, and modF. In some genome are PGL, galM, and galT. In some aspects, the genes aspects, the genes restored to the BL21 or BL21 (DE3) restored to the BL21 or BL21 (DE3) genome are PGL, galM, genome are PGL, galK, galT, and modE. In some aspects, the and galE. In some aspects, the genes restored to the BL21 or 55 genes restored to the BL21 or BL21 (DE3) genome are PGL, BL21 (DE3) genome are PGL, galK, and galT. In some galK, galT, and modA, modB, and modC. In some aspects, aspects, the genes restored to the BL21 or BL21 (DE3) the genes restored to the BL21 or BL21 (DE3) genome are genome are PGL, galK, and galE. In some aspects, the genes PGL, galK, galE, and modF. In some aspects, the genes restored to the BL21 or BL21 (DE3) genome are PGL, galT. restored to the BL21 or BL21 (DE3) genome are PGL, galK, and galE. In some aspects, the genes restored to the BL21 or 60 galE, and modE. In some aspects, the genes restored to the BL21 (DE3) genome are PGL, galM, galK, and galT. In some BL21 or BL21 (DE3) genome are PGL, galK, galE, and aspects, genes restored to the BL21 or BL21 (DE3) genome modA, modB, and modC. In some aspects, the genes restored are PGL, galM, galK, and galE. In some aspects, genes to the BL21 or BL21 (DE3) genome are PGL, galE, galT, and restored to the BL21 or BL21 (DE3) genome are PGL, galK, modF. In some aspects, the genes restored to the BL21 or galT, and galE. 65 BL21 (DE3) genome are PGL, galE, galT, and modE. In some In some aspects, genes restored to the BL21 or BL21 (DE3) aspects, the genes restored to the BL21 or BL21 (DE3) genome are PGL and modF. In some aspects, the genes genome are PGL, galE, galT, and modA, modB, and modC. US 8,455,236 B2 19 20 In some aspects, the genes restored to the BL21 or BL21 (i.e., replacing an amino acid having a basic side chain with (DE3) genome are PGL, galM, galK, galTand modF. In some another amino acid having a basic side chain). A “non-con aspects, the genes restored to the BL21 or BL21 (DE3) servative amino acid substitution' is one in which the amino genome are PGL, galM, galK, galT, and modE. In some acid residue is replaced with an amino acid residue having a aspects, the genes restored to the BL21 or BL21 (DE3) chemically different side chain (i.e., replacing an amino acid genome are PGL, galM, galK, galT, and modA, modB, and having a basic side chain with another amino acid having an modC. In some aspects, the genes restored to the BL21 or aromatic side chain). BL21 (DE3) genome are PGL, galM, galK, galE and modF. In Amino acid substitutions in the PGL polypeptide can be some aspects, the genes restored to the BL21 or BL21 (DE3) introduced to improve the functionality of the molecule. For genome are PGL, galM, galK, galE, and modE. In some 10 example, amino acid substitutions that increase the binding aspects, the genes restored to the BL21 or BL21 (DE3) affinity of the PGL polypeptide for its substrate, or that genome are PGL, galM, galK, galE, and modA, modB, and improve its ability to convert 6-phosphogluconolactone to modC. In some aspects, the genes restored to the BL21 or 6-phosphogluconate can be introduced into the PGL polypep BL21 (DE3) genome are PGL, galE, galK, galT and modF. In tide. In some aspects, the mutant PGL polypeptides contain some aspects, the genes restored to the BL21 or BL21 (DE3) 15 one or more conservative amino acid substitutions. In some genome are PGL, galE. galK, galT, and modE. In some aspects, the mutant PGL polypeptides contain one or more aspects, the genes restored to the BL21 or BL21 (DE3) non-conservative amino acid substitutions. genome are PGL, galE. galK, galT, and modA, modB, and Standard methods, such as those described by A. Sinha and modC. P. K. Maitra, “Induction of specific enzymes of the oxidative In some aspects, the one or more copies of one or more pentose phosphate pathway by glucono-delta-lactone in Sac genes encoded on the 17.257 bp genomic piece (except for charomyces cerevisiae,” J. Gen. Microbiol. 138:1865-1873 PGL) are restored to E. coli BL21 or BL21 (DE3) on a plas (1992), can be used to determine whether a polypeptide has mid. In some aspects, the one or more copies of one or more PGL activity, by measuring the ability of a polypeptide to genes encoded on the 17,257 bp genomic piece are restored to reduce NADP+ to NADPH. In an exemplary assay, PGL E. coli BL21 or BL21 (DE3) on a constitutively expressing 25 activity is assayed by pre-incubating a reaction mixture con plasmid. In some aspects one or more copies of one or more taining 50 LM glucose-6-phosphate 0.5 mMNADP+, and 0.5 genes encoded on the 17,257 bp genomic piece are restored to units glucose-6-phosphate dehydrogenase in 50 mM MES E. coli BL21 or BL21 (DE3) on an inducible plasmid. In some Buffer, pH=6.5, 25 mM KC1, 10 mM MgCl, until the reac aspects, the entire 17.257 bp genomic piece is a plasmid tion was complete. This was followed by addition of 1 unit of which is transfected into E. coli BL21 or BL21 (DE3) cells. In 30 6-phosphogluconate dehydrogenase which resulted in a slow Some aspects, the one or more copies of one or more genes increase in fluorescence due to spontaneous hydrolysis of the encoded on the 17,257 bp genomic piece are restored (e.g., as lactone formed during the earlier reaction. Next, cell-free depicted in FIG. 20) to E. coli BL21 or BL21 (DE3) by chro extracts are added, leading to an increased rate of NADP+ mosomal integration. In some aspects, the entire 17.257 bp reduction to NADPH via the lactonase reaction catalyzed by genomic piece is restored to E. coli BL21 or BL21 (DE3) by 35 PGL. The actual lactonase rate is calculated by subtracting chromosomal integration. the previous blank rate from this final rate. Exemplary PGL Polypeptides and Nucleic Acids Alternatively, conversion of 6-phosphogluconolactone to 6-phosphogluconolactonase (PGL) converts 6-phospho 6-phosphogluconate can be monitored by nuclear magnetic gluconolactone to 6-phosphogluconate. Exemplary PGL resonance (NMR) spectroscopy. See, e.g., E. Miclet et al., polypeptides include polypeptides, fragments of polypep 40 “NMR Spectroscopic Analysis of the First Two Steps of the tides, peptides, and fusion polypeptides that have at least one Pentose-Phosphate Pathway Elucidates the Role of 6-Phos activity of a PGL polypeptide. Exemplary PGL polypeptides phogluconolactonase.J. Biol. Chem. 276(37):34840-34846 and nucleic acids include naturally-occurring polypeptides (2001). and nucleic acids from any of the source organisms described Exemplary PGL nucleic acids include nucleic acids that herein as well as mutant polypeptides and nucleic acids 45 encode a polypeptide, fragment of a polypeptide, peptide, or derived from any of the source organisms described herein fusion polypeptide that has at least one activity of a PGL that have at least one activity of a PGL polypeptide. polypeptide. Exemplary isoprene synthase polypeptides and Mutant PGL polypeptides include those in which one or nucleic acids include naturally-occurring polypeptides and more amino acid residues have undergone an amino acid nucleic acids from any of the source organisms described substitution while retaining PGL activity (i.e., the ability to 50 herein as well as mutant polypeptides and nucleic acids convert 6-phosphogluconolactone to 6-phosphogluconate). derived from any of the source organisms described herein. The amino acid Substitutions may be conservative or non Exemplary PGL nucleic acids include, for example, PGL conservative and Such substituted amino acid residues may or isolated from E. coli K12 MG 1655 or derivatives thereof may not be one encoded by the genetic code. The standard (EcoGene Accession No. EG13231; part of E. coli K12 twenty amino acid “alphabet” has been divided into chemical 55 MG 1655 genomic sequence referenced by GenBank Acces families based on similarity of their side chains. Those fami sion No. U0096; see also UniProtKB/Swiss-Prot Accession lies include amino acids with basic side chains (e.g., lysine, No. P52697 (PGL polypeptide))(see FIG. 40A and SEQ ID arginine, histidine), acidic side chains (e.g., aspartic acid, NO:11); PGL isolated from Pseudomonas aeruginosa strain glutamic acid), uncharged polar side chains (e.g., glycine, PAO1 (Locus Tag PA3182 of GenBank Accession No. asparagine, glutamine, serine, threonine, tyrosine, cysteine), 60 AE004.091); see also GenBank Accession No. AAG06570.1 nonpolar side chains (e.g., alanine, Valine, leucine, isoleu (PGL polypeptide))(see FIG. 40B and SEQID NO:12); and cine, proline, phenylalanine, methionine, tryptophan), beta PGL isolated from Saccharomyces cerevisiae (Locus Tag branched side chains (e.g., threonine, Valine, isoleucine) and YHR163W of GenBank Accession No. NC 001140; see aromatic side chains (e.g., tyrosine, phenylalanine, tryp also UNIProtKB/Swiss-Prot Accession No. P38858 (PGL tophan, histidine). A “conservative amino acid substitution 65 polypeptide))(see FIG. 40C and SEQ ID NO:13). Other is one in which the amino acid residue is replaced with an exemplary PGL nucleic acids can be isolated from any genus amino acid residue having a chemically similar side chain in the family Enterobacteriaceae including, for example, US 8,455,236 B2 21 22 Alishewanella, Alterococcus, Aquamonas, Citrobacter; isolated from any genus in the family Enterobacteriaceae Cronobacter, Edwardsiella, Enterobacter, Klebsiella (e.g., including, for example, Alishewanella, Alterococcus, Aqua Klebsiella pneumoniae), Pantoea (e.g., Pantoea citroea), monas, Citrobacter, Cronobacter, Edwardsiella, Entero Proteus (e.g., Proteus vulgaris), Salmonella, Serratia (e.g., bacter; Klebsiella (e.g., Klebsiella pneumoniae), Pantoea Serratia marcescens), Shigella, and Yersinia (e.g., Yersinia (e.g., Pantoea citroea), Proteus (e.g., Proteus vulgaris), Sal pestis). monella, Serratia (e.g., Serratia marcescens), Shigella, and Exemplary Galactose Metabolism Polypeptides and Nucleic Yersinia (e.g., Yersinia pestis). Acids Exemplary Molybdenum Transporter Polypeptides and Galactose-1-epimerase (galM) catalyzes the conversion of Nucleic Acids B-D-galactose to C-D-galactose. Exemplary galactose-1-epi 10 merase polypeptides include polypeptides, fragments of The polypeptide encoded by the modF gene is an unchar polypeptides, peptides, and fusion polypeptides that have at acterized member of the fused molybdate transporter sub least one activity of a galactose-1-epimerase polypeptide. units of ABC superfamily. Exemplary modF encoded Exemplary galactose-1-epimerase polypeptides and nucleic polypeptides include polypeptides, fragments of polypep acids include naturally-occurring polypeptides and nucleic 15 tides, peptides, and fusion polypeptides that have at least one acids from any of the source organisms described herein as activity of a modF encoded polypeptide. Exemplary modF well as mutant polypeptides and nucleic acids derived from encoded polypeptides and nucleic acids include naturally any of the source organisms described herein that have at least occurring polypeptides and nucleic acids from any of the one activity of a galactose-1-epimerase polypeptide. Source organisms described hereinas well as mutant polypep Galactokinase (galK) catalyzes the phosphorylation of tides and nucleic acids derived from any of the Source organ D-galactose to D-galactose-1-phosphate. Exemplary galac isms described herein that have at least one activity of a modF tokinase polypeptides include polypeptides, fragments of encoded polypeptide. polypeptides, peptides, and fusion polypeptides that have at Repressor of the modABC operon for molybdenum trans least one activity of a galactokinase polypeptide. Exemplary port (modE) polypeptide is a regulatory protein that is galactokinase polypeptides and nucleic acids include natu 25 believed to feedback inhibit the transcription of the modABC rally-occurring polypeptides and nucleic acids from any of operon in the presence of molybdate. Exemplary modE the Source organisms described herein as well as mutant encoded polypeptides include polypeptides, fragments of polypeptides and nucleic acids derived from any of the Source polypeptides, peptides, and fusion polypeptides that have at organisms described herein that have at least one activity of a least one activity of a modE encoded polypeptide. Exemplary galactokinase polypeptide. 30 modE encoded polypeptides and nucleic acids include natu Galactose-1-phosphate uridylyltransferase (galT) cata rally-occurring polypeptides and nucleic acids from any of lyzes the second step of the Leloir pathway of galactose the source organisms described herein as well as mutant metabolism by converting UDP-glucose and galactose polypeptides and nucleic acids derived from any of the source 1-phosphate to glucose 1-phosphate and UDP-galactose. organisms described herein that have at least one activity of a Exemplary galactose-1-phosphate uridylyltransferase 35 modE encoded polypeptide. polypeptides include polypeptides, fragments of polypep The high affinity trimeric molybdenum transporter protein tides, peptides, and fusion polypeptides that have at least one encoded by modA, modB, and modC is a membrane-associ activity of a galactose-1-phosphate uridylyltransferase ated ABC-type transporter system for the uptake of molyb polypeptide. Exemplary galactose-1-phosphate uridylyl denum into the cell. When any one of the modABC genes are transferase polypeptides and nucleic acids include naturally 40 mutated or absent, molybdate transport is accomplished by occurring polypeptides and nucleic acids from any of the the ABC-type Sulfate transport system or by a non-specific Source organisms described hereinas well as mutant polypep anion transporter, but with about 100 times less efficiency. tides and nucleic acids derived from any of the Source organ (Self et al., 2001, Res. Microbiol. 152:311-321). Exemplary isms described herein that have at least one activity of a modABC encoded polypeptides include polypeptides, frag galactose-1-phosphate uridylyltransferase polypeptide. 45 ments of polypeptides, peptides, and fusion polypeptides that UDP-galactose-4-epimerase (galE) catalyzes the revers have at least one activity of a modABC encoded polypeptide. ible conversion of UDP-galactose to UDP-glucose. Exem Exemplary modABC encoded polypeptides and nucleic acids plary UDP-galactose-4-epimerase polypeptides include include naturally-occurring polypeptides and nucleic acids polypeptides, fragments of polypeptides, peptides, and fusion from any of the source organisms described herein as well as polypeptides that have at least one activity of a UDP-galac 50 mutant polypeptides and nucleic acids derived from any of tose-4-epimerase polypeptide. Exemplary UDP-galactose-4- the Source organisms described herein that have at least one epimerase polypeptides and nucleic acids include naturally activity of one of the modABC encoded polypeptides. occurring polypeptides and nucleic acids from any of the Exemplary molybdenum transport nucleic acids include Source organisms described hereinas well as mutant polypep nucleic acids that encode a polypeptide, fragment of a tides and nucleic acids derived from any of the Source organ 55 polypeptide, peptide, or fusion polypeptide that has at least isms described herein that have at least one activity of a one activity of a molybdenum transport polypeptide. Exem UDP-galactose-4-epimerase polypeptide. plary molybdenum transport nucleic acids include, for Exemplary galactose metabolic nucleic acids include example, molybdenum transport genes isolated from E. coli nucleic acids that encode a polypeptide, fragment of a K12 MG 1655 or derivatives thereof molybdenum transport polypeptide, peptide, or fusion polypeptide that has at least 60 genes isolated from Pseudomonas aeruginosa strain PAO1; one activity of a galactose metabolic polypeptide. Exemplary and galactose metabolic genes isolated from Saccharomyces galactose metabolic nucleic acids include, for example, cerevisie. Other exemplary molybdenum transport nucleic galactose metabolic genes isolated from E. coli K12 MG 1655 acids can be isolated from any genus in the family Enterobac or derivatives thereof galactose metabolic genes isolated teriaceae including, for example, Alishewanella, Alterococ from Pseudomonas aeruginosa strain PAO1; and galactose 65 cus, Aquamonas, Citrobacter, Cronobacter; Edwardsiella, metabolic genes isolated from Saccharomyces cerevisie. Enterobacter, Klebsiella (e.g., Klebsiella pneumoniae), Pan Other exemplary galactose metabolic nucleic acids can be toea (e.g., Pantoea citroea), Proteus (e.g., Proteus vulgaris), US 8,455,236 B2 23 24 Salmonella, Serratia (e.g., Serratia marcescens), Shigella, Enterococcus faecalis. In some aspects, the lower MVA path and Yersinia (e.g., Yersinia pestis). way polypeptide is selected from the group consisting of: (i) Exemplary Host Cells mevalonate kinase (MVK); (ii) phosphomevalonate kinase E. coli host cells can be used to express isoprene synthase, (PMK); (iii) diphosphomevalonate decarboxylase (MVD); PGL polypeptide, DXP pathway polypeptides, IDI, and MVA and (iv) isopentenyl diphosphate isomerase (IDI). In some pathway polypeptides in the methods described herein. In one aspects, the lower MVA pathway polypeptide is an MVK aspect, the host cell is a recombinant cell of an Escherichia polypeptide. In some aspects, the MVK polypeptide is from coli (E. coli) strain, or progeny thereof, capable of producing the genus Methanosarcina. In some aspects, the MVK isoprene, the cell comprising: (a) one or more copies of a polypeptide is from Methanosarcina mazei. heterologous nucleic acid(s) encoding a PGL polypeptide 10 wherein the nucleic acid is integrated in the E. coli chromo In some aspects, the one or more copies of a heterologous Some; and (b) one or more heterologous nucleic acid(s) gene encoding a PGL polypeptide with one or more associ encoding isoprene synthase; wherein prior to the integration, ated expression control sequences are chromosomal copies the E. coli cell does not contain nucleic acid(s) encoding a (e.g., integrated into the E. coli chromosome). In some PGL polypeptide, and wherein the resulting recombinant cell 15 aspects, the E. coli cells are in culture. In some aspects, the produces isoprene at a greater titer than that of the same cells bacterial cells are of E. coli strain B. In some aspects, the that do not comprise (a) and (b). In some aspects, the host bacterial strains are of E. coli Strain BL21. In some aspects, cells are bacterial cells of an Escherichia coli strain that does the bacterial cells are of E. coli strain BL21 (DE3). not encode a 6-phosphogluconolactonase (PGL) polypeptide, Exemplary Cell Culture Media further comprising one or more copies of a heterologous gene As used herein, the terms “minimal medium' or “minimal encoding a PGL polypeptide with one or more associated media' refer to growth medium containing the minimum expression control sequences and a nucleic acid encoding a nutrients possible for cell growth, generally, but not always, heterologous polypeptide capable of biological activity. In without the presence of one or more amino acids (e.g., 1,2,3, Some aspects, the bacterial cells produce the heterologous 4, 5, 6, 7, 8, 9, 10, or more amino acids). Minimal medium polypeptide at a specific productivity greater than that of the 25 typically contains: (1) a carbon Source for bacterial growth; same cells lacking one or more copies of a heterologous gene (2) various salts, which may vary among bacterial species and encoding a PGL polypeptide with one or more associated growing conditions; and (3) water. The carbon Source can expression control sequences when the cells are cultured in vary significantly, from simple Sugars like glucose to more minimal medium. In some aspects, the one or more copies of complex hydrolysates of other biomass, such as yeast extract, a heterologous gene encoding a PGL polypeptide with one or 30 as discussed in more detail below. The salts generally provide more associated expression control sequences are chromo essential elements such as magnesium, nitrogen, phosphorus, somal copies (e.g., integrated into the E. coli chromosome). and sulfur to allow the cells to synthesize proteins and nucleic In some aspects, the E. coli cells are in culture. acids. Minimal medium can also be Supplemented with selec In some aspects, the heterologous polypeptide capable of tive agents, such as antibiotics, to select for the maintenance biological activity comprises one or more polypeptides 35 of certain plasmids and the like. For example, if a microor involved in the biosynthesis ofterpenoid (isoprenoid) or caro ganism is resistant to a certain antibiotic, Such as amplicillin or tenoid compounds, and the cells produce a terpenoid or caro tetracycline, then that antibiotic can be added to the medium tenoid at a higher specific productivity than that of the same in order to prevent cells lacking the resistance from growing. cells lacking one or more copies of a heterologous gene Medium can be supplemented with other compounds as nec encoding a PGL polypeptide with one or more associated 40 essary to select for desired physiological or biochemical char expression control sequences when cultured in minimal acteristics, such as particular amino acids and the like. medium. In some aspects, the method further comprises a Any minimal medium formulation can be used to cultivate step of recovering the terpenoid or carotenoid. the host cells. Exemplary minimal medium formulations In some aspects, the host cells are bacterial cells of an include, for example, M9 minimal medium and TM3 minimal Escherichia coli strain that does not encode a 6-phosphoglu 45 medium. Each liter of M9 minimal medium contains (1) 200 conolactonase (PGL) polypeptide, further comprising one or ml sterile M9 salts (64 g NaHPO-7H2O, 15 g KHPO, 2.5 more copies of a heterologous gene encoding a PGL polypep g NaCl, and 5.0 g NHCl per liter); (2) 2 ml of 1 M MgSO tide with one or more associated expression control (sterile); (3) 20 ml of 20% (w/v) glucose (or other carbon sequences and a heterologous nucleic acid encoding an iso source); and (4) 100 ul of 1 M CaCl (sterile). Each liter of prene synthase polypeptide. In some aspects, the bacterial 50 TM3 minimal medium contains (1) 13.6 g. KHPO, (2) 13.6 cells produce isoprene at a specific productivity greater than g KHPO; (3)2 g MgSO47H2O; (4) 2 g Citric Acid Mono that of the same cells lacking one or more copies of a heter hydrate; (5) 0.3 g Ferric Ammonium Citrate; (6) 3.2 g ologous gene encoding a PGL polypeptide with one or more (NH4)2SO; (7) 0.2 g yeast extract; and (8) 1 ml of 1000x associated expression control sequences, when the cells are Trace Elements solution; pH is adjusted to ~6.8 and the solu cultured in minimal medium. 