US009353375B2
(12) United States Patent (10) Patent No.: US 9,353,375 B2 Beck et al. (45) Date of Patent: *May 31, 2016
(54) COMPOSITIONS AND METHODS OF PGL 2009,0203102 A1 8, 2009 Cervin et al. FOR THE INCREASED PRODUCTION OF 2010.0003716 A1 1/2010 Cervin et al. 2010.0048964 A1 2/2010 Calabria et al. ISOPRENE 2010, OO86978 A1 4/2010 Becket al. 2010.0113846 A1 5, 2010 McAuliffe et al. (71) Applicants: Danisco US Inc., Palo Alto, CA (US); 2010.016737O A1 7/2010 Chotani et al. The Goodyear Tire & Rubber 2010.01673.71 A1 7/2010 Chotani et al. Company, Akron, OH (US) 2010, O184178 A1 7/2010 Becket al. 2010.0196977 A1 8, 2010 Chotani et al. (72) Inventors: Zachary Q. Beck, Palo Alto, CA (US); Marguerite A. Cervin, Redwood City, FOREIGN PATENT DOCUMENTS CA (US); Alex T. Nielsen, Kokkedal EP 1510 583 A1 3, 2005 (DK); Caroline M. Peres, Palo Alto, CA SG 181828 A1 8, 2012 (US) WO WO-96,35796 A1 11, 1996 WO WO-98.02550 A2 1, 1998 WO WO-98.02550 A3 1, 1998 (73) Assignees: DANISCO US INC., Palo Alto, CA WO WO-2004/003175 A2 1, 2004 (US): THE GOODYEARTIRE & WO WO-2004/003175 A3 1, 2004 RUBBER COMPANY, Akron, OH WO WO-2004/033646 A2 4/2004 (US) WO WO-2004/033646 A3 4/2004 WO WO-2005/080583 A2 9, 2005 WO WO-2005/080583 A3 9, 2005 (*) Notice: Subject to any disclaimer, the term of this WO WO-2009,076676 A2 6, 2009 patent is extended or adjusted under 35 WO WO-2009,076676 A3 6, 2009 U.S.C. 154(b) by 0 days. WO WO-2009,132220 A9 10, 2009 WO WO-2010/OO3OO7 A2 1, 2010 This patent is Subject to a terminal dis WO WO-2010/OO3OO7 A3 1, 2010 claimer. WO WO-2010/O14825 A1 2, 2010 WO WO-2010/078457 A2 T 2010 (21) Appl. No.: 14/509,000 WO WO-2010/078457 A3 T 2010 WO WO-2010, 124146 A2 10, 2010 WO WO-2010, 124146 A3 10, 2010 (22) Filed: Oct. 7, 2014 WO WO-2010, 148144 A1 12, 2010 (65) Prior Publication Data WO WO-2011 O75748 A1 6, 2011 US 2015/O104853 A1 Apr. 16, 2015 OTHER PUBLICATIONS Anderson, M.S. et al., (1989). “Isopentenyl Diphosphate: Dimethlal Related U.S. Application Data lyl Diphosphate Isomerare. An Improved Purification of the Enzyme (62) Division of application No. 13/888,248, filed on May andn Isolation of the Gen From Saccharomyces cerevisia.” J. Biol. 6, 2013, now Pat. No. 8,889,387, which is a division of Chem. 264(32): 19169-19175. Aon, J. et al., (2008) "Suppressing Posttranslational Gluconoylation application No. 12/978.324, filed on Dec. 23, 2010, of Heterologous Proteins by Metabolic Engineering of Escherichia now Pat. No. 8,455,236. coli.” Appl. Environ. Microbiol. 74:950-958. (60) Provisional application No. 61/289.959, filed on Dec. Baba, T. et al., (2006) “Construction of Escherichia coli K-12 In 23, 2009. Frame, Single-Gene Knockout Mutants: The Keio Collection.” Mol. Syst. Biol. 2: 2006.0008. (51) Int. Cl. Balbas et al. (Jun. 12, 1996). Gene 172(1):65-69. CI2N 15/52 (2006.01) Bouvier, F. et al., (2005) “Biogenesis, Moleculars Regulation and Function of Plant Isoprenoids.” Progress in Lipid Res. 44:357-429. CI2N 15/63 (2006.01) Campbell, E. et al., (1989) “Improved Transformation Efficiency of CI2N 15/70 (2006.01) Aspregillus Niger Using Homologous niaD Gene for Nitrate CI2P 5/00 (2006.01) Reductase. Curr. Genet. 