55 tion is filter sterilized. Each liter of 1000x Trace Elements In some aspects, the cells further comprise an MVA path contains: (1) 40 g Citric Acid Monohydrate; (2) 30 g way polypeptide. In some aspects, the MVA pathway MnSO*HO; (3) 10 g NaCl; (4) 1 g FeSO.7HO; (4) 1 g polypeptide is an upper MVA pathway polypeptide. In some CoCl*6HO; (5) 1 g ZnSO*7HO; (6) 100 mg aspects, the MVA pathway polypeptide is a lower MVA path CuSO4.5H2O; (7) 100 mg HBO; and (8) 100 mg way polypeptide. In some aspects, the upper MVA pathway 60 NaMoO2H.O; pH is adjusted to -3.0. polypeptide is selected from the group consisting of: (i) an Any carbon Source can be used to cultivate the host cells. acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) The term "carbon source” refers to one or more carbon a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase containing compounds capable of being metabolized by a polypeptide; and (iii) a 3-hydroxy-3-methylglutaryl-Coen host cell or organism. For example, the cell medium used to Zyme A reductase polypeptide. In some aspects, the upper 65 cultivate the host cells may include any carbon source Suitable MVA pathway polypeptide is from the genus Enterococcus. for maintaining the viability or growing the host cells. In In Some aspects, the upper MVA pathway polypeptide is from Some aspects, the carbon Source is a carbohydrate (such as US 8,455,236 B2 25 26 monosaccharide, disaccharide, oligosaccharide, or polysac tions for greater than or about 5, 10, 15, 20, 25, 30,35, 40, 50, charides), or invert Sugar (e.g., enzymatically treated Sucrose 60, 70,80,90, 95, or 100% of the total length of time the cells syrup). are cultured. While not intending to be bound by any particu Exemplary monosaccharides include glucose and fructose; lar theory, it is believed that limited glucose conditions may exemplary oligosaccharides include lactose and Sucrose, and allow more favorable regulation of the cells. exemplary polysaccharides include starch and cellulose. In Some aspects, the carbon source includes yeast extractor Exemplary carbohydrates include C6 Sugars (e.g., fructose, one or more components of yeast extract. In some aspects, the mannose, galactose, or glucose) and C5 Sugars (e.g., Xylose or concentration of yeast extract is 0.1% (w/v), 0.09% (w/v), arabinose). 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% Exemplary Cell Culture Conditions 10 (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast Materials and methods suitable for the maintenance and extract. In some aspects, the carbon Source includes both growth of the recombinant cells of the invention are described yeast extract (or one or more components thereof) and infra, e.g., in the Examples section. Other materials and meth another carbon Source, such as glucose. ods suitable for the maintenance and growth of bacterial In Some aspects, the E. coli cells are grown in batch culture. cultures are well known in the art. Exemplary techniques may 15 In some aspects, the E. coli cells are grown in fed-batch be found in International Publication No. WO 2009/076676, culture. In some aspects, the E. coli cells are grown in con U.S. patent application Ser. No. 12/335,071 (U.S. Publ. No. tinuous culture. In some aspects, the E. coli cells are cultured 2009/0203102), WO 2010/003007, US Publ. No. 2010/ in minimal medium. In some aspects, the minimal medium is 0048964, WO 2009/132220, US Publ. No. 2010/0003716, M9 medium or TM3 medium. In some aspects, the minimal Manual of Methods for General Bacteriology Gerhardt et al., medium is M9 medium. In some aspects, the minimal eds), American Society for Microbiology, Washington, D.C. medium is TM3 medium. In some aspects, the minimal (1994) or Brock in Biotechnology: A Textbook of Industrial medium is supplemented with 1.0% (w/v) glucose or less. In Microbiology, Second Edition (1989) Sinauer Associates, Some aspects, the minimal medium is Supplemented with 1% Inc., Sunderland, Mass. In some aspects, the cells are cultured (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% in a culture medium under conditions permitting the expres 25 (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) sion of one or more isoprene synthase, DXS. IDI, DXP path glucose. In certain aspects, the minimal medium is Supple way polypeptides or MVA pathway polypeptides encoded by mented 0.1% (w/v) or less yeast extract. In some aspects, the a nucleic acid inserted into the host cells. minimal medium is supplemented with 0.1% (w/v), 0.09% Standard cell culture conditions can be used to culture the (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), cells (see, for example, WO 2004/033646 and references 30 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast cited therein). In some aspects, cells are grown and main extract. In some aspects, the minimal medium is Supple tained at an appropriate temperature, gas mixture, and pH mented with 1% (w/v) glucose or less and 0.1% (w/v) or less. (such as at about 20°C. to about 37°C., at about 6% to about In some aspects, the minimal medium is Supplemented with 84% CO, and at a pH between about 5 to about 9). In some 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), aspects, cells are grown at 35° C. in an appropriate cell 35 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% medium. In some aspects, the pH ranges for fermentation are (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% between about pH 5.0 to about pH 9.0 (such as about pH 6.0 (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), to about pH 8.0 or about 6.5 to about 7.0). Reactions may be 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. In performed under aerobic, anoxic, or anaerobic conditions Some aspects, the heterologous gene encoding a PGL based on the requirements of the host cells. 40 polypeptide is from E. coli strain K12 MG 1655. In some Standard culture conditions and modEs of fermentation, aspects, the heterologous gene encoding a PGL polypeptide is Such as batch, fed-batch, or continuous fermentation, that can from a derivative of E. coli strain K12 MG 1655. In some be used are described in International Publication No. WO aspects, the heterologous gene encoding a PGL polypeptide is 2009/076676, U.S. patent application Ser. No. 12/335,071 from the genus Pseudomonas. In some aspects, the (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. 45 Pseudomonas is Pseudomonas aeruginosa. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/ The invention encompasses recombinant cell(s) of an 0003716. Batch and Fed-Batch fermentations are common Escherichia coli (E. coli) strain capable of producing iso and well known in the art and examples may be found in prene, the cell(s) comprising: (a) one or more copies of a Brock, Biotechnology: A Textbook of Industrial Microbiol heterologous nucleic acid(s) encoding a PGL polypeptide ogy, Second Edition (1989) Sinauer Associates, Inc. 50 wherein the nucleic acid(s) is/are integrated in the E. coli In some aspects, the cells are cultured under limited glu chromosome; and (b) one or more heterologous nucleic cose conditions. By “limited glucose conditions' is meant acid(s) encoding isoprene synthase; wherein prior to the inte that the amount of glucose that is added is less than or about gration, the E. coli cell does not contain nucleic acid(s) that 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, encode(s) a encoding a PGL polypeptide, and wherein the 40%, 30%, 20%, or 10%) of the amount of glucose that is 55 resulting recombinant cell(s) produce(s) isoprene at a greater consumed by the cells. In particular aspects, the amount of titer than that of the same cell(s) that does/do not comprise (a) glucose that is added to the culture medium is approximately and (b). the same as the amount of glucose that is consumed by the In some aspects, the host cells are bacterial cells of an cells during a specific period of time. In some aspects, the rate Escherichia coli strain that do not encode a 6-phosphoglu of cell growth is controlled by limiting the amount of added 60 conolactonase (PGL) polypeptide, further comprising one or glucose such that the cells grow at the rate that can be Sup more copies of a heterologous gene encoding a PGL polypep ported by the amount of glucose in the cell medium. In some tide with one or more associated expression control aspects, glucose does not accumulate during the time the cells sequences and a heterologous nucleic acid encoding an iso are cultured. In various aspects, the cells are cultured under prene synthase polypeptide. In some aspects, the host cells limited glucose conditions for greater than or about 1, 2, 3, 5, 65 are bacterial cells of an Escherichia coli strain that does not 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In various encode a 6-phosphogluconolactonase (PGL) polypeptide, aspects, the cells are cultured under limited glucose condi polypeptides transcribed from genes for galactose metabo US 8,455,236 B2 27 28 lism (for example, galM, galK, galT, and galE), or polypep Enterococcus faecalis. In some aspects, the lower MVA path tides transcribed from genes for molybdate transport (for way polypeptide is selected from the group consisting of: (i) example, modF, modE, modA, modB, and modC) further mevalonate kinase (MVK); (ii) phosphomevalonate kinase comprising one or more copies of a heterologous gene encod (PMK); (iii) diphosphomevalonate decarboxylase (MVD); ing a PGL polypeptide with one or more associated expres and (iv) isopentenyl diphosphate isomerase (IDI). In some sion control sequences, a heterologous nucleic acid encoding aspects, the lower MVA pathway polypeptide is an MVK an isoprene synthase polypeptide, a heterologous nucleic acid polypeptide. In some aspects, the MVK polypeptide is from encoding one or more copies of one or more galactose the genus Methanosarcina. In some aspects, the MVK metabolism polypeptides, and a heterologous nucleic acid polypeptide is from Methanosarcina mazei. encoding one or more copies of one or more molybdate trans 10 The recombinant bacterial cells described herein have the porter polypeptides. In some aspects, the one or more copies ability to produce isoprene at a specific productivity greater of the heterologous gene encoding a PGL polypeptide with than that of the same cells lacking one or more copies of a one or more associated expression control sequences are heterologous gene encoding a PGL polypeptide with one or chromosomal copies (e.g., integrated into the E. coli chromo more associated expression control sequences when cultured Some). In some aspects, the one or more copies of the heter 15 in minimal medium. In some cases, the heterologous gene ologous gene encoding a PGL polypeptide, the one or more encoding a PGL polypeptide is a heterologous nucleic acid copies of the heterologous gene encoding one or more galac encoding a PGL polypeptide that is integrated into the host tose metabolism polypeptides, and/or the one or more copies cell's chromosome. In some aspects, the bacterial cells pro of the heterologous gene encoding one or more molybdate duce isopreneata specific productivity greater than that of the transport polypeptides are chromosomal copies (e.g., inte same cells lacking one or more copies of a heterologous gene grated into the E. coli chromosome). encoding a PGL polypeptide with one or more associated In some aspects, the bacterial cells are of E. coli strain B. In expression control sequences, one or more copies of a heter some aspects, the bacterial strains are of E. coli strain BL21. ologous gene encoding one or more galactose metabolism In some aspects, the bacterial cells are of E. coli strain BL21 polypeptides, and/or one or more copies of a heterologous (DE3). In some aspects, the minimal medium is Supple 25 gene encoding one or more molybdate transport polypeptides mented with 0.1% (w/v) yeast extractor less. In some aspects, when cultured in minimal medium. the minimal medium is supplemented with 1.0% (w/v) glu In some aspects, the E. coli cells have a specific productiv cose or less. In some aspects, the minimal medium is Supple ity greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), mg/OD/hr of isoprene. In some aspects, the E. coli cells have 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), 30 a specific productivity greater than about 15 mg/OD/hr of or 0.1% (w/v) glucose. In certain aspects, the minimal isoprene. In some aspects, the E. coli cells have a specific medium is supplemented 0.1% (w/v) or less yeast extract. In productivity greater than about 16 mg/OD/hr of isoprene. In Some aspects, the minimal medium is Supplemented with Some aspects, the E. coli cells have a specific productivity 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% greater than about 17 m mg/OD/hr of isoprene. In some (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), 35 aspects, the E. coli cells have a specific productivity greater or 0.01% (w/v) yeast extract. In some aspects, the minimal than about 18 mg/OD/hr of isoprene. In some aspects, the E. medium is Supplemented with 1% (w/v) glucose or less and coli cells have a specific productivity greater than about 19 0.1% (w/v) or less. In some aspects, the minimal medium is mg/OD/hr of isoprene. In some aspects, the E. coli cells have supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% a specific productivity greater than about 20 mg/L/hr of (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% 40 isoprene. In some aspects, the E. coli cells have a specific (w/v), or 0.1% (w/v) glucose and with 0.1% (w/v), 0.09% productivity greater than about 21 mg/OD/hr of isoprene. In (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), Some aspects, the E. coli cells have a specific productivity 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast greater than about 22 mg/OD/hr ofisoprene. In some aspects, extract. In some aspects, the minimal medium is M9 medium the E. coli cells have a specific productivity greater than about or TM3 medium. In some aspects, the minimal medium is M9 45 23 mg/OD/hr of isoprene. In some aspects, the E. coli cells medium. In some aspects, the minimal medium is TM3 have a specific productivity greater than about 24 mg/OD/hr medium. In some aspects, the minimal medium is M9 of isoprene. In some aspects, the E. coli cells have a specific medium. In some aspects, the minimal medium is TM3 productivity greater than about 25 mg/OD/hr of isoprene. medium. In some aspects, the heterologous gene encoding a In other aspects, the E. coli cells have an upper limit of PGL polypeptide is from E. coli strain K12 MG 1655. In some 50 specific productivity of about 25, 24, 23, 22, 21, 20, 19, 18, aspects, the heterologous gene encoding a PGL polypeptide is 17, 16, 15, 14, 13, 12, 11, 10,9,8,7,6, or 5 mg/OD/hr of from a derivative of E. coli strain K12 MG 1655. In some isoprene. In other aspects, the E. coli cells have a lower limit aspects, the heterologous gene encoding a PGL polypeptide is of specific productivity of about 5, 6,7,8,9, 10, 11, 12, 13, 14, from the genus Pseudomonas. In some aspects, the 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg/OD/hr of Pseudomonas is Pseudomonas aeruginosa. 55 isoprene. In some aspects, the cells further comprise an MVA path In Some aspects, the heterologous nucleic acid encoding an way polypeptide. In some aspects, the MVA pathway isoprene synthase polypeptide is operably linked to a pro polypeptide is an upper MVA pathway polypeptide. In some moter, and the cells have a specific productivity greater than aspects, the MVA pathway polypeptide is a lower MVA path about 15 mg/OD/hr of isoprene. In some aspects, the heter way polypeptide. In some aspects, the upper MVA pathway 60 ologous nucleic acid encoding an isoprene synthase polypep polypeptide is selected from the group consisting of: (i) an tide is operably linked to a promoter, and the cells have a acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) specific productivity greater than about 16 mg/OD/hr of iso a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase prene. In some aspects, the heterologous nucleic acid encod polypeptide; and (iii) a 3-hydroxy-3-methylglutaryl-Coen ing an isoprene synthase polypeptide is operably linked to a Zyme A reductase polypeptide. In some aspects, the upper 65 promoter, and the cells have a specific productivity greater MVA pathway polypeptide is from the genus Enterococcus. than about 17 mg/OD/hr of isoprene. In some aspects, the In Some aspects, the upper MVA pathway polypeptide is from heterologous nucleic acid encoding an isoprene synthase US 8,455,236 B2 29 30 polypeptide is operably linked to a promoter, and the cells incorporated by reference in their entirety, especially with have a specific productivity greater than about 18 mg/OD/hr respect to isoprene synthases, variants thereof and/or iso of isoprene. In some aspects, the heterologous nucleic acid prene synthase mutants). encoding an isoprene synthase polypeptide is operably linked Methods for the Increased Production of Isoprene to a promoter, and the cells have a specific productivity Genetically engineered cell cultures in bioreactors have greater than about 19 mg/OD/hr ofisoprene. In some aspects, produced isoprene more efficiently, in larger quantities, in the heterologous nucleic acid encoding an isoprene synthase higher purities and/or with unique impurity profiles, and polypeptide is operably linked to a promoter, and the cells methods of producing commercially useful quantities of iso have a specific productivity greater than about 20 mg/OD/hr prene from renewable resources are described and exempli of isoprene. In some aspects, the heterologous nucleic acid 10 fied, for example, in International Patent Application Publi encoding an isoprene synthase polypeptide is operably linked cation No. WO2009/076676 A2, U.S. Patent Application Publication Nos. US2009/0203102 A1, US2010/0003716 to a promoter, and the cells have a specific productivity A1, US2010/0048964 A1, US2010/0086978 A1, US2010/ greater than about 21 mg/OD/hr ofisoprene. In some aspects, 0167370 A1, US2010/0113846 A1, US2010/0184178 A1, the heterologous nucleic acid encoding an isoprene synthase 15 US2010/0167371 A1, US2010/0196977 A1, US2010/ polypeptide is operably linked to a promoter, and the cells 0196977 A1; U.S. Provisional Patent Application Nos. have a specific productivity greater than about 22 mg/OD/hr 61/187,930, 61/187,941 and 61/187,959. of isoprene. In some aspects, the heterologous nucleic acid Also provided herein are improved methods for the pro encoding an isoprene synthase polypeptide is operably linked duction of isoprene. In some aspects, the improved method to a promoter, and the cells have a specific productivity for producing isoprene comprises: (a) culturing a composi greater than about 23 mg/OD/hr ofisoprene. In some aspects, tion comprising recombinant cell(s) of an Escherichia coli (E. the heterologous nucleic acid encoding an isoprene synthase coli) strain, or progeny thereof, capable of producing iso polypeptide is operably linked to a promoter, and the cells prene, the cell comprising: (i) one or more copies of a heter have a specific productivity greater than about 24 mg/OD/hr ologous nucleic acid(s) encoding a PGL polypeptide wherein of isoprene. In some aspects, the heterologous nucleic acid 25 the nucleic acid is integrated in the E. coli chromosome; and encoding an isoprene synthase polypeptide is operably linked (ii) one or more heterologous nucleic acid(s) encoding iso to a promoter, and the cells have a specific productivity prene synthase; wherein prior to the integration, the E. coli greater than about 25 mg/OD/hr of isoprene. cell does not contain nucleic acid(s) encoding a PGL In some aspects, the E. coli cells further comprise a heter polypeptide, and wherein the resulting recombinant cell pro 30 duces isoprene at a greater titer than that of the same cells that ologous nucleic acid encoding an IDI polypeptide. In some do not comprise (i) and (ii) and (b) producing the isoprene. In aspects, the E. coli cells further comprise a chromosomal some aspects, the improved method of producing isoprene copy of an endogenous nucleic acid encoding an IDI polypep comprises the steps of: (a) culturing bacterial cells of an tide. In some aspects, the E. coli cells further comprise a Escherichia coli strain that does not encode a 6-phosphoglu heterologous nucleic acid encoding a DXS polypeptide or 35 conolactonase (PGL) polypeptide in minimal medium, other DXP pathway polypeptides. In some aspects, the E. coli wherein the E. coli cells comprise one or more copies of a cells further comprise a chromosomal copy of an endogenous heterologous gene encoding a PGL polypeptide with one or nucleic acid encoding a DXS polypeptide or other DXP path more associated expression control sequences and a heterolo way polypeptides. In some aspects, the E. coli cells further gous nucleic acid encoding an isoprene synthase polypeptide; comprise one or more nucleic acids encoding an IDI polypep 40 and (b) producing isoprene, wherein the E. coli cells have a tide and a DXS polypeptide or other DXP pathway polypep specific productivity ofisoprene greater than that of the same tides. In some aspects, one nucleic acid encodes the isoprene cells lacking one or more copies of a heterologous gene synthase polypeptide, IDI polypeptide, and DXS polypeptide encoding a PGL polypeptide with one or more associated or other DXP pathway polypeptides. In some aspects, one expression control sequences, when the cells are cultured in plasmid encodes the isoprene synthase polypeptide, IDI 45 minimal medium. In some aspects, the one or more copies of polypeptide, and DXS polypeptide or other DXP pathway a heterologous gene encoding a PGL polypeptide with one or polypeptides. In some aspects, multiple plasmids encode the more associated expression control sequences are chromo isoprene synthase polypeptide, IDI polypeptide, and DXS Somal copies (e.g., integrated into the E. coli chromosome). polypeptide or other DXP pathway polypeptides. In some aspects, the improved method of producing isoprene In some aspects, the E. coli cells further comprise a heter 50 further comprises a step of recovering the isoprene. ologous nucleic acid encoding an isoprene synthase polypep In some aspects, the improved method of producing iso tide. In some cases, the isoprene synthase polypeptide can be prene comprises the steps of culturing the recombinant cells one or more copies of an endogenous isoprene synthase. In described herein under conditions suitable for the production Some aspects, the isoprene synthase polypeptide is a plant of isoprene and allowing the recombinant cells to produce isoprene synthase polypeptide. In some aspects, the isoprene 55 isoprene. In some aspects, the improved method of producing synthase polypeptide is a naturally-occurring polypeptide isoprene further comprises a step of recovering the isoprene. from the genus Pueraria. In some aspects, the isoprene Syn Without being bound by theory, recombinant cells having thase polypeptide is a naturally-occurring polypeptide from chromosomally integrated heterologous nucleic acids encod Pueraria montana. In some aspects, the isoprene synthase ing PGL polypeptide produce isoprene at a higher titer and a polypeptide is a naturally-occurring polypeptide from the 60 higher specific productivity than cells where a heterologous genus Populus. In some aspects, the isoprene synthase PGL nucleic acid is on a plasmid. Surprisingly, recombinant polypeptide is a naturally-occurring polypeptide from Popu cells comprising one or more copies of chromosomally inte lus alba. Other isoprene synthase polypeptides or isoprene grated PGL polypeptide, and optionally with one or more synthase variants that can be used to practice the invention copies of one or more polypeptides encoded by chromosoma include, but is not limited to, the isoprene synthases, variants 65 lly integrated galactose metabolism genes (for example, thereof and/or isoprene synthase mutants as described in WO galM, galK, galT and galE), and/or one or more copies of one 2009/132220 or WO 2010/124146 (the contents of which are or more polypeptides encoded by chromosomally integrated US 8,455,236 B2 31 32 molybdenum transport genes (for example, modF, modE. cerevisiae. In some aspects, the lower MVA pathway modA, modB, and modC) conveya substantial growth benefit polypeptide is IDI. In some aspects, the lower MVA pathway to the cells, a higher titer of isoprene production, and/or a polypeptide is from the genus Saccharomyces. In some higher specific production of isoprene versus cells compris aspects, the Saccharomyces is Saccharomyces cerevisiae. ing a heterologous PGL nucleic acid on a plasmid. In some aspects, the isoprene synthase polypeptide is from Therefore, in one aspect the improved method of produc a plant. In some aspects, the plant is kudzu. In some aspects, ing isoprene comprises the steps of: (a) culturing bacterial the plant is poplar (Populus alba x tremula CAC35696). In cells of an Escherichia coli strain that does not encode a Some aspects, the plant is aspen (Populus tremuloides). In 6-phosphogluconolactonase (PGL) polypeptide, one or more Some aspects, the plantis English oak (Quercus robur). In one polypeptides encoded by genes for galactose metabolism (for 10 aspect, the plant is Populus alba. Other isoprene synthase example, galM, galK, galT and galE), and/or one or more polypeptides or isoprene synthase variants that can be used to polypeptides encoded by genes for molybdenum transport practice the invention include, but is not limited to, the iso (for example, modF, modE. modA, modB, and modC). prene synthases, variants thereof and/or isoprene synthase wherein the E. coli cells comprise one or more copies of a mutants as described in WO 2009/132220 or WO 2010/ chromosomally integrated heterologous gene encoding a 15 124146 (the contents of which are incorporated by reference PGL polypeptide with one or more associated expression in their entirety, especially with respect to isoprene synthases, control sequences, a heterologous nucleic acid encoding an variants thereof and/or isoprene synthase mutants). isoprene synthase polypeptide, one or more copies of a chro In some aspects, the E. coli cells further comprise a heter mosomally integrated heterologous nucleic acid encoding ologous nucleic acid encoding an IDI polypeptide. n some one or more galactose metabolism polypeptides and/or one or aspects, the E. coli cells further comprise one or more copies more molybdenum transport polypeptides; and (b) producing of an endogenous nucleic acid encoding an IDI polypeptide. isoprene, wherein the E. coli cells have a higher specific In some aspects, the E. coli cells further comprise a chromo growth rate, specific productivity of isoprene and/or titer Somal copy of an endogenous nucleic acid encoding an IDI production ofisoprene than that of the same cells wherein the polypeptide. In some aspects, the E. coli cells further com heterologous gene encoding PGL is located on a plasmid. 25 prise a heterologous nucleic acid encoding a DXS polypep In some aspects, the cells further comprise an MVA path tide or other DXP pathway polypeptides. In some aspects, the way polypeptide. In such cases, the invention contemplates E. coli cells further comprise a chromosomal copy of an compositions and methods for producing mevalonate as well. endogenous nucleic acid encoding a DXS polypeptide or The methods for producing mevalonate using a chromosoma other DXP pathway polypeptides. In some aspects, the E. coli lly integrated PGL host cell system can optionally include 30 cells further comprise one or more nucleic acids encoding an recovery of the mevalonate. In some aspects, the MVA path IDI polypeptide and a DXS polypeptide or other DXP path way polypeptide is an upper MVA pathway polypeptide. In way polypeptides. In some aspects, one nucleic acid encodes some aspects, the MVA pathway polypeptide is a lower MVA the isoprene synthase polypeptide, IDI polypeptide, and DXS pathway polypeptide. polypeptide. In some aspects, one plasmid encodes the iso In some aspects, the upper MVA pathway polypeptide is 35 prene synthase polypeptide, IDI polypeptide, and DXS selected from the group consisting of: (i) an acetoacetyl polypeptide or other DXP pathway polypeptides. In some Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hy aspects, multiple plasmids encode the isoprene synthase droxy-3-methylglutaryl-Coenzyme A synthase polypeptide; polypeptide, IDI polypeptide, and DXS polypeptide or other and (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reduc DXP pathway polypeptides. tase polypeptide. In some aspects, the upper MVA pathway 40 In some aspects, the heterologous gene encoding a PGL polypeptide is acetoacetyl-Coenzyme A synthase (thiolase). polypeptide is from E. coli strain K12 MG 1655. In some In some aspects, the upper MVA pathway polypeptide is aspects, the heterologous gene encoding a PGL polypeptide is 3-hydroxy-3-methylglutaryl-Coenzyme A synthase polypep from a derivative of E. coli strain K12 MG 1655. In some tide. In some aspects, the upper MVA pathway polypeptide is aspects, the E. coli K12 strain MG 1655 polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. In 45 PGL activity is SEQID NO:11. In some aspects, the E. coli Some aspects, the upper MVA pathway polypeptide is from a K12 strain MG 1655 polypeptide having PGL activity com bacterium. In some aspects, the bacterium is from the genus prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Enterococcus. In some aspects, bacterium is from Enterococ 19, 20, or more amino acid substitutions compared to SEQID cus faecalis. NO:11. In some aspects, the amino acid Substitutions are In some aspects, the lower MVA pathway polypeptide is 50 conservative. In some aspects, the amino acid Substitutions selected from the group consisting of: (i) mevalonate kinase are non-conservative. In some aspects, the E. coli K12 strain (MVK); (ii) phosphomevalonate kinase (PMK); (iii) diphos MG1655 polypeptide having PGL activity has 99%, 98%, phomevalonate decarboxylase (MVD); and (iv) isopentenyl 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, diphosphate isomerase (IDI). In some aspects, the lower 87%. 86%, or 85% amino acid sequence identity to SEQID MVA pathway polypeptide is MVK. In some aspects, the 55 NO:11. MVK is from the genus Methanosarcina. In some aspects, the In some aspects, the heterologous gene encoding a PGL Methanosarcina is Methanosarcina mazei. In some aspects, polypeptide is from the genus Pseudomonas. In some aspects, the lower MVA pathway polypeptide is PMK, MVD, or IDI. the Pseudomonas is Pseudomonas aeruginosa. In some In some aspects, the PMK. MVD, or IDI is from the genus aspects, the Paeruginosa polypeptide having PGL activity is Saccharomyces. In some aspects, the Saccharomyces is Sac 60 SEQID NO:12. In some aspects, the Paeruginosa polypep charomyces cerevisiae. In some aspects, the lower MVA path tide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, way polypeptide is PMK. In some aspects, the PMK is from 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid the genus Saccharomyces. In some aspects, the Saccharomy substitutions compared to SEQID NO:12. In some aspects, ces is Saccharomyces cerevisiae. In some aspects, the lower the amino acid substitutions are conservative. In some MVA pathway polypeptide is MVD. 65 aspects, the amino acid Substitutions are non-conservative. In In some aspects, the MVD is from the genus Saccharomy some aspects, the E. coli K12 strain MG 1655 polypeptide ces. In some aspects, the Saccharomyces is Saccharomyces having PGL activity has 99%, 98%, 97%, 96%. 95%, 95%, US 8,455,236 B2 33 34 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino greater than about 24 mg/OD/hr ofisoprene. In some aspects, acid sequence identity to SEQID NO:12. the E. coli cells have a specific productivity greater than about In some aspects, the heterologous gene encoding a PGL 25 mg/OD/hr of isoprene. polypeptide is from the genus Saccharomyces. In some In Some aspects, the heterologous nucleic acid encoding an aspects, the Saccharomyces is Saccharomyces cerevisiae. In isoprene synthase polypeptide is operably linked to a pro Some aspects, the S. cerevisiae polypeptidehaving PGL activ moter and the E. coli cells have a specific productivity greater ity is SEQ ID NO:13. In some aspects, the S. cerevisiae than about 15 mg/OD/hr of isoprene. In some aspects, the polypeptide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, heterologous nucleic acid encoding an isoprene synthase 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino polypeptide is operably linked to a promoter and the E. coli 10 cells have a specific productivity greater than about 16 acid substitutions compared to SEQ ID NO:13. In some mg/OD/hr of isoprene. In some aspects, the heterologous aspects, the amino acid substitutions are conservative. In nucleic acid encoding an isoprene synthase polypeptide is Some aspects, the amino acid Substitutions are non-conserva operably linked to a promoter and the E. coli cells have a tive. In some aspects, the E. coli K12 strain MG 1655 polypep specific productivity greater than about 17 mg/OD/hr of iso tide having PGL activity has 99%, 98%, 97%, 96%. 95%, 15 prene. In some aspects, the heterologous nucleic acid encod 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% ing an isoprene synthase polypeptide is operably linked to a amino acid sequence identity to SEQID NO:13. promoter and the E. coli cells have a specific productivity In some aspects, the bacterial cells of an Escherichia coli greater than about 18 mg/OD/hr ofisoprene. In some aspects, strain that does not encode a PGL polypeptide are of E. coli the heterologous nucleic acid encoding an isoprene synthase strain B. In some aspects, the bacterial cells are of E. coli polypeptide is operably linked to a promoter and the E. coli strain BL21. In some aspects, the bacterial cells are of E. coli cells have a specific productivity greater than about 19 strain BL21 (DE3). mg/OD/hr of isoprene. In some aspects, the heterologous In some aspects, the E. coli cells are cultured in minimal nucleic acid encoding an isoprene synthase polypeptide is medium. In some aspects, the E. coli cells of E. coli strain B operably linked to a promoter and the E. coli cells have a are cultured in minimal medium. In some aspects, the E. coli 25 specific productivity greater than about 20 mg/OD/hr of iso cells of E. coli strain BL21 are cultured in minimal medium. prene. In some aspects, the heterologous nucleic acid encod In some aspects, the E. coli cells of E. coli strain BL21 (DE3) ing an isoprene synthase polypeptide is operably linked to a are cultured in minimal medium. In some aspects, the mini promoter and the E. coli cells have a specific productivity mal medium is Supplemented with 1% (w/v) or less glucose. greater than about 21 mg/OD/hr ofisoprene. In some aspects, 30 the heterologous nucleic acid encoding an isoprene synthase In some aspects, the minimal medium is Supplemented with polypeptide is operably linked to a promoter and the E. coli 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), cells have a specific productivity greater than about 22 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% mg/OD/hr of isoprene. In some aspects, the heterologous (w/v) glucose. In certain aspects, the minimal medium is nucleic acid encoding an isoprene synthase polypeptide is supplemented 0.1% (w/v) or less yeast extract. In some 35 operably linked to a promoter and the E. coli cells have a aspects, the minimal medium is Supplemented with 0.1% specific productivity greater than about 23 mg/OD/hr of iso (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), prene. In some aspects, the heterologous nucleic acid encod 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or ing an isoprene synthase polypeptide is operably linked to a 0.01% (w/v) yeast extract. In some aspects, the minimal promoter and the E. coli cells have a specific productivity medium is Supplemented with 1% (w/v) glucose or less and 40 greater than about 24 mg/OD/hr ofisoprene. In some aspects, 0.1% (w/v) or less. In some aspects, the minimal medium is the heterologous nucleic acid encoding an isoprene synthase supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% polypeptide is operably linked to a promoter and the E. coli (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% cells have a specific productivity greater than about 25 (w/v), or 0.1% (w/v) glucose and with 0.1% (w/v), 0.09% mg/OD/hr of isoprene. In some aspects, the heterologous (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 45 nucleic acid encoding an isoprene synthase polypeptide is 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast operably linked to a promoter and the E. coli cells have a extract. In some aspects, the minimal medium is M9 medium specific productivity greater than about 25 mg/OD/hr of iso or TM3 medium. In some aspects, the minimal medium is M9 prene to about 100 mg/OD/hr of isoprene. In some aspects, medium. In some aspects, the minimal medium is TM3 the heterologous nucleic acid encoding an isoprene synthase medium. 