16:53-56. CI2P 7/42 (2006.01) (52) U.S. Cl. (Continued) CPC ...... CI2N 15/52 (2013.01); C12N 15/635 (2013.01); C12N 15/70 (2013.01); C12P5/007 Primary Examiner — Christian Fronda (2013.01); CI2P 7/42 (2013.01) (74) Attorney, Agent, or Firm — Mintz, Levin, Cohn, Ferris, (58) Field of Classification Search Glovsky Popeo, P.C. None See application file for complete search history. (57) ABSTRACT (56) References Cited Provided herein are improved compositions and methods for the increased production of isoprene. Also provided herein U.S. PATENT DOCUMENTS are improved compositions and methods for the increased 7,132,527 B2 1 1/2006 Payne et al. production of heterologous polypeptides capable of biologi 8,455,236 B2* 6/2013 Beck ...... C12N 15,52 cal activity. 435/146 8,889,387 B2 11/2014 Becket al. 2008.0038805 A1 2/2008 Melis 14 Claims, 66 Drawing Sheets US 9,353,375 B2 Page 2
(56) References Cited Oulmouden, A. et al., (1991). "Nucleotide Sequence of the ERG 12 Gene of Saccharomyces cerevisiae EncodinO Mevalonate Kinase.” OTHER PUBLICATIONS Curr Genet. 19:9-14. Rohdich, F. et al., (1999). "Cytidine 5'-Triphosphate-Dependent Cherepanov, P.P. et al., (1995)“Gene Disruption in Escherichia coli: Biosynthesis of Isoprenoids:YgbP Protein of Escherichia coli Cata TcRand KmR Cassettes with the Option of FIp-Catalyzed Excision lyzes the Formation of 4-Diphosphocytidyl-D-2-C- of the Antibiotic-Resistance Determinant.” Gene 158(1):9-14. Methylerythritol.” PNAS 96(21): 11758-11763. Chica, R.A. et al. (Aug. 2005). "Semi-Rational Approaches to Engi Rohdich, F. et al., (2000). "Biosynthesis of Terpenoids: neering Enzyme Activity: Combining the Benefits of Directed Evo 4-Diphosphocytidly-2C-Methyl-o-Erythritol Synthase of lution and Rational Design.” Curr: Opin. Biotechnol. 16(4):378-384. Arabidopsis Thaliana.” PNAS97(12):6451-6456. Datsenko, K., et al., (2000) "One-Step Inactivation of Chromosomal Self, W.T. et al., (2001). “Molybdate Transport.” Res Microbiol. Genes in Escherichia coli K-12 Using PCR Products.” Proc. Nat. 152:311-321. Acad. Sci. USA 97:6640-6645. Sasaki et al. (Apr. 25, 2005). FEBS Lett. 579(11):2514-2518. Dhe-Paganon, S. et al., (1994). “Mechanism of Mevalonate Sen, S. et al. (Dec. 2007). “Developments in Directed Evolution for Pyrophosphate Decarboxylase: Evidence for a Carbocationic Tran Improving Enzyme Functions.” Appl. Biochem. Biotechnol. sition State.” Biochemistry 33(45): 13355-13362. 143(3):212-223. EcoGene Accession No. EG13231, located at FG. A. A PAY OX PATAY 1's. 2. -sca Acetyl-CoA Acetyl-CoA ro-o is ---. O Glyceraldehyde Pyruvate 3-phosphate ---.. Acetoacetyl-CoA s (s A:st-ce. -. 38 stro--o{}i Six Exp bap X af hio, HO, i 3 s. o--o f se ... . . fig, e. it. As 3. Sg Af ises -ko,-- c. o-i-o--o- 3. 