50 polypeptide is operably linked to a promoter and the E. coli In some aspects, the E. coli cells have a specific productiv cells have a specific productivity greater than about 15 ity greater than about 15 mg/L/hr of isoprene. In some mg/OD/hr of isoprene to about 100 mg/OD/hr of isoprene. aspects, the E. coli cells have a specific productivity greater The invention also provides for recombinant E. coli cells than about 16 mg/OD/hr of isoprene. In some aspects, the E. with PGL integration that have been engineered to produce coli cells have a specific productivity greater than about 17 55 isoprene that also have better growth due to their increased mg/OD/hr of isoprene. In some aspects, the E. coli cells have overall fitness. One of skill in the art can appreciate that a specific productivity greater than about 18 mg/OD/hr of increased growth rate can lead to enhanced production of isoprene. In some aspects, the E. coli cells have a specific isoprene, Such as higher specific activity, more isoprene pro productivity greater than about 19 mg/OD/hr of isoprene. In duced over a period of time, or higher isoprene titers. In one Some aspects, the E. coli cells have a specific productivity 60 aspect, the recombinant E. coli cells with PGL integration that greater than about 20 mg/OD/hr ofisoprene. In some aspects, have been engineered to produce isoprene has at least 10% the E. coli cells have a specific productivity greater than about increased growth as compared to those cells without PGL 21 mg/OD/hr of isoprene. In some aspects, the E. coli cells integration and/or the restoration of the 17,257 base pair piece have a specific productivity greater than about 22 mg/OD/hr as described herein (see, for example, FIG. 20). In other of isoprene. In some aspects, the E. coli cells have a specific 65 aspects, the recombinant E. coli cells with PGL integration productivity greater than about 23 mg/OD/hr of isoprene. In that have been engineered to produce isoprene has at least Some aspects, the E. coli cells have a specific productivity about 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, US 8,455,236 B2 35 36 20%, 21%. 22%, 23%, 24%, or 25% growth as compared to 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, those cells without PGL integration and/or the restoration of 87%. 86%, or 85% amino acid sequence identity to SEQID the 17,257 base pair piece as described herein. NO:11. Methods for the Increased Production of Other Heterologous In some aspects, the heterologous gene encoding a PGL Polypeptides Capable of Biological Activity polypeptide is from the genus Pseudomonas. In some aspects, Also provided herein are improved methods for the pro the Pseudomonas is Pseudomonas aeruginosa. In some duction of other heterologous polypeptides capable of bio aspects, the Paeruginosa polypeptide having PGL activity is logical activity or other products. One non-limiting example SEQID NO:12. In some aspects, the Paeruginosa polypep of a product is mevalonate. One of skill in the art can produce tide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid mevalonate by: (a) culturing a composition comprising the substitutions compared to SEQID NO:12. In some aspects, recombinant cell of an Escherichia coli (E. coli) strain, or the amino acid substitutions are conservative. In some progeny thereof, capable of producing isoprene, the cellcom aspects, the amino acid Substitutions are non-conservative. In prising: (i) one or more copies of a heterologous nucleic Some aspects, the P. aeruginosa polypeptide having PGL acid(s) encoding a PGL polypeptide wherein the nucleic acid 15 activity has 99%, 98%, 97%, 96%. 95%, 95%, 93%, 92%, is integrated in the E. coli chromosome; (ii) one or more 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino acid heterologous nucleic acid(s) encoding isoprene synthase; sequence identity to SEQID NO:12. amd (iii) (c) a heterologous nucleic acid encoding an upper In some aspects, the heterologous gene encoding a PGL mevalonate (MVA) pathway polypeptide and/or a lower polypeptide is from the genus Saccharomyces. In some MVA pathway polypeptide; wherein prior to the integration, aspects, the Saccharomyces is Saccharomyces cerevisiae. In the E. coli cell does not contain nucleic acid(s) encoding a Some aspects, the S. cerevisiae polypeptide having PGL activ PGL polypeptide, and wherein the resulting recombinant cell ity is SEQ ID NO:13. In some aspects, the S. cerevisiae produces isoprene at a greater titer than that of the same cells polypeptide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, that do not comprise (i) and (ii) under Suitable culture condi 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino tions for the production of mevalonate and (b) producing 25 acid substitutions compared to SEQ ID NO:13. In some mevalonate. aspects, the amino acid Substitutions are conservative. In In some aspects, the improved method of producing heter Some aspects, the amino acid Substitutions are non-conserva ologous polypeptides capable of biological activity com tive. In some aspects, the E. coli K12 strain MG 1655 polypep prises the steps of: (a) culturing cells of an Escherichia coli tide having PGL activity has 99%, 98%, 97%, 96%. 95%, strain that does not encode a 6-phosphogluconolactonase 30 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% (PGL) polypeptide, further comprising one or more copies of amino acid sequence identity to SEQID NO:13. a heterologous gene encoding a PGL polypeptide with one or In some aspects, the bacterial cells of an Escherichia coli more associated expression control sequences and a nucleic strain that does not encode a 6-phosphogluconolactonase acid encoding a heterologous polypeptide capable of biologi (PGL) polypeptide are cultured in minimal medium. In some 35 aspects, the bacterial cells of E. coli strain B are cultured in cal activity; and (b) producing the heterologous polypeptide, minimal medium. In some aspects, the bacterial cells of E. wherein the E. coli cells have a specific productivity of the coli strain BL21 are cultured in minimal medium. In some heterologous polypeptide greater than that of the same cells aspects, the bacterial cells of E. coli strain BL21 (DE3) are lacking one or more copies of a heterologous gene encoding cultured in minimal medium. In some aspects, the minimal a PGL polypeptide with one or more associated expression 40 medium is supplemented with 1% (w/v) or less glucose. In control sequences, when the cells are cultured in minimal Some aspects, the minimal medium is Supplemented with 1% medium. In some aspects, the one or more copies of a heter (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% ologous gene encoding a PGL polypeptide with one or more (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) associated expression control sequences are chromosomal glucose. In certain aspects, the minimal medium is Supple copies (e.g., integrated into the E. coli chromosome). In some 45 mented 0.1% (w/v) or less yeast extract. In some aspects, the aspects, the E. coli cells are in culture. In some aspects, the minimal medium is supplemented with 0.1% (w/v), 0.09% improved method of producing heterologous polypeptides (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), capable of biological activity further comprises a step of 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast recovering the polypeptide. extract. In some aspects, the minimal medium is Supple In some aspects, the bacterial cells of an Escherichia coli 50 mented with 1% (w/v) glucose or less and 0.1% (w/v) or less. strain that does not encode a PGL polypeptide are of E. coli In some aspects, the minimal medium is Supplemented with strain B. In some aspects, the bacterial cells are of E. coli 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), strain BL21. In some aspects, the bacterial cells are of E. coli 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% strain BL21 (DE3). (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% In some aspects, the heterologous gene encoding a PGL 55 (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), polypeptide is from E. coli strain K12 MG 1655. In some 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. In aspects, the heterologous gene encoding a PGL polypeptide is some aspects, the minimal medium is M9 medium or TM3 from a derivative of E. coli strain K12 MG 1655. In some medium. In some aspects, the minimal medium is M9 aspects, the E. coli K12 strain MG 1655 polypeptide having medium. In some aspects, the minimal medium is TM3 PGL activity is SEQID NO:11. In some aspects, the E. coli 60 medium. K12 strain MG 1655 polypeptide having PGL activity com Also provided herein are improved methods for the pro prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, duction of other heterologous polypeptides capable of bio 19, 20, or more amino acid substitutions compared to SEQID logical activity. In some aspects, the improved method of NO:11. In some aspects, the amino acid substitutions are producing heterologous polypeptides capable of biological conservative. In some aspects, the amino acid Substitutions 65 activity comprises the steps of: (a) culturing cells of an are non-conservative. In some aspects, the E. coli K12 strain Escherichia coli strain that does not encode a 6-phosphoglu MG1655 polypeptide having PGL activity has 99%, 98%, conolactonase (PGL) polypeptide, a gene that encodes one or US 8,455,236 B2 37 38 more galactose metabolism polypeptides (for example, galM, native Paeruginosa polypeptide. In some aspects, the amino galK, galT, and galE), and/or a gene that encodes one or more acid substitutions are conservative. In some aspects, the molybdenum transporter polypeptides (for example, modF. amino acid substitutions are non-conservative. In some modE. modA, modB, and modC) further comprising one or aspects, the Paeruginosa polypeptide having PGL activity, more copies of a heterologous gene encoding a PGL polypep galactose metabolic activity, and/or molybdenum transport tide with one or more associated expression control activity has 99%, 98%, 97%, 96%. 95%, 95%, 93%, 92%, sequences and a nucleic acid encoding a heterologous 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino acid polypeptide capable of biological activity, one or more copies sequence identity to the native P. aeruginosa polypeptide. of a heterologous gene encoding one or more galactose In some aspects, the heterologous gene encoding a PGL metabolism polypeptides, and/or one or more copies of a 10 heterologous gene encoding one or more molybdenum trans polypeptide, one or more galactose metabolism polypeptides, port polypeptides; and (b) producing the heterologous and/or one or more molybdenum transport polypeptides is polypeptide, wherein the E. coli cells have a specific produc from the genus Saccharomyces. In some aspects, the Saccha tivity of the heterologous polypeptide greater than that of the romyces is Saccharomyces cerevisiae. In some aspects, the S. same cells lacking one or more copies of a heterologous gene 15 cerevisiae polypeptide having PGL activity, galactose meta encoding a PGL polypeptide with one or more associated bolic activity, and/or molybdenum transport activity com expression control sequences, one or more copies of a heter prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, ologous gene encoding one or more galactose metabolism 19, 20, or more amino acid substitutions compared to the polypeptides, and/or one or more copies of a heterologous native Saccharomyces cerevisiae. In some aspects, the amino gene encoding one or more molybdenum transport polypep acid substitutions are conservative. In some aspects, the tides when the cells are cultured in minimal medium. In some amino acid substitutions are non-conservative. In some aspects, the one or more copies of a heterologous gene encod aspects, the Saccharomyces cerevisiae polypeptide having ing a PGL polypeptide with one or more associated expres PGL activity has 99%, 98%, 97%, 96%. 95%, 95%, 93%, sion control sequences, the one or more copies of a heterolo 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino acid gous gene encoding one or more galactose metabolism 25 sequence identity to the native Saccharomyces cerevisiae polypeptides, and/or the one or more copies of a heterologous polypeptide. gene encoding one or more molybdenum transporter In some aspects, the bacterial cells of an Escherichia coli polypeptides are chromosomal copies (e.g., integrated into strain that does not encode a 6-phosphogluconolactonase the E. coli chromosome). In some aspects, the E. coli cells are (PGL) polypeptide, one or more galactose metabolism in culture. In some aspects, the improved method of produc 30 polypeptides, and/or one or more molybdenum transport ing heterologous polypeptides capable of biological activity polypeptides are cultured in minimal medium. In some further comprises a step of recovering the polypeptide. aspects, the bacterial cells of E. coli strain B are cultured in In some aspects, the bacterial cells of an Escherichia coli minimal medium. In some aspects, the bacterial cells of E. strain that does not encode a PGL polypeptide, one or more coli strain BL21 are cultured in minimal medium. In some galactose metabolic genes, and/or one or more molybdenum 35 aspects, the bacterial cells of E. coli strain BL21 (DE3) are transport genes are of E. coli Strain B. In some aspects, the cultured in minimal medium. In some aspects, the minimal bacterial cells are of E. coli strain BL21. In some aspects, the medium is supplemented with 1% (w/v) or less glucose. In bacterial cells are of E. coli strain BL21 (DE3). Some aspects, the minimal medium is Supplemented with 1% In some aspects, the heterologous gene encoding a PGL (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% polypeptide, one or more galactose metabolic genes, and/or 40 (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) one or more molybdenum transport genes is from E. coli glucose. In certain aspects, the minimal medium is Supple strain K12 MG 1655. In some aspects, the heterologous gene mented 0.1% (w/v) or less yeast extract. In some aspects, the encoding a PGL polypeptide, one or more galactose meta minimal medium is supplemented with 0.1% (w/v), 0.09% bolic genes, and/or one or more molybdenum transport genes (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), is from a derivative of E. coli strain K12 MG 1655. In some 45 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast aspects, the E. coli K12 strain MG 1655 polypeptide having extract. In some aspects, the minimal medium is Supple PGL activity, galactose metabolic activity, and/or molybde mented with 1% (w/v) glucose or less and 0.1% (w/v) or less. num transportactivity comprises 1,2,3,4,5,6,7,8,9, 10, 11, In some aspects, the minimal medium is Supplemented with 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substi 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), tutions compared to the native E. coli K12 strain MG 1655 50 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% polypeptide. In some aspects, the amino acid substitutions are (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% conservative. In some aspects, the amino acid Substitutions (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), are non-conservative. In some aspects, the E. coli K12 strain 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. In MG 1655 polypeptide having PGL activity, galactose meta some aspects, the minimal medium is M9 medium or TM3 bolic activity, and/or molybdenum transport activity has 99%, 55 medium. In some aspects, the minimal medium is M9 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, medium. In some aspects, the minimal medium is TM3 88%, 87%. 86%, or 85% amino acid sequence identity to the medium. native E. coli K12 strain MG 1655 polypeptide. In some aspects, the heterologous polypeptide capable of In some aspects, the heterologous gene encoding a PGL biological activity comprises one or more polypeptides polypeptide, one or more galactose metabolism polypeptides, 60 involved in the biosynthesis ofterpenoid (isoprenoid) or caro and/or one or more molybdenum transport polypeptides is tenoid compound(s), and the cells produce a terpenoid or from the genus Pseudomonas. In some aspects, the carotenoid at a higher specific productivity than that of the Pseudomonas is Pseudomonas aeruginosa. In some aspects, same cells lacking one or more copies of a heterologous gene the Paeruginosa polypeptide having PGL activity, galactose encoding a PGL polypeptide with one or more associated metabolic activity, and/or molybdenum transport activity 65 expression control sequences when cultured in minimal comprises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17. medium. In some aspects, the method further comprises a 18, 19, 20, or more amino acid substitutions compared to the step of recovering the terpenoid or carotenoid. US 8,455,236 B2 39 40 As used herein, the term “terpenoid” or "isoprenoid refers citrea, the genus Pseudomonas, such as P alcaligenes, P to a large and diverse class of naturally-occurring organic putida, or Pfluorescens, the genus Streptomyces, such as S. chemicals similar to terpenes. Terpenoids are derived from lividans, S. coelicolor; S. griseus, or S. rubiginosus, the genus five-carbon isoprene units assembled and modified in a vari Corynebacterium, Such as Corynebacterium glutamicum, the ety of ways, and are classified in groups based on the number 5 genus Rhodopseudomonas, such as Rhodopseudomonas of isoprene units used in group members. Hemiterpenoids palustris, or the genus Escherichia. Such as E. coli. In some have one isoprene unit. Monoterpenoids have two isoprene aspects, the bacterium is selected from group consisting of B. units. Sesquiterpenoids have three isoprene units. Diterpe subtilis, B. licheniformis, B. lentus, B. brevis, B. Stearother noids have four isoprene units. Sesterterpenoids have five mophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, isoprene units. Triterpenoids have six isoprene units. Tetrater 10 B. halodurans, B. megaterium, B. coagulans, B. circulans, B. penoids have eight isoprene units. Polyterpenoids have more lautus, and B. thuringiensis. than eight isoprene units. One of ordinary skill in the art In some aspects, the source organism is a plant. Such as a would be able to identify heterologous polypeptides capable plant from the family Fabaceae, such as the Faboideae sub of biological activity, e.g., capable of making terpenoids of family. In some aspects, the Source organism is kudzu, poplar various classes by assembling the appropriate number of 15 (such as Populus alba or Populus albax tremula CAC35696), isoprene units and modifying them as appropriate. aspen (Such as Populus tremuloides), or Quercus robur. In As used herein, the term “carotenoid refers to a group of Some aspects, the Source organism is an algae, such as a green naturally-occurring organic pigments produced in the chlo algae, red algae, glaucophytes, chlorarachniophytes, roplasts and chromoplasts of plants, of some other photosyn euglenids, chromista, or dinoflagellates. In some aspects, the thetic organisms, such as algae, in some types of fungus, and Source organism is a cyanobacterium, Such as cyanobacte in Some bacteria. Carotenoids include the oxygen-containing rium classified into any of the following groups based on Xanthophylls and the non-oxygen-containing carotenes. morphology: Chlorococcales, Pleurocapsales, Oscillatori In some aspects, the terpenoids are selected from the group ales, Nostocales, or Stigonematales. consisting of hemiterpenoids, monoterpenoids, sesquiterpe Isoprene Compositions Produced from Renewable Resources noids, diterpenoids, sesterterpenoids, triterpenoids, tetrater 25 Isoprene compositions produced from renewable penoids, and higher polyterpenoids. In some aspects, the resources are distinguished from petro-isoprene composi hemiterpenoid is prenol (i.e., 3-methyl-2-buten-1-ol), isopre tions in that isoprene produced from renewable resources is nol (i.e., 3-methyl-3-buten-1-ol), 2-methyl-3-buten-2-ol, or produced with other biological byproducts (compounds isovaleric acid. In some aspects, the monoterpenoid is geranyl derived from the biological sources and/or associated the pyrophosphate, eucalyptol, limonene, or pinene. In some 30 biological processes that are obtained together with isoprene) aspects, the sesquiterpenoid is farnesyl pyrophosphate, arte that are not present or present in much lower levels in petro misinin, or bisabolol. In some aspects, the diterpenoid is isoprene compositions, such as alcohols, aldehydes, ketone geranylgeranyl pyrophosphate, retinol, retinal, phytol, taxol. and the like. The biological byproducts may include, but are forskolin, or aphidicolin. In some aspects, the triterpenoid is not limited to, ethanol, acetone, methanol, acetaldehyde, squalene or lanosterol. In some aspects, the tetraterpenoid is 35 methacrolein, methyl vinylketone, 2-methyl-2-vinyloxirane, lycopene or carotene. In some aspects, the carotenoids are cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol selected from the group consisting of Xanthophylls and car (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), otenes. In some aspects, the Xanthophyll is lutein or Zeaxan 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyri thin. In some aspects, the carotene is C-carotene, B-carotene, dine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, Y-carotene, B-cryptoxanthin or lycopene. 40 methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl In some aspects, the source organism for the heterologous 3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl polypeptide capable of biological activity is a fungus. In some 1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, aspects, the fungus is a species of Aspergillus such as A. 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, Oryzae and A. niger, a species of Saccharomyces such as S. butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl cerevisiae, a species of Schizosaccharomyces such as S. 45 acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol. pombe, or a species of Trichoderma Such as Treesei. In some 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dim aspects, the Source organism for the heterologous polypeptide ethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen capable of biological activity is a filamentous fungal cell. In 1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1, Some aspects, the filamentous fungal cell is from Tricho 3.6-octatriene, or a linear isoprene polymer (Such as a linear derma longibrachiatum, T. viride, T. koningii, T. harzianum, 50 isoprene dimer or a linear isoprene trimer derived from the Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, polymerization of multiple isoprene units). Products derived Chrysosporium sp., C. lucknowense, Gliocladium sp., from isoprene produced from renewable resources contain Aspergillus sp., Such as A. Oryzae, A. niger; A Sojae, A. japoni one or more of the biological byproducts or compounds cus, A. nidulans, or A. awamori, Fusarium sp., Such as F. derived from any of the by-products. In addition, products roseum, F. graminum F. cerealis, F. Oxysporuin, or F. venena 55 derived from isoprene produced from renewable resources tum, Neurospora sp., Such as N. crassa, Hypocrea sp., Mucor may contain compounds formed from these by-products dur sp., Such as M. miehei, Rhizopus sp. or Emericella sp. In some ing Subsequent chemical conversion. Examples of such com aspects, the fungus is A. nidulans, A. awamori, A. Oryzae, A. pounds include those derived from Diels-Alder cycloaddition aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. of dienophiles to isoprene, or the oxidation of isoprene. Oxysporum, or F. Solani. In some aspects, the source organism 60 Isoprene compositions produced from renewable is a yeast, Such as Saccharomyces sp., Schizosaccharomyces resources, including particular byproducts or impurities, are sp., Pichia sp., or Candida sp. In some aspects, the Saccha described in more detail in U.S. Provisional Patent Applica romyces sp. is Saccharomyces cerevisiae. tion No. 61/187,959 and WO 2010/14825. In some aspects, the source organism for the heterologous The amount of isoprene produced by cells can be greatly polypeptide capable of biological activity is a bacterium. In 65 increased by introducing a heterologous nucleic acid encod Some aspects, the bacterium is of the genus Bacillus, such as ing an isoprene synthase polypeptide (e.g., a plant isoprene B. lichenformis or B. subtilis, the genus Pantoea, such as P synthase polypeptide), a DXS polypeptide, other DXP path US 8,455,236 B2 41 42 way polypeptide, and/or an MVA pathway polypeptide into DXP pathway nucleic acids include nucleic acids that encode the cells, e.g., as described in International Patent Application a polypeptide, fragment of a polypeptide, peptide, or fusion Publication No. WO2009/076676A2, U.S. patent application polypeptide that has at least one activity of a DXP pathway Ser. No. 12/335,071, U.S. patent application Ser. Nos. polypeptide. Exemplary DXP pathway polypeptides and 12/429,143, 12/496,573, 12/560,390, 12/560,317, 12/560, nucleic acids include naturally-occurring polypeptides and 370, 12/560,305, and 12/560,366; U.S. Provisional Patent nucleic acids from any of the source organisms described Application Nos. 61/187,930: 61/187,941: 61/187,959; U.S. herein as well as mutant polypeptides and nucleic acids Pub1. No. 2010/O196977 and WO 201OFO78457. derived from any of the Source organisms described herein. Exemplary isoprene synthase polypeptide (e.g., a plant Exemplary DXS polypeptides include polypeptides, frag isoprene synthase polypeptide), a DXS, a DXP pathway, oran 10 ments of polypeptides, peptides, and fusions polypeptides MVA pathway polypeptides and nucleic acids include natu that have at least one activity of a DXS polypeptide. Standard rally-occurring polypeptides and nucleic acids from any of methods (such as those described herein) can be used to the Source organisms described herein as well as mutant determine whether a polypeptide has DXS polypeptide activ polypeptides and nucleic acids derived from any of the Source ity by measuring the ability of the polypeptide to convert organisms described herein. 15 pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy Exemplary Isoprene Synthase Polypeptides and Nucleic D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo. Acids Exemplary DXS polypeptides and nucleic acids and methods In some aspects, the E. coli cells comprise a heterologous of measuring DXS activity are described in more detail in nucleic acid encoding an isoprene synthase polypeptide. In International Publication No. WO 2009/076676, U.S. patent Some aspects, the isoprene synthase polypeptide or nucleic application Ser. No. 12/335,071 (US Publ. No. 2009/ acid is from the family Fabaceae, such as the Faboideae 0203102), WO 2010/003007, US Publ. No. 2010/0048964, Subfamily. In some aspects, the isoprene synthase polypep WO 2009/132220, and US Publ. No. 2010/0003716. tide or nucleic acid is a polypeptide or nucleic acid from In particular, DXS polypeptides convert pyruvate and Pueraria montana (kudzu) (Sharkey et al., Plant Physiology D-glyceraldehyde 3-phosphate into 1-deoxy-d-xylulose 137:700-712, 2005), Pueraria lobata, poplar (such as Popu 25 5-phosphate (DXP). Standard methods can be used to deter lus alba, Populus migra, Populus trichocarpa, or Populus alba mine whether a polypeptide has DXS polypeptide activity by x tremula (CAC35696) Miller et al., Planta 213:483-487, measuring the ability of the polypeptide to convert pyruvate 2001) aspen (such as Populus tremuloides) Silver et al., JBC and D-glyceraldehyde 3-phosphate in vitro, in a cell extract, 270(22): 13010-1316, 1995), or English Oak (Quercus robur) or in vivo. (Zimmer et al., WO 98/02550). In some aspects, the isoprene 30 DXR polypeptides convert 1-deoxy-d-xylulose 5-phos synthase polypeptide or nucleic acid is a naturally-occurring phate (DXP) into 2-C-methyl-D-erythritol 4-phosphate isoprene synthase polypeptide or nucleic acid. In some (MEP). Standard methods can be used to determine whether aspects, the isoprene synthase polypeptide or nucleic acid is a polypeptide has DXR polypeptides activity by measuring not a naturally-occurring isoprene synthase polypeptide or the ability of the polypeptide to convert DXP in vitro, in a cell nucleic acid. Exemplary isoprene synthase polypeptides and 35 extract, or in vivo. nucleic acids and methods of measuring isoprene synthase MCT polypeptides convert 2-C-methyl-D-erythritol activity are described in more detail in International Publica 4-phosphate (MEP) into 4-(cytidine 5'-diphospho)-2-methyl tion No. WO 2009/076676, U.S. patent application Ser. No. D-erythritol (CDP-ME). Standard methods can be used to 12/335,071 (US Publ. No. 2009/0203102), WO 2010/ determine whether a polypeptide has MCT polypeptides 003007, US Publ. No. 2010/0048964, WO 2009/132220, and 40 activity by measuring the ability of the polypeptide to convert US Pub1. No. 201O/OOO3716. MEP in vitro, in a cell extract, or in vivo. Exemplary DXS Polypeptides and Nucleic Acids CMK polypeptides convert 4-(cytidine 5'-diphospho)-2-C- Exemplary DXS polypeptides include polypeptides, frag methyl-D-erythritol (CDP-ME) into 2-phospho-4-(cytidine ments of polypeptides, peptides, and fusions polypeptides 5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP). Stan that have at least one activity of a DXS polypeptide. Standard 45 dard methods can be used to determine whether a polypeptide methods (such as those described herein) can be used to has CMK polypeptides activity by measuring the ability of determine whether a polypeptide has DXS polypeptide activ the polypeptide to convert CDP-ME in vitro, in a cell extract, ity by measuring the ability of the polypeptide to convert or in vivo. pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy MCS polypeptides convert 2-phospho-4-(cytidine D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo. 50 5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into Exemplary DXS polypeptides and nucleic acids and methods 2-C-methyl-D-erythritol 2.4-cyclodiphosphate (ME-CPP or of measuring DXS activity are described in more detail in cMEPP). Standard methods can be used to determine whether International Publication No. WO 2009/076676, U.S. patent a polypeptide has MCS polypeptides activity by measuring application Ser. No. 12/335,071 (US Publ. No. 2009/ the ability of the polypeptide to convert CDP-MEP in vitro, in 0203102), WO 2010/003007, US Publ. No. 2010/0048964, 55 a cell extract, or in vivo. WO 2009/132220, and US Publ. No. 2010/0003716. HDS polypeptides convert 2-C-methyl-D-erythritol 2,4- Exemplary DXP Pathway Polypeptides and Nucleic Acids cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl Exemplary DXP pathways polypeptides include, but are diphosphate (HMBPP or HDMAPP). Standard methods can not limited to any of the following polypeptides: DXS be used to determine whether a polypeptide has HDS polypeptides, DXR polypeptides, MCT polypeptides, CMK 60 polypeptides activity by measuring the ability of the polypep polypeptides, MCS polypeptides, HDS polypeptides, HDR tide to convert ME-CPP in vitro, in a cell extract, or in vivo. polypeptides, IDI polypeptides, and polypeptides (e.g., HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2- fusion polypeptides) having an activity of one, two, or more en-1-yl diphosphate into isopentenyl diphosphate (IPP) and of the DXP pathway polypeptides. In particular, DXP path dimethylallyl diphosphate (DMAPP). Standard methods can way polypeptides include polypeptides, fragments of 65 be used to determine whether a polypeptide has HDR polypeptides, peptides, and fusions polypeptides that have at polypeptides activity by measuring the ability of the polypep least one activity of a DXP pathway polypeptide. Exemplary tide to convert HMBPP in vitro, in a cell extract, or in vivo. US 8,455,236 B2 43 44 IDI polypeptides convert isopentenyl diphosphate into tions and methods described in. Such polypeptides and dimethylallyl diphosphate. Standard methods can be used to nucleic acids are described in U.S. Publ. No. 2010/0196977 determine whether a polypeptide has IDI polypeptides activ and WO 2010/078457. ity by measuring the ability of the polypeptide to convert Exemplary Methods for Isolating Nucleic Acids isopentenyl diphosphate in vitro, in a cell extract, or in vivo. Isoprene synthase, DXS. IDI, DXP pathway, MVA path Exemplary IDI Polypeptides and Nucleic Acids way, PGL, hydrogenase, hydrogenase maturation and/or tran Isopentenyl diphosphate isomerase polypeptides (isopen Scription factor nucleic acids can be isolated using standard tenyl-diphosphate delta-isomerase or IDI) catalyses the inter methods. Methods of obtaining desired nucleic acids from a conversion of isopentenyl diphosphate (IPP) and dimethyl Source organism of interest (such as a bacterial genome) are allyl diphosphate (DMAPP) (e.g., converting IPP into 10 common and well known in the art of molecular biology (see, for example, WO 2004/033646 and references cited therein). DMAPP and/or converting DMAPP into IPP). Exemplary Standard methods of isolating nucleic acids, including PCR IDI polypeptides include polypeptides, fragments of amplification of known sequences, synthesis of nucleic acids, polypeptides, peptides, and fusions polypeptides that have at screening of genomic libraries, screening of cosmid libraries least one activity of an IDI polypeptide. Standard methods 15 are described in International Publication No. WO 2009/ (such as those described herein) can be used to determine 076676, U.S. patent application Ser. No. 12/335,071 (US whether a polypeptide has IDI polypeptide activity by mea Publ. No. 2009/0203102), WO 2010/003007, US Publ. No. suring the ability of the polypeptide to interconvert IPP and 2010/0048964, WO 2009/132220, and US Publ. No. 2010/ DMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI OOO3716. polypeptides and nucleic acids and methods of measuring IDI Exemplary Promoters and Vectors activity are described in more detail in International Publica Any of the isoprene synthase, DXS. DXP pathway, IDI, tion No. WO 2009/076676, U.S. patent application Ser. No. MVA pathway, PGL, hydrogenase, hydrogenase maturation, 12/335,071 (US Publ. No. 2009/0203102), WO 2010/ transcription factor, galactose metabolic, and/or molybde 003007, US Publ. No. 2010/0048964, WO 2009/132220, and num transport nucleic acids described herein can be included US Pub1. No. 201O/OOO3716. 25 in one or more vectors. Accordingly, also described hereinare Exemplary MVA Pathway Polypeptides and Nucleic Acids vectors with one more nucleic acids encoding any of the Exemplary MVA pathway polypeptides include acetyl isoprene synthase, DXS. IDI, DXP pathway, MVA pathway, CoA acetyltransferase (AA-CoA thiolase) polypeptides, PGL, hydrogenase, hydrogenase maturation, transcription 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA syn factor polypeptides, galactose metabolic polypeptides, and/ thase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA 30 or molybdenum transport polypeptides that are described reductase (HMG-CoA reductase) polypeptides, mevalonate herein. In some aspects, the vector contains a nucleic acid kinase (MVK) polypeptides, phosphomevalonate kinase under the control of an expression control sequence. In some (PMK) polypeptides, diphosphomevalonate decarboxylase aspects, the expression control sequence is a native expres (MVD) polypeptides, phosphomevalonate decarboxylase sion control sequence. In some aspects, the expression con 35 trol sequence is a non-native expression control sequence. In (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) Some aspects, the vector contains a selective marker or select polypeptides, IDI polypeptides, and polypeptides (e.g., able marker. In some aspects, an isoprene synthase, DXS. fusion polypeptides) having an activity of two or more MVA IDI, MVA pathway, PGL, hydrogenase, hydrogenase matu pathway polypeptides. In particular, MVA pathway polypep ration, transcription regulatory, galactose metabolic, and/or tides include polypeptides, fragments of polypeptides, pep 40 molybdenum transport nucleic acid integrates into a chromo tides, and fusions polypeptides that have at least one activity some of the cells without a selectable marker. In some of an MVA pathway polypeptide. Exemplary MVA pathway aspects, an isoprene synthase, DXS. IDI, DXP pathway, polypeptides and nucleic acids and methods of measuring IDI MVA pathway, PGL, hydrogenase, hydrogenase maturation, activity are described in more detail in International Publica transcription regulatory, galactose metabolic, and/or molyb tion No. WO 2009/076676, U.S. patent application Ser. No. 45 denum transport nucleic acid integrates into a chromosome of 12/335,071 (US Publ. No. 2009/0203102), WO 2010/ the cells with a selectable marker. 003007, US Publ. No. 2010/0048964, WO 2009/132220, and Suitable vectors are those which are compatible with the US Pub1. No. 201O/OOO3716. host cell employed. Suitable vectors can be derived, for In Some aspects, the cells contain the upper MVA pathway, example, from a bacterium, a virus (such as bacteriophage T7 which includes AA-CoA thiolase, HMG-CoA synthase, and 50 or a M-13 derived phage), a cosmid, a yeast, or a plant. HMG-CoA reductase nucleic acids. In some aspects, the cells Suitable vectors can be maintained in low, medium, or high contain the lower MVA pathway, which includes MVK, copy number in the host cell. Protocols for obtaining and PMK, MVD, and IDI nucleic acids. In some aspects, the cells using Such vectors are known to those in the art (see, for contain an entire MVA pathway that includes AA-CoA thio example, Sambrook et al., Molecular Cloning: A Laboratory lase, HMG-CoA synthase, HMG-CoA reductase, MVK, 55 Manual. 2" ed., Cold Spring Harbor, 1989). Suitable vectors PMK, MVD, and IDI nucleic acids. In some aspects, the cells compatible with the cells and methods described herein are contain an entire MVA pathway that includes AA-CoA thio described in International Publication No. WO 2009/076676, lase, HMG-CoA synthase, HMG-CoA reductase, MVK, U.S. patent application Ser. No. 12/335,071 (US Publ. No. PMDC, IPK, and IDI nucleic acids. 