8 - s He, p. se 3. Hoor co : K. tewaterate se a- iy s s 1 o i s cats. ics c.g. e. C.Sr. c. 2.so ote:* as Mewaterate phosphate - i. 8-pe f : o-i-o--o' HC, 8. l s: * Hooc Cho--0--0- 4- . ls- is ap AOP p, s C Mevalorate diphosphate pp. p. U.S. Patent May 31, 2016 Sheet 2 of 66 US 9,353,375 B2 F.G. B. 2 CH3CO-S-CoA acetyl-CoA ' O O. H3C-C-CHg-ScoA acetoacetyl-CoA 2 CH3CO-S-CoA CO-S-CoA CH2 HC-C-OH G-CoA 866HCH 3. CHOH CH HCCOH evatoric acid 866HCH 4. A. CHO-P CH2 Hogg 5-phosphomevaionic acid 866H 548 co, NSTP CHO-P CHO-PP isopentenyl-P HacgCH2 Hscg3CH2 appsphomevalonic ATP s 666H 9 - CO2 CHO-PP H She isopentenyl-PP ÖH, 7. CHO-PPCH dimethylallyl-PPs HCC CH3 U.S. Patent May 31, 2016 Sheet 3 of 66 US 9,353,375 B2 FG, 2. terminator f1 origin P. a a GS kan sequence s * s * pET24 P alba HGS 6957 bp T7 promoter lac operator N-CoIE1 per322 origin U.S. Patent May 31, 2016 Sheet 6 of 66 US 9,353,375 B2 F.G. 4. ac trc promoter RBS /minicistron ORF Nicol site acic pTrchis2B 4404 bp. Amp resistance pBR322 ori U.S. Patent May 31, 2016 Sheet 7 of 66 US 9,353,375 B2 F.G. 5, lac operator re-2. Ptric promoter P.alba HGS EWL230 (pTrc P.alba) 6068 bp - pBR322 rrn terminator U.S. Patent May 31, 2016 Sheet 10 of 66 US 9,353,375 B2 FG. I. ac operator Ptric promoter Para S. Pst site EWL230 (pTrc P.alba) ... CTGCA 6068 bp ...G ^ Nsil Site Pine T. pBR322 Pst (aioi) ACGTA. M. mazei MVK Pne (2203) rr terrirator ba U.S. Patent May 31, 2016 Sheet 11 of 66 US 9,353,375 B2 F.G. 8. lac operator lac s ra| Ptrc promoter P.alba HGS / EWL244 (pTrc P.alba-mMVK) 6906 bp pBR322 s RBS M. mazei MVK ORF rrn terminator U.S. Patent May 31, 2016 Sheet 14 of 66 US 9,353,375 B2 U.S. Patent May 31, 2016 Sheet 17 of 66 US 9,353,375 B2 FG. A. MVKNP 633786 EK recognition site T7 reverse primer Xpress epitope T7 transcription terminator Xpress forward prime 6xisNWY 2Y- bla promoter initiation ATG Kan(R) RBS T7 promote lac operatof T7 primer pBR322 origin lac MCM376 - MMK from M. 22e andheal LowerinpET200D 7 U.S. Patent May 31, 2016 Sheet 20 of 66 US 9,353,375 B2 FG, 2. RBS ATG M3 Reverse pBBRCMPG|1.5pg| 7519 bp CDS 1 CMP-G1.5-pg| lacz alpha M13 Forward CDS2: Gentamycin resistance gene; CDS1: E. coli replication protein U.S. Patent May 31, 2016 Sheet 23 of 66 US 9,353,375 B2 F.G. 4A. 1 OOO 1 O O ---- 1O 1 wro ro ro rr »;«& roorgorov8 O 5 10 15 20 25 3O 35 4O 45 5O 55 6.O 65 Time (hr) U.S. Patent May 31, 2016 Sheet 24 of 66 US 9,353,375 B2 F.G. 4B. 5 O 4. O 23 OO 1 O O O 5 1 O 15 20 25 3O 35 A.O 45 5O 55 6.O 65 Time (hr) F.G. 4C. O 5 O 15 20 25 3O 35 4 O 45 5O 55 6O 65 Time (hr) U.S. Patent May 31, 2016 Sheet 25 of 66 US 9,353,375 B2 FG, 4D, 500 cc 45O 4OO - 350 - Sts 3OO - 25O 200 150 - 1 OO 50 - O O 5 10 15 20 25 30 35 40 45 50 55 6.O 65 Time (hr) FG, 4E. 2.3. 5O 2. O Average = 1.4 g/L hr between 19 and 59 hrs 1 5 r 1 O Average = 1.0 g/Lhr between and 40 hrs O .5 O. O O 5 1 O 15 20 25 3O 35 4 O 45 5O 55 6.O 65 Time (hr) U.S. Patent May 31, 2016 Sheet 26 of 66 US 9,353,375 B2 FG, 4F. s s O 5 1 O 15 20 25 30 35 40 45 50 55 6O 65 Time (hr) U.S. Patent May 31, 2016 Sheet 27 of 66 US 9,353,375 B2 F.G. 5, SEM : torr Faraday: torr A RC RGA 20 2854 1.6e-08 g 1.4e-08 - s t 1.2e-08 . . . . i . s - - - -- I - , , i OOOD O2: 4: 5:OO 8:00 10:00 2:00 14:00 16:00 Time miss .' Hydrogen / CO2 M Oxygen isoprene U.S. Patent May 31, 2016 Sheet 28 of 66 US 9,353,375 B2 F.G. 6A. Primary Secondary carbon filter carbor filter Off-gas out Fo eer s t Dehumidifier toCooled sate ws reservoir Off-gas in Sample point B U.S. Patent May 31, 2016 Sheet 29 of 66 US 9,353,375 B2 Figure 16B. Steam in Water-cooled condenser Glycol-cooled Cold trap condenser (-78°C) (40°C) 1 (0°C) Carbon trap NS/ Gas out Carbon filter Stearn isoprene condenser collection U.S. Patent May 31, 2016 Sheet 31 of 66 US 9,353,375 B2 Abundance FG, 8A, 28 2. 