2009/0203102), WO 2010/003007, US Publ. No. 2010/ The E. coli cells described herein can also be used for 60 0048964, WO 2009/132220, and US Publ. No. 2010/ improved methods of producing isoprene and a co-product, OOO3716. Such as hydrogen, ethanol, or propanediol (e.g., 1,2-pro Promoters are well known in the art. Any promoter that panediol or 1,3-propanediol). Exemplary hydrogenase functions in the host cell can be used for expression of an polypeptides and nucleic acids, polypeptides and nucleic isoprene synthase, DXS. IDI, DXP pathway, MVA pathway, acids for genes related to production of fermentation side 65 PGL, hydrogenase, hydrogenase maturation, transcription products, and polypeptides and nucleic acids for genes relat factor, galactose metabolic and/or molybdenum transport ing to hydrogen reuptake can also be used with the composi nucleic acid in the host cell. Initiation control regions or US 8,455,236 B2 45 46 promoters, which are useful to drive expression of isoprene pathway or contains both the MVA and DXP pathways. IDI synthase, DXS. IDI, DXP pathway, MVA pathway, PGL, and isoprene synthase nucleic acids can be obtained, e.g., hydrogenase, hydrogenase maturation, transcription factor, from any organism that contains the MVA pathway, DXP galactose metabolic and/or molybdenum transport nucleic pathway, or both the MVA and DXP pathways. MVA pathway acids in various host cells are numerous and familiar to those nucleic acids can be obtained, e.g., from any organism that skilled in the art (see, for example, WO 2004/033646 and contains the MVA pathway or contains both the MVA and references cited therein). Virtually any promoter capable of DXP pathways. Hydrogenase nucleic acids can be obtained, driving these nucleic acids can be used including a glucose e.g., from any organism that oxidizes hydrogen or reduces isomerase promoter (see, for example, U.S. Pat. No. 7,132, hydrogen ions. Fermentation side product genes can be 527 and references cited therein). Suitable promoters com 10 obtained or identified, e.g., from any organism that undergoes patible with the cells and methods described herein are described in International Publication No. WO 2009/076676 oxygen-limited or anaerobic respiration, such as glycolysis. A2 and U.S. Patent Application Publication No. US2009/ The nucleic acid sequence of the isoprene synthase, DXP O2O31 O2A1. pathway, IDI, MVA pathway, PGL, hydrogenase, hydroge In some aspects, the expression vector also includes a 15 nase maturation, transcription factor, galactose metabolic termination sequence. Termination control regions may also and/or molybdenum transport nucleic acids can be isolated be derived from various genes native to the host cell. In some from a bacterium, fungus, plant, algae, or cyanobacterium. aspects, the termination sequence and the promoter sequence Exemplary Source organisms include, for example, yeasts, are derived from the same source. Suitable termination Such as species of Saccharomyces (e.g., S. cerevisiae), bacte sequences compatible with the cells and methods described ria, Such as species of Escherichia (e.g., E. coli), or species of herein are described in International Publication No. WO Methanosarcina (e.g., Methanosarcina mazei), plants, such 2009/076676A2 and U.S. Patent Application Publication No. as kudzu or poplar (e.g., Populus alba or Populus alba x US2O09/O2O31 O2 A1 tremula CAC35696) or aspen (e.g., Populus tremuloides). An isoprene synthase, DXS. IDI, DXP pathway, MVA Exemplary host organisms are described in International pathway, PGL, hydrogenase, hydrogenase maturation, tran 25 Publication No. WO 2009/076676, U.S. patent application Scription factor, galactose metabolic and/or molybdenum Ser. No. 12/335,071 (US Publ. No. 2009/0203102), WO nucleic acid can be incorporated into a vector, Such as an 2010/003007, US Publ. No. 2010/0048964, WO 2009/ expression vector, using standard techniques (Sambrook et 132220, and US Publ. No. 2010/0003716. al., Molecular Cloning: A Laboratory Manual, Cold Spring Exemplary Transformation Methods Harbor, 1982). Suitable techniques compatible with the cells 30 Isoprene synthase. DXP pathway, IDI, MVA pathway, and methods described herein are described in International PGL, hydrogenase, hydrogenase maturation, transcription Publication No. WO 2009/076676A2 and U.S. Patent Appli factor, galactose metabolic and/or molybdenum transport cation Publication No. US2009/0203102 A1. nucleic acids or vectors containing them can be inserted into In some aspects, it may be desirable to over-express iso a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a prene synthase, DXP pathway, IDI, MVA pathway, PGL, 35 bacterial cell described herein) using standard techniques for hydrogenase, hydrogenase maturation, transcription factor, introduction of a DNA construct or vector into a host cell, galactose metabolic and/or molybdenum transport nucleic Such as transformation, electroporation, nuclear microinjec acids at levels far higher than currently found in naturally tion, transduction, transfection (e.g., lipofection mediated or occurring cells. In some aspects, it may be desirable to under DEAE-Dextrin mediated transfection or transfection using a express (e.g., mutate, inactivate, or delete) isoprene synthase, 40 recombinant phage virus), incubation with calcium phos DXP pathway, IDI, MVA pathway, PGL, hydrogenase, phate DNA precipitate, high velocity bombardment with hydrogenase maturation, transcription factor polypeptide, DNA-coated microprojectiles, and protoplast fusion. General galactose metabolic polypeptide and/or molybdenum trans transformation techniques are known in the art (see, e.g., port polypeptide-encoding nucleic acids at levels far below Current Protocols in Molecular Biology (F. M. Ausubel et al. that those currently found in naturally-occurring cells. Suit 45 (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A able methods for over- or under-expressing the isoprene Syn Laboratory Manual, 2" ed., Cold Spring Harbor, 1989; and thase, DXP pathway, IDI, MVA pathway, PGL, hydrogenase, Campbell et al., Curr. Genet. 16:53-56, 1989). The intro hydrogenase maturation, transcription factor, galactose meta duced nucleic acids may be integrated into chromosomal bolic and/or molybdenum transport nucleic acids compatible DNA or maintained as extrachromosomal replicating with cells and methods described herein are described in 50 sequences. Transformants can be selected by any method International Publication No. WO 2009/076676 A2 and U.S. known in the art. Suitable methods for selecting transfor Patent Application Publication No. US2009/0203102A1. mants are described in International Publication No. WO Exemplary Source Organisms 2009/076676, U.S. patent application Ser. No. 12/335,071 Isoprene synthase. DXP pathway, IDI, MVA pathway, (US Publ. No. 2009/0203102), WO 2010/003007, US Publ. PGL, hydrogenase, hydrogenase maturation, transcription 55 No. 2010/0048964, WO 2009/132220, and US Publ. No. factor, galactose metabolic and/or molybdenum transport 2O1 O/OOO3716. nucleic acids (and their encoded polypeptides) can be Exemplary Purification Methods obtained from any organism that naturally contains isoprene In some aspects, any of the methods described herein fur synthase, DXP pathway, IDI, MVA pathway, PGL, hydroge ther include a step of recovering the compounds produced. In nase, hydrogenase maturation, transcription factor, galactose 60 some aspects, any of the methods described herein further metabolic and/or molybdenum transport nucleic acids. As include a step of recovering the isoprene. In some aspects, the noted above, isoprene is formed naturally by a variety of isoprene is recovered by absorption stripping (see, e.g., U.S. organisms, such as bacteria, yeast, plants, and animals. Prov. 61/288,142 or U.S. application Ser. No. 12/969,440). In Organisms contain the MVA pathway, DXP pathway, or both some aspects, any of the methods described herein further the MVA and DXP pathways for producing isoprene (FIGS. 65 include a step of recovering the heterologous polypeptide. In 1A and 1B). Thus, DXP pathway nucleic acids can be some aspects, any of the methods described herein further obtained, e.g., from any organism that contains the DXP include a step of recovering the terpenoid or carotenoid. US 8,455,236 B2 47 48 Suitable purification methods are described in more detail rated phenol pH7.9 (Ambion Inc.) and chloroform were in U.S. Patent Application Publication US2010/0196977 A1; added. The tubes were mixed well and microcentrifuged for 5 and U.S. Provisional Patent Application No. 61/187,959. minutes. A second extraction was done with 400 ul chloro Other Techniques form and the aqueous layer was transferred to a new eppen Additional examples of efficient methods for the produc dorf tube. The genomic DNA was precipitated by the addition tion and recovery ofisoprene and a coproduct, such as hydro of 1 ml of 100% ethanol and centrifugation for 5 minutes. The gen, are described in U.S. Patent Application Publication No. genomic DNA pellet was washed with 1 ml 70% ethanol. The US2010/O196977. ethanol was removed and the genomic DNA pellet was Examples of other techniques (e.g., decoupling isoprene allowed to air dry briefly. The genomic DNA pellet was production from cell growth, methods of producing isoprene 10 resuspended with 200 ul TE. within safe operating ranges, cell viability at high isoprene Using Pfu Ultra II DNA polymerase (Stratagene) and 200 titers, efficient methods for the production and recovery of ng/ul of genomic DNA as template, 2 different sets of PCR isoprene and a co-product (e.g., hydrogen, ethanol, or 1.3- reaction tubes were prepared according to manufacturers propanediol)) that can be used with the cells and methods protocol. For set 1, primers MCM130 and GBCm-Rev (Table described herein are described in International Patent Publi 15 1) were used to ensure transductants were successfully inte cation No. WO 2009/076676 A2; U.S. Patent Application grated into the attTnT locus. PCR parameters for set 1 were Publication Nos. US2010/0048964 A1, US2010/0086978 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, A1, US2010/0113846 A1, US2010/0184178 A1 and 55° C. for 25 seconds, 72°C. for 25 seconds (repeat steps 2-4 US2010/0167371 A1, US2010/0196977 A1; U.S. Provi for 28 cycles), 72° C. for 1 minute. For set 2, primers MVD sional Patent Application Nos. 61/187,930, 61/187,959, and For and MVD Rev (Table 1) were used to ensure that the 61/187,941; and International Patent Application Publication gi1.2-KKDyI operon integrated properly. PCR parameters NoS. WO 2004/033646 A2 and WO 1996/035796 A2. for set 2 were 95°C. for 2 minutes (first cycle only),95°C. for The invention can be further understood by reference to the 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds following examples, which are provided by way of illustra (repeat steps 2-4 for 28 cycles), 72°C. for 1 minute. Analysis tion and are not meant to be limiting. 25 of PCR amplicons on a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant clones were correct. One was picked EXAMPLES and designated as strain EWL201. (ii) Construction of Strain EWL204 (BL21, Loopout-GI 1.2- Example 1 KKDyI) 30 The chloramphenicol marker was looped out of strain Construction of E. Coli Strains Expressing the S. EWL201 using plasmid pCP20 as described by Datsenko and Cerevisiae gi1.2KKDyI Operon, P alba Isoprene Wanner (2000) (Datsenko et al., Proc Natl. Acad. Sci. USA Synthase, M. Mazei Mevalonate Kinase, pCL Upper 97:6640-6645, 2000). EWL201 cells were grown in L. Broth MVA (E. Faecalis mvaE and mvaS) and ybhE (pgl) to midlog phase and then washed three times in ice-cold, 35 sterile water. An aliquot of 50 ul of cell suspension was mixed (i) Construction of strain EWL201 (BL21, Cm-GI1.2- with 1 ul of pCP20 and the cell suspension mixture was KKDyI) electroporated in a 2 mm cuvette (Invitrogen Corp.) at 2.5 E. coli BL21 (Novagenbrand, EMD Biosciences, Inc.) was Volts and 25 uFd using a Gene Pulser Electroporator (Bio a recipient strain, transduced with MCM331 P1 lysate (lysate Rad Inc.). 1 ml ofLB was immediately added to the cells, then prepared according to the method described in Ausubel, et al., 40 transferred to a 14 ml polypropylene tube (Sarstedt) with a Current Protocols in Molecular Biology. John Wiley and metal cap. Cells were allowed to recover by growing for 1 Sons, Inc.). MCM331 cells contain chromosomal construct hour at 30°C. Transformants were selected on L. Agar and 20 gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, ug/ul chloramphenicol and 50 g/ul carbenicillin and incu mevalonate phosphate kinase, mevalonate pyrophosphate bated at 30° C. overnight. The next day, a single clone was decarboxylase, and IPP isomerase (i.e., the gi1.2-KKDyI 45 grown in 10 ml L. Broth and 50 ug/ul carbenicillin at 30° C. operon from S. cerevisiae; construction of which is described until early log phase. The temperature of the growing culture in Example 10 of International Publication No. WO 2009/ was then shifted to 42° C. for 2 hours. Serial dilutions were 076676 A2 and U.S. patent application Ser. No. 12/335,071 made, the cells were then spread onto LA plates (no antibiotic (US Publ. No. 2009/0203102)). Transductants were selected selection), and incubated overnight at 30°C. The next day, 20 for by spreading cells onto L. Agar and 20 ug/ul chloram 50 colonies were picked and patched onto LAgar (no antibiotics) phenicol. The plates were incubated overnight at 30° C. and LA and 201gful chloramphenicol plates. Plates were then Analysis of transductants showed no colonies on control incubated overnight at 30° C. Cells able to grow on LA plates, plates (water+cells control plate for reversion and water and but not LA and 20 ugful chloramphenicol plates, were P1 lysate control plate for lysate contamination. deemed to have the chloramphenicol marker looped out Four transductants were picked and used to inoculate 5 mL. 55 (picked one and designated as strain EWL204). L. Broth and 20 g/ul chloramphenicol. The cultures were (iii) Construction of Plasmid pEWL230 (pTrc P alba) grown overnight at 30°C. with shaking at 200 rpm. To make Generation of a synthetic gene encoding Populus alba genomic DNA preparations of each transductant for PCR isoprene synthase (P alba HGS) was outsourced to DNA2.0 analysis, 1.5 mL of overnight cell culture were centrifuged. Inc. (Menlo Park, Calif.) based on their codon optimization The cell pellet was resuspended with 400 ul Resuspension 60 method for E. coli expression. The synthetic gene was custom Buffer (20 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 7.5) and cloned into plasmid pET24a (Novagen brand, EMD Bio 4 ul RNase, DNase-free (Roche) was added. The tubes were sciences, Inc.) and delivered lyophilized (FIGS. 2, 3A-B; incubated at 37°C. for 30 minutes followed by the addition of SEQID NO:1). 4 Jul 10% SDS and 4 ul of 10 mg/ml Proteinase K stock A PCR reaction was performed to amplify the P alba solution (Sigma-Aldrich). The tubes were incubated at 37°C. 65 isoprene synthase (P alba HGS) gene using pET24 P alba for 1 hour. The cell lysate was transferred into 2 ml Phase HGS as the template, primers MCM182 and MCM192, and Lock Light Gel tubes (Eppendorf) and 200 ul each of satu Herculase II Fusion DNA polymerase (Stratagene) according US 8,455,236 B2 49 50 to manufacturer's protocol. PCR conditions were as follows: mazei MVK PCR product was purified using QIAquick PCR 95°C. for 2 minutes (first cycle only), 95°C. for 25 seconds, Purification Kit (Qiagen Inc.). 55° C. for 20 seconds, 72° C. for 1 minute, repeat for 25 The M. mazei MVKPCR product was then digested in a 40 cycles, with final extension at 72° C. for 3 minutes. The P. ul reaction containing 8 ul PCR product, 2 ul PmeI endonu alba isoprene synthase PCR product was purified using clease (New England Biolabs), 4 Jul 10xNEB Buffer 4, 4 ul QIAquick PCR Purification Kit (Qiagen Inc.). 10xNEB BSA, and 22 ul of ddHO. The reaction was incu P alba isoprene synthase PCR product was then digested bated for 3 hours at 37°C. The digested PCR fragment was in a 20 ul reaction containing 1 ul BspHI endonuclease (New then purified using the QIAquick PCR Purification Kit. A England Biolabs) with 2 ul 10xNEB Buffer 4. The reaction secondary restriction digest was performed in a 47 ul reaction 10 containing 2 ul NsiI endonuclease (Roche), 4.7 ul 10x Buffer was incubated for 2 hours at 37°C. The digested PCR frag H, and 40 ul of Pme digested M. mazei MVK fragment. The ment was then purified using the QIAquick PCR Purification reaction was incubated for 3 hours at 37°C. The digested PCR Kit. A secondary restriction digest was performed in a 20 fragment was then gel purified using a 1.2% E-gel and reaction containing 1 Jul PstI endonuclease (Roche) with 2 ul 10x Buffer H. The reaction was incubated for 2 hours at 37° extracted using the QIAquick Gel Extraction Kit. Plasmid 15 EWL230 was digested in a 40 ul reaction containing 10 ul C. The digested PCR fragment was then purified using the plasmid, 2 ul PmeI endonuclease, 4 ul 10xNEB Buffer 4, 4 ul QIAquick PCR Purification Kit. Plasmid pTrcHis2B (Invit 10xNEB BSA, and 20 ul of ddHO. The reaction was incu rogen Corp.) was digested in a 20 reaction containing 1 ul bated for 3 hours at 37°C. The digested PCR fragment was NcoI endonuclease (Roche), 1 ul PstI endonuclease, and 2 ul then purified using the QIAquick PCR Purification Kit. A 10x Buffer H. The reaction was incubated for 2 hours at 37° secondary restriction digest was performed in a 47 ul reaction C. The digested pTrcHis2B vector was gel purified using a containing 2 ulPstI endonuclease, 4.7 ul 10x Buffer H, and 40 1.2% E-gel (Invitrogen Corp.) and extracted using the ul of PmeI digested EWL230 linear fragment. The reaction QIAquick Gel Extraction Kit (Qiagen) (FIG. 4). Using the was incubated for 3 hours at 37°C. The digested PCR frag compatible cohesive ends of BspHI and NcoI sites, a 20 ul ment was then gel purified using a 1.2% E-gel and extracted ligation reaction was prepared containing 5 ul P alba iso 25 using the QIAquick Gel Extraction Kit (FIG. 7). Using the prene synthase insert, 2 ul pTrc vector, 1 ul T4 DNA ligase compatible cohesive ends of NsiI and PstI sites, a 20 ul (New England Biolabs), 2 Jul 10x ligase buffer, and 10 ul ligation reaction was prepared containing 8 ul M. mazei MVK ddHO. The ligation mixture was incubated at room tempera insert, 3 ul. EWL230 plasmid, 1 ul T4 DNA ligase, 2 Jul 10x ture for 40 minutes. The ligation mixture was desalted by ligase buffer, and 6 ul ddHO. The ligation mixture was floating a 0.025 um nitrocellulose membrane filter (Milli 30 incubated overnight at 16°C. The next day, the ligation mix pore) in a petri dish of ddH2O and applying the ligation ture was desalted by floating a 0.025 um nitrocellulose mem mixture gently on top of the nitrocellulose membrane filter brane filter in a petridish of ddHO and applying the ligation for 30 minutes at room temperature. MCM446 cells were mixture gently on top of the nitrocellulose membrane filter grown in LB to midlog phase and then washed three times in for 30 minutes at room temperature. MCM446 cells were ice-cold, sterile water. An aliquot of 50 ul of cell suspension 35 grown in LB to midlog phase and then washed three times in was mixed with 5 ul of desalted pTrc P alba HGS ligation ice-cold, sterile water. An aliquot of 50 ul of cell suspension mix. The cell Suspension mixture was electroporated in a 2 was mixed with 5ul of desalted pTrc P alba-mMVK ligation mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser mix. The cell Suspension mixture was electroporated in a 2 Electroporator. 1 ml of LB was immediately added to the mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser cells, then transferred to a 14 ml polypropylene tube 40 Electroporator. 1 ml of LB is immediately added to the cells, (Sarstedt) with a metal cap. Cells were allowed to recover by then the cells are transferred to a 14 ml polypropylene tube growing for 2 hour at 30°C. Transformants were selected on with a metal cap. Cells were allowed to recover by growing LAgar and 50 ug/ul carbenicillin and 10 mM mevalonic acid for 2 hour at 30°C. Transformants were selected on LA and and incubated at 30°C. The next day, 6 transformants were 50 ug/ul carbenicillin and 5 mM mevalonic acid plates and picked and grown in 5 ml L. Broth and 50 lug/ul carbenicillin 45 incubated at 30°C. The next day, 6 transformants were picked tubes overnight at 30° C. Plasmid preps were performed on and grown in 5 ml LB and 50 lug/ul carbenicillin tubes over the overnight cultures using QIAquick Spin Miniprep Kit night at 30° C. Plasmid preps were performed on the over (Qiagen). Due to the use of BL21 cells for propagating plas night cultures using QIAquick Spin Miniprep Kit. Due to the mids, a modification of washing the spin columns with PB use of BL21 cells for propagating plasmids, a modification of Buffer 5x and PE Buffer 3x was incorporated to the standard 50 washing the spin columns with PB Buffer 5x and PE Buffer manufacturer's protocol for achieving high quality plasmid 3x was incorporated to the standard manufacturer's protocol DNA. Plasmids were digested with PstI in a 20 ul reaction to for achieving high quality plasmid DNA. Plasmids were ensure the correct sized linear fragment. All 6 plasmids were digested with PstI in a 20 ul reaction to ensure the correct the correct size and shipped to Quintara Biosciences (Berke sized linear fragment. Three of the 6 plasmids were the cor ley, Calif.) for sequencing with primers MCM65, MCM66, 55 rect size and shipped to Quintara BioSciences for sequencing EL1000 (Table 1). DNA sequencing results showed all 6 with primers MCM65, MCM66, EL1000, EL1003, and plasmids were correct. One plasmid was picked designated as EL 1006 (Table 1). DNA sequencing results showed all 3 plasmid EWL230 (FIGS. 5, 6A-B: SEQID NO:2). plasmids were correct. One was picked and designated as iv) Construction of PlasmidpEWL244 (pTrc P alba-mMVK) plasmid EWL244 (FIGS. 8 and 9A-B; SEQID NO:3). A PCR reaction was performed to amplify the Methanosa 60 v) Construction of Plasmid MCM376 MVK from M. mazei rcina mazei (M. mazei) MVK gene using MCM376 as the archaeal Lower in pET200D. template, primers MCM165 and MCM177 (see Table 1), and The MVK ORF from the M. mazei archaeal Lower Path Pfu Ultra II Fusion DNA polymerase (Stratagene) according way operon (FIGS. 10A-C: SEQID NO:4) was PCR ampli to manufacturer's protocol. PCR conditions were as follows: fied using primers MCM161 and MCM162 (Table 1) using 95°C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 65 the Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28 was combined with 1 uL template, 1 of each primer at 10M, cycles, with final extension at 72° C. for 1 minute. The M. and 2 ul, water. The reaction was cycled as follows: 94°C. for US 8,455,236 B2 51 52 2:00 minutes; 30 cycles of 94° C. for 0:30 minutes, 55° C. for TABLE 1 - continued 0:30 minutes and 68°C. for 1:15 minutes; and then 72°C. for 7:00 minutes, and 4°C. until cool. 3 uI of this PCR reaction Primer Sequences was ligated to Invitrogen pET200D plasmid according to the Primer manufacturer's protocol. 3 ul of this ligation was introduced 5 ale Primer sequence into Invitrogen TOP10 cells, and transformants were selected on LA/kans0. A plasmid from a transformant was isolated MCM65 ACAATTTCACACAGGAAACAGC (SEQ ID NO: 2O) and the insert sequenced, resulting in MCM376 (FIGS. 11A MCM66 CCAGGCAAATTCTGTTTTATCAG (SEO ID NO: 21) C). vi) Construction of Strain EWL251 (BL21 (DE3), Cm-GI1.2- 10 EL1OOO GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 22) KKDyI, pTrc P alba-mMVK) MCM165 GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCT MCM331 cells (which contain chromosomal construct GTTCTGCGCCGGGTAAGATTTACCTG gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, (SEQ ID NO: 23) mevalonate phosphate kinase, mevalonate pyrophosphate 15 MCM177 GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCA decarboxylase, and IPP isomerase) were grown in LB to GACCTTGC (SEO ID NO. 24) midlog phase and then washed three times in ice-cold, sterile water. Mixed 50 ul of cell suspension with 1 ul of plasmid EL10O3 GATAGTAACGGCTGCGCTGCTACC (SEO ID NO: 25) EWL244. The cell suspension mixture was electroporated in EL1OO6 GACAGCTTATCATCGACTGCACG (SEO ID NO: 26) a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, MCM161 CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 27) and then the cells were transferred to a 14 ml polypropylene MCM162 TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 28) tube with a metal cap. Cells were allowed to recover by growing for 2 hours at 30°C. Transformants were selected on LA and 50 carbenicillin and 5 mM mevalonic acid plates and viii) Construction of Strain RM111608-2 (Cm-GI1.2- incubated at 37°C. One colony was selected and designated KKDyI, pTrc P alba-mMVK, pCL Upper MVA, pBBRC as Strain EWL251. MPGI1.5-pgl) vii) Construction of Strain EWL256 (BL21 (DE3), The BL21 strain of E. coli producing isoprene (EWL256) Cm-GI1.2-KKDyI, pTrc P alba-mMVK, pCL Upper MVA) was constructed with constitutive expression of the ybhE EWL251 cells were grown in LB to midlog phase and then gene (encoding E. coli 6-phosphogluconolactonase) on a rep washed three times in ice-cold, sterile water. Mixed 50 ul of 30 licating plasmid pBBR1MCS5 (Gentamycin) (obtained from cell suspension with 1 ul of plasmid MCM82 (comprising Dr. K. Peterson, Louisiana State University). pCL PtrcUpperPathway (also known as “pCLUpper MVA), FRT-based recombination cassettes, and plasmids for Red/ encoding E. faecalis mvaE and mvaS). Plasmid pCL Ptrc ET-mediated integration and antibiotic marker loopout were Upper Pathway was constructed as described in Example 8 of obtained from Gene Bridges GmbH (Germany). Procedures International Publication No. WO 2009/076676 A2 and U.S. using these materials were carried out according to Gene patent application Ser. No. 12/335,071 (US Publ. No. 2009/ Bridges protocols. Primers Pgl-F (SEQ ID NO:29) and 0203102). The cell suspension mixture was electroporated in PglGI1.5-R (SEQID NO:30) were used to amplify the resis a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB was immediately added to the tance cassette from the FRT-gb2-Cm-FRT template using cells. Cells were then transferred to a 14 ml polypropylene 40 Stratagene Herculase II Fusion kit according to the manufac tube with a metal cap. Cells were allowed to recover by turer's protocol. The PCR reaction (50 uL final volume) con growing for 2 hours at 30°C. Transformants were selected on tained: 5uL buffer, 1 uL template DNA (FRT-gb2-Cm-F from LA and 50 ug/ul carbenicillin and 50 g/ul spectinomycin Gene Bridges), 10 pmols of each primer, and 1.5 uL 25 mM plates and incubated at 37° C. One colony was picked and dNTP mix, made to 50 uL with dHO. The reaction was designated as strain EWL256. 45 cycled as follows: 1x2 minutes, 95°C. then 30 cycles of (30 seconds at 95°C.; 30 seconds at 63°C.: 3 minutes at 72° C.). TABLE 1. The resulting PCR product was purified using the QIAquick R. PCR Purification Kit (Qiagen) and electropo Primer Sequences 50 rated into electrocompetent MG 1655 cells harboring the Primer pRed-ET recombinase-containing plasmid as follows. Cells ale Primer sequence were prepared by growing in 5 mLs of L broth to and OD600-0.6 at 30°C. The cells were induced for recombinase MCM13 O ACCAATTGCACCCGGCAGA (SEQ ID NO: 14) expression by the addition of 4% arabinose and allowed to GB Cm GCTAAAGCGCATGCTCCAGAC (SEO ID NO: 15) 55 grow for 30 minutes at 30° C. followed by 30 minutes of Rew growth at 37° C. An aliquot of 1.5 mLs of the cells was MVD GACTGGCCTCAGATGAAAGC (SEQ ID NO: 16) washed 3-4 times in ice cold dELO. The final cell pellet was For resuspended in 40 uL of ice cold dHO and 2-5 uL of the PCR product was added. The electroporation was carried out in MVD CAAACATGTGGCATGGAAAG (SEQ ID NO: 17) 60 1-mm gap cuvettes, at 1.3 kV in a Gene Pulser Electroporator Rew (Bio-Rad Inc.). Cells were recovered for 1-2 hours at 30°C. MCM182 GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTT and plated on Lagar containing chloramphenicol (5ug/mL). CAAACGGCAGAA (SEQ ID NO: 18) Five transformants were analyzed by PCR and sequencing MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA using primers flanking the integration site (2 primer sets: pgl (SEQ ID NO: 19) 65 and 49 rev and 3' EcoRV-pglstop; Bottom Pgb2 and Top GB’s CMP (946)). A correct transformant was selected and this strain was designated MG1655 GI1.5-pgl::CMP. US 8,455,236 B2 53 54 The chromosomal DNA of MG1655 GI1.5-pgl:CMP was control strain expressing ybhE at wild-type levels (i.e., used as template to generate a PCR fragment containing the EWL256). The gene ybhE (pgl) encodes E. coli 6-phospho FRT-CMP-FRT-GI1.5-ybhE construct. This construct was gluconolactonase that Suppresses posttranslational glucony cloned into pBBR1MCS5 (Gentamycin) as follows. The frag lation of heterologously expressed proteins and improves ment, here on referred to as CMP-GI1.5-pgl, was amplified product solubility and yield while also improving biomass using the 5' primer Pglconfirm-F (SEQ ID NO:31) and 3' yield and flux through the pentose phosphate pathway (Aonet primer 3' EcoRV-pglstop (SEQ ID NO:32). The resulting al., Applied and Environmental Microbiology 74(4): 950 fragment was cloned using the Invitrogen TOPO-Blunt clon 958, 2008). ing kit into the plasmid vector pCR-Blunt II-TOPO as sug i) Small Scale Analysis gested from the manufacturer. The Nsil fragment harboring 10 Media Recipe (per liter fermentation media): KHPO 13.6 the CMP-GI1.5-pgl fragment was cloned into the PstI site of g, KHPO 13.6 g. MgSO7HO 2 g, citric acid monohy pBBR1MCS5 (Gentamycin). A 20 Jul ligation reaction was drate 2 g, ferric ammonium citrate 0.3 g. (NH4)2SO 3.2 g, prepared containing 5 ul CMP-GI1.5-pgl insert, 2 ul yeast extract 1 g, 1000x Trace Metals Solution 1 ml. All of the pBBR1MCS5 (Gentamycin) vector, 1 ul T4 DNA ligase components were added together and dissolved in diFIO. (New England Biolabs), 2 Jul 10x ligase buffer, and 10 ul 15 The pH was adjusted to 6.8 with ammonium hydroxide (30%) ddH2O. The ligation mixture was incubated at room tempera and brought to volume. Media was filter-sterilized with a 0.22 ture for 40 minutes then 2-4 uL were electroporated into micron filter. Glucose 5.0 g and antibiotics were added after electrocompetent Top10 cells (Invitrogen) using the param sterilization and pH adjustment. eters disclosed above. Transformants were selected on Lagar 1000x Trace Metal Solution (per liter fermentation media): containing 10 g/ml chloramphenicol and 5 g/ml Gentamy Citric Acid'HO 40 g, MnSOHO 30 g, NaCl 10 g, cin. The sequence of the selected clone was determined using FeSO.7HO 1 g, CoCl*6HO 1 g, ZnSO*7HO 1 g, a number of the primers described above as well as with the CuSO4.5H2O 100 mg., HBO 100 mg. NaMoO2HO 100 in-house T3 and Reverse primers provided by Sequetech, mg. Each component was dissolved one at a time in diFIO. Calif. This plasmid was designated pBBRCMPGI1.5-pg| The pH was adjusted to 3.0 with HCl/NaOH, and then the (FIGS. 12, 13 A-B and SEQID NO:6). 25 solution was brought to volume and filter-sterilized with a Plasmid pPBRCMPGI1.5-pgl was electroporated into 0.22 micron filter. EWL256, as described herein and transformants were plated (a) Experimental Procedure on Lagar containing Chloramphenicol (10 ug/mL), Genta Isoprene production was analyzed by growing the strains in mycin (5 g/mL), spectinomycin (50 ug/mL), and carbenicil a CelleratorTM from MicroReactor Technologies, Inc. The lin (50 ug/mL). One transformant was selected and desig 30 working volume in each of the 24 wells was 4.5 mL. The nated Strain RM1 11608-2. temperature was maintained at 30° C., the pH setpoint was Primers: 7.0, the oxygen flow setpoint was 20 sccm and the agitation rate was 800 rpm. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with (SEQ ID NO: 29) 35 antibiotics) and incubated at 30° C. A single colony was 5'-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAA inoculated into media with antibiotics and grown overnight. CCCTCACTAAAGGGCGGCCGC-3' The bacteria were diluted into 4.5 mL of media with antibi PglGI1.5-R otics to reach an optical density of 0.05 measured at 550 nm. (SEQ ID NO: 30) Off-gas analysis of isoprene was performed using a gas s' - GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTAC 40 chromatograph-mass spectrometer (GC-MS) (Agilent) head CTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGG space assay. Sample preparation was as follows: 100 L of ATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACT CACTATAG whole broth was placed in a sealed GC vial and incubated at GGCTCGAG-3 30° C. for a fixed time of 30 minutes. Following a heat kill 3' EcoRV-pglstop: step, consisting of incubation at 70° C. for 5 minutes, the (SEQ ID NO: 31) 45 sample was loaded on the GC.. s' - CTT GAT ATC TTA. GTG TGC GTT AAC CAC CAC Optical density (OD) at a wavelength of 550 nm was pgl +49 rev: obtained using a microplate reader (Spectramax) during the (SEQ ID NO: 32) course of the run. Specific productivity was obtained by CGTGAATTTGCTGGCTCTCAG dividing the isoprene concentration (ug/L) by the ODreading 50 Bottom Pgb2: and the time (hour). (SEQ ID NO: 33) The two strains EWL256 and RM11608-2 were assessed at GGTTTAGTTCCT CACCTTGTC 200 and 400 uM IPTG induction levels. Samples were ana lyzed for isoprene production and cell growth (OD550) at 1, Top GB's CMP (946) : (SEQ ID NO: 34) 2.5, 4.75, and 8 hours post-induction. Samples were done in ACTGAAACGTTTTCATCGCTC 55 duplicate. (b) Results Pglconfirm-F The experiment demonstrated that at 2 different concen (SEO ID NO : 35) trations of IPTG the strain expressing the ybhE (pgl) had a 5'-ACCGCCAAAAGCGACTAATTTTAGCT-3' dramatic 2-3 fold increase in specific productivity ofisoprene 60 compared to the control strain. Example 2 ii) Isoprene Fermentation from E. coli Expressing Cm-GI1.2- KKDyI. M. mazei Mevalonate Kinase, P alba Isoprene Syn Improvement of Isoprene Production by Constitutive thase, and ybhE (pgl) (RM111608-2) and Grown in Fed Expression of ybhE (pgl) from a Plasmid in E. coli Batch Culture at the 15-L Scale 65 Medium Recipe (per liter fermentation medium): KHPO This example shows production of isoprene in a strain 7.5 g. MgSO47H2O2 g, citric acid monohydrate 2 g, ferric constitutively expressing E. coli ybhE (pgl) compared to a ammonium citrate 0.3 g yeast extract 0.5g, 1000x Modified US 8,455,236 B2 55 56 Trace Metal Solution 1 ml. All of the components were added 83%) was adsorbed by the carbon filters. Following the steam together and dissolved in diHO. This solution was auto desorption/condensation step, the amount of liquid isoprene claved. The pH was adjusted to 7.0 with ammonium hydrox recovered was 810 g, corresponding to an overall recovery ide (30%) and q.s. to volume. Glucose 10g, thiamine HC1 yield of 70%. The recovered isoprene was analyzed for the 0.1 g, and antibiotics were added after sterilization and pH presence of impurities. adjustment. Analysis and Impurity Profile of Isoprene Liquid 1000x Modified trace Metal Solution: Citric Acids*HO Recovered isoprene liquid was analyzed by GC/MS and 40 g, MnSOHO 30 g, NaCl 10 g, FeSO.7HO 1 g, gas chromatography/flame ionization detection (GC/FID) to CoCl*6HO 1 g, ZnSO*7HO 1 g, CuSO4.5H2O 100 mg, determine the nature and levels of impurities. The product HBO 100 mg. NaMoO2H2O 100 mg. Each component 10 was determined to be >99.5% pure and contained several was dissolved one at a time in Di HO, pH to 3.0 with HCl/ dominant impurities in addition to many minor components. NaOH, then q.s. to volume and filter sterilized with a 0.22 The GC/FID chromatogram is depicted in FIG. 17, and the micron filter typical levels of impurities are shown in Table 19. The impu Fermentation was performed in a 15-L bioreactor with rity profile was similar to other isoprene batches produced on BL21 (DE3) E. coli cells containing the upper mevalonic acid 15 this scale. (MVA) pathway (pCL Upper), the integrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonate TABLE 2 kinase from M. mazei and isoprene synthase from P alba (pTrc Alba-mMVK), and high expression of E. coli pgl Summary of the nature and levels of impurities seen in several (pBBR-pgl). This experiment was carried out to monitor iso batches of isoprene produced from renewable resources prene formation from glucose at the desired fermentation pH Retention Time (min 7.0 and temperature 34°C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract Compound GCMS GCFID Conc. Range medium. After the inoculum grew to OD 1.0, measured at 550 Ethanol 1.59 11.89 ppm nm, 500 mL was used to inoculate a 15-L bioreactor bringing 25 Acetone 1624 12.673 <100 ppm the initial volume to 5-L. Methacrolein 1851 15.369 <200 ppm Methyl vinyl ketone 1923 16.333 <2O ppm Glucose was fed at an exponential rate until cells reached Ethyl acetate 2.037 17.145 100 to 800 ppm the stationary phase. After this time the glucose feed was 3-Methyl-1,3-pentadiene 2.27 18.875 SO to SOO ppm decreased to meet metabolic demands. The total amount of Methyl vinyl oxirane 2.548 19.931 <100 ppm glucose delivered to the bioreactor during the 40 hour (59 30 Isoprenol 2.962 21.583 <500 ppm 3-methyl-1-butanol 2.99 21.783 ppm hour) fermentation was 3.1 kg (4.2 kg at 59 hour). Induction 3-hexen-1-ol 4.019 24.819 <100 ppm was achieved by adding IPTG. The IPTG concentration was Isopentenyl acetate 4.466 25.733 2OO to 1 OOO ppm brought to 110 uM when the optical density at 550 nm 3-hexen-1-yl acetate 5.339 27.223 <400 ppm (ODsso) reached a value of 4. The IPTG concentration was limonene 5.715 27.971 <500 ppm raised to 192 uM when ODsso reached 150. The ODsso profile 35 Other cyclics S.SO-6.SO 27.5-28.0 <200 ppm within the bioreactor over time is shown in FIG. 14A. The isoprene level in the off gas from the bioreactor was deter Purification of Isoprene Produced from Renewable mined using a Hiden mass spectrometer. The isoprene titer Resources by Treatment with Adsorbents increased over the course of the fermentation to a maximum Adsorbents are widely used by industry for the removal of value of 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 40 trace impurities from hydrocarbon feedstocks. Suitable 14B). The isoprene titer increased over the course of the adsorbents include Zeolite, alumina and silica-based materi fermentation to a maximum value of 40.0 g/L at 40 hours als. Isoprene produced from renewable resources can be Sub (60.5 g/L at 59 hours) (FIG. 14C). The total amount of iso stantially purified by passage over silica gel, and to a lesser prene produced during the 40-hour (59-hour) fermentation extent with alumina. FIG. 18 shows the GC/FID chromato was 281.3 g (451.0 g at 59 hours) and the time course of 45 grams of an isoprene sample before (A) and after treatment production is shown in FIG. 14D. The time course of volu with alumina (B) or silica (C). The SelexsorbTM adsorbent metric productivity is shown in FIG. 14E and shows that an products from BASF is one of the adsorbents of choice for the average rate of 1.0 g/L/hr was maintained between 0 and 40 removal of polar impurities from isoprene produced from hours (1.4 g/L/hour between 19 and 59 hour). The metabolic renewable resources. Specifically, the SelexsorbTM CD and activity profile, as measured by CER, is shown in FIG. 14F. 50 CDX products are preferred given their proven utility for The molar yield of utilized carbon that went into producing removal of polar impurities from isoprene and butadiene isoprene during fermentation was 19.6% at 40 hours (23.6% feedstocks. at 59 hours). The weight percent yield of isoprene from glu cose was 8.9% at 40 hours (10.7% at 59 hours). Example 4 55 Example 3 Construction of Strains MCM518-521 and 528-531: Lambda Promoters Driving Integrated mKKDyI Recovery of Isoprene Produced from Renewable Resources P1 transduction enables movement of up to 100kb of DNA 60 between bacterial strains (Thomasonetal. 2007). A 17,257 bp Isoprene was recovered from a set of four 14-L scale fer deletion in E. coli BL21 (DE3) (see FIG. 20) was replaced by mentations in a two-step operation involving stripping of moving a piece of the bacterial chromosome from E. coli K12 isoprene from the fermentation off-gas stream by adsorption MG1655 to E. coli BL21 (DE3) using P1 transduction. to activated carbon, followed by off-line steam desorption Two strategies were used employing different selectable and condensation to give liquid isoprene (FIGS. 16A and 65 markers to identify colonies containing the recombined bac 16B). The total amount of isoprene produced by the four terial chromosome. First, an antibiotic marker in a gene close fermentors was 1150 g (16.9 mol), of which 953 g (14 mol, to the 17,257 bp sequence to be transferred, whose deletion US 8,455,236 B2 57 58 was not likely to be detrimental to the strain, was inserted. A carbenicillin and kanamycin (i.e., which could not grow on strain containing that antibiotic marker would likely have the carb50 and kan10) were cultured in liquid LB and frozen as 17.257 bp piece of bacterial chromosome transduced at the Strains MCM528-MCM531. same time as the marker. In this case, a gene encoding kana mycin resistance (“kan”) was inserted into the ybgS gene, TABLE 3 encoding a 126 amino acid protein of unknown function. Second, since it is known that a number of genes involved in E. coli strains utilization of galactose are close topgl in the 17,257 bp piece Strain Description Parent to be transduced into E. coli BL21 (DE3), colonies transduced 10 MCMSO8 BL21 gi1.6-mKKDyI+ predet.-carb MCM446 with a P1 lysate obtained from E. coli K12 MG1655 (which MCMS.18 BL21 neo-PL.6-mKKDyI, clone 10 MCMSO8 contains the 17,257 bp sequence deleted in E. coli BL21 MCMS 19 BL21 neo-PL.O-mKKDyI, clone 11 MCMSO8 MCMS2O BL21 neo-PL.0-mKKDyI (bad RBS in front MCM508 (DE3)) and isolated in M9 medium (6 g/L NaHPO, 3 g/L of mMVK), clone 13 KHPO, 0.5g/L NaCl, 0.5 g/L NHCl, 0.1 mM CaCl2 mM MCMS21 BL21 neo-PL.2-mKKDyI, clone 15 MCMSO8 MgSO) containing 0.4% (w/v) galactose would likely con MCMS28 BL21 PL.6-mKKDyI, neo looped out MCMS18 15 MCMS29 BL21 PLO-mKKDyI, neo looped out MCMS 19 tain the 17,257 bp piece of bacterial chromosome. MCMS30 BL21 PL.0-mKKDyI (bad RBS in front MCMS2O Primers MCM120 (SEQ ID NO:36) and MCM224 (SEQ of mMVK), neo looped out ID NO:37) were used to amplify the chloramphenicol resis MCMS31 BL21 PL.2-mKKDyI, neo looped out MCMS21 tance ("Cm”) cassette from the GeneBridges FRT-gb2-Cm FRT template using the Stratagene HerculaseTM II Fusion kit (Agilent Technologies, Stratagene Products Division, La Jolla, Calif.) according to the manufacturer's protocol. Four TABLE 4 50 uL PCR reactions were cycled as follows: 95°C./2 min Primer sequences utes; 30 cycles of 95°C./20 seconds, 55° C./20 seconds, 72° Primer C./1 minute; and 72° C./3 minutes. Reactions were then 25 ale Sequence (5' -> 3') cooled to 4°C. The four reactions were pooled, loaded onto a MCM120 aaagtagc.cgaagatgacggitttgtca catggagttggcagga Qiagen PCR column according to the manufacturer's proto tgtttgattaaaagcaattaac cct cactaaaggg.cgg col and eluted with 60 uL elution buffer (“EB) at 55° C. Plasmid pRedET-carbenicillin' (GeneBridges, Heidel (SEQ ID NO: 36) 30 MCM224 taaatcttacccggcgcagaac aggataccatgtttittitt acc berg, Germany) was electroporated into E. coli BL21 (DE3) t cctttgcaccitt catggtggt cagtgcgt.cctgctgatgtgc strain MCM446 (Cm', gi1.6mKKDyI A1-3) using standard tcagt at Caccgc.ca.gtggt atttalNgtcaiacaccgc.ca.gaga. procedures. Transformants were recovered by shaking for taattitat caccgcagatggittatctg tatgttttittatatga one hour in SOC medium at 30° C. and then selected on atttaatacgact cactatagggct cq (SEQ ID NO : 37) (where N can be a, t, c, or g) LB+50 ug/mL carbenicillin (“LB/carb50) plates at 30° C. 35 overnight. A carbenicillin-resistant colony was frozen as GB-DW aaagaccgaccaag.cgacgtctga (SEQ ID NO: 38) Strain MCM508. Strain MCM508 was grown from a fresh streak in 5 mL MCM2O8 gctctgaatagtgatagagt ca (SEQ ID NO: 39) LB/carb50 at 30° C. to an ODoof-0.5. At that point, 40 mM L-arabinose was added, and the culture was incubated at 37° 40 The assemblies integrated into the chromosomes of strains C. for 1.5 hours. Cells were then harvested by centrifugation, MCM518-MCM521 include new P. promoters derived from electroporated with 3 uI of purified amplicons as described bacteriophage lambda (W) and the very beginning of the above, and then recovered in 500 uL SOC medium at 37° C. mMVK ORF, with sequences from the Gene Bridges FRT for 1.5-3 hours. Transformants were selected on LB+ 10 gb2-Cm-FRT cassette integrated upstream of the promoter/ ug/mL, kanamycin (LB/kan10) plates at 37° C. 45 mMVK assembly, as well as the remainder of the mMVK Recombination of the amplicon at the target locus was ORF followed by the rest of the lower MVA pathway integron confirmed by PCR with primers GB-DW (SEQ ID NO:38) from Strain MCM508. and MCM208 (SEQ ID NO:39). The resulting amplicons were sequenced to identify four clones having the sequences listed below. Four carbenicillin-sensitive clones were frozen 50 Promoter/mMVK sequence integrated into MCM518 as Strains MCM518-MCM521. (SEQ ID NO: 40) : Strains MCM518-MCM521 were re-streaked onto aaagaccgaccaa.gcgacgtctgaga.gctic cctggc gaatticggtacca LB/kan 10 and grown overnight at 37° C. Colonies of strains ataaaagagctittattitt catgatctgttgttgttggtttttgttgttgcggc MCM518-MCM521 were picked, cultured in LB/kan 10 at 37° C. and electrotransformed with plasmid pCP20, which 55 gcqgaagttcc tatt ct ctagaaagtataggaact tcct cqagcc citat encodes the yeast Flp recombinase, chloramphenicol and ampicillin resistance genes and confers temperature sensitive agtgagt cqt attaaatt catataaaaaacatacagataac catctg.cg replication on host cells (Cherepanov, P. P. et al., Gene 158 gtgataaattatct ctggcggtgttgacataaataccactggcggtgat (1):9-14 (1995)). Cells were recovered in 500 uL SOC actgagcacat cago aggacgcactgaccaccatgaaggtgcaaaggag medium by shaking at 30°C. for 1 hour. Transformants were 60 selected on LB/carb50 plates at 30°C. overnight. The follow gtaaaaaaacatggitat cotgttctg.cgc.cgggtaagatttacctgttc ing morning a colony from each plate was grown at 30°C. in LB/carb50 medium until visibly turbid. The culture was then ggtgaacacgc.cgtagttt atggcgaaactgcaattgcgtgtgcggtgg shifted to 37°C. for at least 3 hours. Cells were streaked from aactg.cgtaccc.gtgttcgc.gcggaact caatgactictato act attca that culture onto LB plates and grown overnight at 37° C. 65 The following day colonies were patched to LB, gagc LB/carb50 and LB/kan10. Clones that were sensitive to both US 8,455,236 B2 59 60 - Continued pTrc-P alba(MEA)-mMVK (described in Example 10 of U.S. patent application Ser. No. 12/335,071). The PCR reac Promoter/mMVK sequence integrated into MCM519 (SEQ ID NO: 41) : tion contained the following components: 1 ul pEWL244 aaagaccgacca agcgacgtctgagagctic cctggc gaatticggtacca (encoding pTrc P alba-mMVK), 5 Jul 10xPful Jltra High Fidelity buffer, 1 ul 100 mM dNTPs, 1 ul 50 uMQC EWL244 ataaaagagctittattitt catgatctgttgttgttggtttttgttgttgcggc MEAF primer (SEQ ID NO:44), 1 ul 50 uM QC EWL244 gcqgaagttcct attctictagaaagtataggaact tcc to gagcc citat MEA R primer (SEQID NO:45), 2 ul DMSO, 1 ul Pful Jltra High Fidelity polymerase (Agilent Technologies, Stratagene agtgagt cqt attaaatt catataaaaaacatacagataaccatctg.cg 10 Products Division, La Jolla, Calif.), and 39 ul diHO. The gtgataaattat ct ctggcggtgttgacct aaataccactggcggtgat PCR reaction was cycled as follows: 95°C./1 minute; and 18 cycles of 95° C./30 seconds, 55° C./1 minute, 68° C./7.3 actgagcacat cagcaggacgcactgaccaccatgaaggtgcaaaggag minutes. The reaction was then cooled to 4°C. gtaaaaaaacatggitat cotgttctg.cgc.cgggtaagatttacctgttc The PCR product was visualized by gel electrophoresis 15 using an E-gel (Invitrogen, Carlsbad, Calif.), and then treated ggtgaacacgc.cgtagttt atggcgaaactgcaattgcgtgtgcggtgg with 1 ul DpnI restriction endonuclease (Roche, South San aactg.cgtaccc.gtgttcgc.gcggaact caatgactictat cactatt ca Francisco, Calif.) for three hours at 37° C. Tenul of the PCR product were then de-salted using a microdialysis membrane gagc (MilliPore, Billerica, Mass.) and transformed into electro Promoter/mMVK sequence integrated into MCM52O competent E. coli strain MCM531 (prepared as described (SEQ ID NO: 42): above) using standard molecular biology techniques. Cells aaagaccgacca agcgacgtctgagagctic cctggc gaatticggtacca were recovered in one ml of LB medium for 1.5 hours at 30° C., plated onto LB-agar plates containing 50 ug/ml carbeni ataaaagagctittattitt catgatctgttgttgttggtttttgttgttgcggc cillin and 5 mM mevalonic acid, and then incubated overnight gcqgaagttcct attctictagaaagtataggaact tcc to gagcc citat 25 at 37° C. The next day, positive colonies (of strain DW195, see below) were selected for growth, plasmid purification agtgagt cqt attaaatt catataaaaaacatacagataaccatctg.cg (Qiagen, Valencia, Calif.), confirmed by DNA sequencing gtgataaattat ct ctggcggtgttgacct aaataccactggcggtgat (Quintara Biosciences, Berkeley, Calif.) with the primers listed below. The final plasmid, pIDW34 (FIG. 19 A; SEQID actgagcacat cagcaggacgcactgaccaccatgaaggtgcaaaggta 30 NO:7), was confirmed to carry the open reading frame that encodes the truncated version of P alba IspS. aaaaaa catggt at CCtgttctg.cgc.cgggtaagatttacctgttcggit Strain DW199 was generated by transformation of pDW34 gaac acgc.cgtagtttatggcgaaactgcaattgcgtgtgcggtggaac and pMCM82 (described in Example 10 of U.S. patent appli cation Ser. No. 12/335,071) into electrocompetent MCM531 tgcgtaccc.gtgttcgc.gcggaact caatgactictato act attcagag 35 (prepared as described above). Cells were recovered in 1 ml of LB medium for 1 hour at 37°C., plated on LBagar plates c containing 50 ug/ml spectinomycin and 50 ug/ml carbenicil Promoter/mMVK sequence integrated into MCM521 lin, and then incubated overnight at 37° C. The next day, (SEQ ID NO : 43) : antibiotic resistant colonies of strain DW199 were chosen for aaagaccgacca agcgacgtctgagagctic cctggc gaatticggtacca 40 further study. ataaaagagctittattitt catgatctgttgttgttggtttttgttgttgcggc Strain DW202 was generated by transformation of pBBRCMPGI1.5-pgl (described in example 1) into electro gcqgaagttcct attctictagaaagtataggaact tcc to gagcc citat competent DW 199 (prepared as described above). Cells were agtgagt cqt attaaatt catataaaaaacatacagataaccatctg.cg recovered in 1 ml of LB medium for 1 hour at 37°C., plated 45 on LB agar plates containing 50 ug/ml spectinomycin, 50 gtgataaattat ct ctggcggtgttgacgtaaataccactggcggtgat ug/ml carbenicillin and 5 ug/ml gentamycin, and then incu bated overnight at 37° C. The next day, antibiotic resistant actgagcacat cagcaggacgcactgaccaccatgaaggtgcaaaggag colonies of strain DW202 were chosen for further study. gtaaaaaaacatggitat cotgttctg.cgc.cgggtaagatttacctgttc 50 TABLE 5 ggtgaacacgc.cgtagttt atggcgaaactgcaattgcgtgtgcggtgg Primers aactg.cgtaccc.gtgttcgc.gcggaact caatgactictat cactatt ca Primer Name Sequence (5' -> 3') gagc 55 QC EWL244 MEA F gaggaataalaccatggaagct cqtcgttct Example 5 (SEQ ID NO: 44) QC EWL244 MEA R agaacgacgagct tccatggtttatt cotc Construction of Strains DW199 and DW202 (SEQ ID NO: 45) EL-1 OO6 gacagott at catcgactgcacg This example describes the construction of an isoprene 60 producing E. coli strain harboring the truncated version of P (SEQ ID NO: 26) alba isoprene synthase (the MEA variant) under control of the EL-1 OOO gcactgtc.tttcc.gtctgctgc PTrc promoter. (SEQ ID NO: 22) The plasmid harboring truncated P alba isoprene synthase A-rew Ctcgtacaggct caggatag (IspS) was constructed by QuikchangeTM (Agilent Technolo 65 (SEQ ID NO: 48) gies, Stratagene Products Division, La Jolla, Calif.) PCR mutagenesis from the template pEWL244 (also referred to as US 8,455,236 B2 62 TABLE 5 - continued lower T" of the primer pair. The size of the resulting PCR fragment was determined on a pre-cast 0.8% E-gel(R) (Invit Primers rogen, Carlsbad, Calif.), using DNA Molecular Weight Marker X (75-12,216 bp) (Roche Diagnostics, Mannheim, Primer Name Sequence (5' -> 3') Germany) as size marker. Successful transduction was also A.-rew-2 ttacgt.cccaacgct caact confirmed by the ability of strain CMP215 to grow on galac (SEQ ID NO: 49) tOSe. Alternatively, a lysate of E. coli MG 1655 was used to QB1493 Cttctgcaacgcatggaaat (SEO ID NO : 50) transduce strain BL21 (as described in Example 1 above). A 10 colony selected on M9 medium supplemented with 0.4% MCM2O8 gctctgaatagtgatagagt ca (w/v) galactose was named CMP258. Presence of the 17,257 (SEO ID NO. 39) bp region containing pgl was confirmed by PCR using prim MCM66 (aka c caggcaaattctgttittat cag ers galM R (SEQ ID NO:9) and galM F (SEQ ID NO:8), pTrc Reverse) (SEQ ID NO: 21) essentially as described above. Strain CMP215 was cotransformed by electroporation with plasmids pCLPtrcUpperPathway expressing mvaE and TABLE 6 Strains Strain Background Plasmid Resistance Genotype DW195 MCMS31 pDW34 Carb BL21 (Novagen) PL.2mKKDyI, pTrc-P. alba(MEA)-mMVK DW199 MCMS31 pDW34 Carb/Spec BL21 (Novagen) PL.2mKKDyI, pTrc-P. MCM82 alba (MEA)-mMVK, pCL pTrc-Upper DWO2 MCMS31 pDW34 Carb/SpeciGm BL21 (Novagen) PL.2mKKDyI, pTrc-P. MCM82 alba (MEA)-mMVK, pCL pTrc-Upper, pBBRCM pBBRCMPGI1.5-pg| PGI1.5-pg| 30 Example 6 mvaS (described in Example 8 of U.S. patent application Ser. No. 12/335,071) and pL)W34 (containing a truncated P alba Construction of E. coli BL21 Strains CMP215, isoprene synthase and M. mazei mevalonate kinase, as CMP258 and CMP234 described above). Transformants were selected on LB agar 35 plates including 50 g/ml carbenicillin+50 ug/ml spectino This example describes the construction of E. coli strains mycin. One colony was picked and named CMP234. derived from BL21 transduced with P1 phage containing E. coli MG 1655 genomic DNA and selected for recombination of a 17.257 bp piece present in MG 1655 but absent in BL21 Example 7 and BL21 (DE3). 40 AP1 lysate was made of strain JW0736, in which theybgS gene was replaced with a kanamycin resistance gene Construction of E. coli BL21 Strains CMP269 and (“Kan')(i.e., ybgS:Kan' mutation) from the Keio collec CMP312 tion (Baba et al. 2006). That lysate was used to infect strain MCM531 (described above), producing strain CMP215. The 45 This example describes the construction of E. coli strains genotype of CMP215 was confirmed by PCR using primers derived from BL21 transduced with P1 phage containing E. galMR (5'-GTCAGGCTG GAA TACTCTTCG-3'; SEQID coli MG 1655 genomic DNA and selected for recombination NO:8) and galM F (5'-GAC GCT TTC GCC AAG TCA of a 17.257 bp piece present in MG 1655 but absent in BL21 GG-3'; SEQ ID NO:9). Those primers anneal to the galM and BL21 (DE3). The marker used for selection has been gene, as shown on FIG. 20, but only produce a PCR product 50 looped out. from E. coli BL21 (DE3) chromosomal DNA having the 17,257 bp deletion. Strain CMP215 (described above) was transformed with Integration of the 17,257 bp fragment following P1 trans pCP20 (Cherepanov, P. P. et al., 1995, Gene 158(1):9-14: duction was verified by PCR with the following protocol. One Datsenko and Wanner, 2000, Proc. Nat'l Acad. Sci. USA, bacterial colony was stirred in 30 ul H2O and heated to 95°C. 55 97(12):6645) and the kan marker contained in theybgS gene for 5 minutes. The resulting solution was spun down and 2 ul was looped out according to a previously described procedure of the supernatant used as template in the following PCR (Datsenko and Wanner, Proc. Nat'l Acad. Sci. USA, 97(12): reaction: 2 ul colony in H2O, 5ul Herculase(R) Buffer, 1 Jul 100 6645 (2000)). Marker loopout was verified by PCR as mM dNTPs, 1 Jul 10 uM Forward primer, 1 ul 10 uM Reverse described above, but usingybgSAmp F primer (5'-CCTGGA primer, 0.5 ul of Herculase(R) Enhanced DNA Polymerase 60 ATT AGC AAG AAA AAC GC-3'; SEQ ID NO:52) and (Agilent Technologies, Stratagene Products Division, La ybgSAmp R primer (5'-GTG AAA ATT GCA CGG CGA Jolla, Calif.), and 39.5 ul diHO. The PCR reaction was GTA GG-3'; SEQ ID NO:53). That strain was designated cycled in a PCR Express Thermal Cycler (Thermo Hybaid, CMP269. Strain CMP269 was cotransformed by electropo Franklin, Mass.) as follows: 95°C./2 minutes; 30 cycles of ration with plasmids pCLPtrcUpperPathway (expressing 95°C/30 seconds, 52°C/30 seconds, 72°C./60 seconds; and 65 mvaE and mvaS) and plW34 (see FIG. 19A) containing a 72°C./7 minutes. The reaction was then cooled to 4°C. The truncated P alba IspS and M. mazei MVK to produce strain annealing temperature of 52° C. was 3° C. lower than the CMP3.12. US 8,455,236 B2 63 64 Example 8 TABLE 7-continued Construction of E. coli BL21 Strains CMP296, Description of strains CMP315 and CMP323 Strain Description Parent CMP258 BL21 tpg| BL21 This example describes the construction of strains derived (Nowagen) from E. coli BL21 transduced with P1 phage containing E. CMP234 BL21 PL.2-mKKDyI tybgS::Kan, CMP215 coli MG 1655 genomic DNA and selected for recombination pCLPtrcUpperPathway, plDW34 of a 17.257 bp piece present in MG 1655 but deleted in BL21 CMP269 BL21 PL2-mKKDyI tybgSML CMP215 10 CMP296 BL21 PL2-mKKDyI tybgSML, pRedETAmp CMP269 and BL21 (DE3), thereby restoring a functional copy of pgl to CMP312 BL21 PL.2-mKKDyI tybgSML, CMP269 the E. coli BL21 and BL21 (DE3) derived strains. A strain in pCLPtrcUpperPathway, plDW34 which the restored pgl gene has been precisely knocked out CMP315 BL21 PL.2-mKKDyI tybgSML rpg|ML CMP296 by inserting a kanamycin cassette which was Subsequently CMP323 BL21 PL.2-mKKDyl tybgSML rpg| ML, CMP315 looped out was also constructed. pCLPtrcUpperPathway, plDW34 A PCR product containing a copy of pgl/ybhE in which a 15 kan' gene has been inserted (pgl/ybhE::kan') was amplified References cited: Aon et al., 2008, “Suppressing posttrans from E. coli strain JW0750 from the Keio collection using the lational gluconoylation of heterologous proteins by meta primer pair pg|Ampf (5'-Cagcaaatagcaggtgtatccagc-3'; SEQ bolic engineering of Escherichia coli, 'Appl. Environ. Micro ID NO:54) and pg|AmpR (5'-GCA ACC GAC TGT TGA biol. 74:950-958; Baba et al., 2006, “Construction of TAG AAC AAC-3'; SEQ ID NO:55). That primer pair pro Escherichia coli K-12 in-frame, single-gene knockout duces a fragment containing pg/ybhE::kan' plus ~350 bp of mutants: the Keio collection.” Mol. Syst. Biol. 2: 2006.0008: flanking sequence from each side of the mutation. PCR tem Cherepanov, P. P. et al., 1995, “Gene disruption in Escheri plate was prepared as follows: one colony of E. coli JW0750 chia coli: TcP and KmR cassettes with the option of Flp carrying pgl/ybhE::kan“ was stirred in 30 ul H2O and heated catalyzed excision of the antibiotic-resistance determinant.” to 95°C. for 5 minutes. The resulting solution was spun down 25 and 2 ul of the supernatant was used as the template in a PCR Gene 158(1): 9-14: Datsenko, K., and Wanner, B., 2000, reaction performed as follows: 2 ul colony in HO, 5ul Pfu “One-step inactivation of chromosomal genes in Escherichia Ultra II Buffer, 1 ul 100 mM dNTPs, 1 ul 10 uM Forward coli K-12 using PCR products, Proc. Nat. Acad. Sci. USA primer, 1 Jul 10 uM Reverse primer, 1 ul of Pfu Ultra II 97:6640-6645; Neidhart, F., Ingraham, J., and Schaechter, polymerase (Agilent Technologies, Stratagene Products 30 M., 1990, Physiology of the bacterial cell: a molecular Division, La Jolla, Calif.), and 39 ul H2O. The PCR reaction approach (Sinauer Associates, Inc. Sunderland, Mass.); Tho was cycled in a PCR Express Thermal Cycler (Thermo mason, L., Court, D., Datta, A., Khanna, R. and Rosner, J., Hybaid, Franklin, Mass.) as follows: 95°C./2 minutes; 30 2004, “Identification of the Escherichia coli K-12 ybhE gene cycles of 95°C./20 seconds, 53.4° C./20 seconds, 72° C./40 as pgl, encoding 6-phosphogluconolactonase. J. Bact. 186: seconds; 72°C./3 minutes. The reactions were then cooled to 35 8248-8253; Thomason, L., Costantino, N., Court, D., 2007, 40 C. “E. coli genome manipulation by P1 transduction. Curr. The size of the resulting PCR fragments was determined on Protocols Mol. Biol. Chapter 1, Unit 1.17; Studier F., Dae a pre-cast 0.8% E-gel(R) (Invitrogen, Carlsbad, Calif.), using gelen, P. Lenski, R., Maslov, S., Kim, J. F., 2009, “Under DNA Molecular Weight Marker X (75-12,216 bp)(Roche standing the differences between genome sequences of Diagnostics, Mannheim, Germany) as size marker. The PCR 40 Escherichia coli B strains REL606 and BL21 (DE3) and com reaction was purified using the QIAquick R. PCR Purification parison of the E. coli B and K-12 genomes. J. Mol. Biol. Kit (Qiagen, La Jolla, Calif.). 394(4):653-80, 2009). Plasmid pRedETAmp (GeneBridges Gmbh, Heidelberg, Germany) was electroporated into CMP269 to form Example 9 CMP296. CMP296 was grown and induced with L-arabinose 45 according to the manufacturers instructions (GeneBridges) Isoprene Production in a BL21 Strain Transduced and transformed with the pgl/ybhE::kan“ PCR product with the 17,257 bp Chromosomal Fragment described in this example. Transformants were selected on Encoding pgl LB agar including 20 ppm kanamycin. One colony was picked, its genotype checked by PCR with Herculase(R) poly 50 This example demonstrates that high specific productivity merase using pg|Ampf (5'-cagcaaatagcaggtgtaticcagc-3', of isoprene in 4.5-mL batch mini-fermentations by E. coli SEQ ID NO:54) and pg|RecCheck (5'-GGTTACAAAATG harboring the mevalonic acid pathway requires the restora ATT GGC GTA CGC-3'; SEQ ID NO:56) and named tion of pgl to the bacterial chromosome. CMP298. The marker was removed as described above in Medium Recipe (per liter fermentation medium): 13.6 g. Example 2 to form strain CMP315. Plasmids pCLPtrcUpper 55 KHPO, 13.6 g. KHPO, 2 g citric acid monohydrate, 0.3g Pathway and plW34 (see Example 1) were introduced in ferric ammonium citrate, 3.2 g (NH)SO 1 ml 1000x Trace Metals Solution were added together and dissolved in diHO. CMP315 as described above in Examples 4-5 to form strain The pH was adjusted to 6.8 with 28% (w/v) ammonium CMP323. hydroxide and brought up to final volume. The medium was 60 filter-sterilized with a 0.22 micron filter. Glucose (10 g for TABLE 7 overnight culture and 5.0 g for main culture) and appropriate Description of strains antibiotics were added after sterilization and pH adjustment, followed by 1 g of yeast extract from a 100 g/L stock solution Strain Description Parent and 1 g of MgSO from a 1 M MgSO solution. MCMS31 BL21 PL.2-mKKDyI 65 1000x Trace Metal Solution (per liter fermentation CMP215 BL21 PL.2-mKKDyI tybgS::Kan MCMS31 medium): 40 g Citric Acid HO, 30 g MnSOHO, 10 g NaCl, 1 g FeSO.7H2O, 1 g CoCl*6HO, 1 g ZnSO*7H2O,