24. 22 E. 2O s 2 Tie-X 8 O. 2 . S. 8, 2, 2 A. S. F.G. SB, s s 2. Inex ...... 8. 2, 2, 2, 2. F.G. S.C. re- in 20 in so go on 22 on 25 U.S. Patent May 31, 2016 Sheet 32 of 66 US 9,353,375 B2 FIG. 19A. Map of plasmid pDW34 lac operator ?Ptric promoter f MCM65 ax Ptric, complete sigp10 RBS --- reinitiation RBS Truncated Paiba HGS (MEA) pDW34 (MEA Truncation in pirc P. 6858 bp p3R322 M, mazei VK ORF rrn terminator U.S. Patent May 31, 2016 Sheet 36 of 66 US 9,353,375 B2 000'26) 000222000'922 £100: U.S. Patent May 31, 2016 Sheet 37 of 66 US 9,353,375 B2 F.G. 21. Time (Hrs) -- C32 OC -e- C323 O. CN A Sample Point U.S. Patent May 31, 2016 Sheet 38 of 66 US 9,353,375 B2 F.G. 22. Specific Productivity 2 2 s s Time (Hrs) -- CMP312 S.Prod (ug/L/ODIh) -e-CMP323 S. Prod (ug/LOD/h) A Sample Point U.S. Patent May 31, 2016 Sheet 39 of 66 US 9,353,375 B2 F.G. 23. 8 : O 5 1O 15 20 25 30 35 4O 45 50 Time (hr) U.S. Patent May 31, 2016 Sheet 40 of 66 US 9,353,375 B2 F.G. 24. O 5 1O 15 20 25 30 35 40 45 50 Time (hr) --DW 199 -o- CMP312 -A-CMP323 U.S. Patent May 31, 2016 Sheet 41 of 66 US 9,353,375 B2 FG. 25. 700 - 6OO - 34.5 OOO OOO 2 O O 1 OO - O 5 1O 15 20 25 30 35 4O 45 50 Time (hr) --DW 199 -o- CMP312 -a-CMP323 U.S. Patent May 31, 2016 Sheet 42 of 66 US 9,353,375 B2 F.G. 26. 1223 5O5O 10 O 5 10 15 20 25 30 35 40 45 50 Time (hr) -Dw199 -e-CMP312 - CMP323 U.S. Patent May 31, 2016 Sheet 43 of 66 US 9,353,375 B2 F.G. 27. 180 - 16O - 140 – 12O - 1 OO - 2 O O Time (hr) - - pg -O- pg|- U.S. Patent May 31, 2016 Sheet 44 of 66 US 9,353,375 B2 FG, 28. : O 5 10 15 2O 25 Time (hr) U.S. Patent May 31, 2016 Sheet 45 of 66 US 9,353,375 B2 FG, 29. 10 O 5 1O 15 2O 25 Time (hr) -O- pg -O- pg U.S. Patent May 31, 2016 Sheet 46 of 66 US 9,353,375 B2 FG, 30, 6 53 100 s 10 S. O 1 & & O 5 1O 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent May 31, 2016 Sheet 47 of 66 US 9,353,375 B2 F.G. 3, 3 5 3 O 2 5 2 O 1 5 1O O 5 1O 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent May 31, 2016 Sheet 48 of 66 US 9,353,375 B2 F.G. 32. O 5 1O 15 20 25 30 35 40 45 50 Time (hr) U.S. Patent May 31, 2016 Sheet 49 of 66 US 9,353,375 B2 FG, 33. 1 O 8 6 4 O T T 8 : 8. O 5 1O 15 20 25 3O 35 4O 45 50 Time (hr) U.S. Patent May 31, 2016 Sheet 50 of 66 US 9,353,375 B2 FIG. 34A. Map of plasmid pDW15. Aacc1 (Gent Resistance) 3. M13 Reverse / RBS? 1 ATG. Laczalph Pro :* mvaE M13 Forw ard U.S. Patent May 31, 2016 Sheet 54 of 66 US 9,353,375 B2 FG. 35. Specific Productivity 16 3. 8. S --- 8 . 8 21 + po 8.2 pg. + pC B 21 + ps3R B21 pg. + p88R BL2+ proupper 821 pg - pTrcupper ProJpper Procupper Pircupper Prepper U.S. Patent May 31, 2016 Sheet 55 of 66 US 9,353,375 B2 F.G. 36. O 1 OO 2OO 3OO 400 Time (min) --MCM872BL21 ptrc-upper -E-MCM876BL21 pg. pirc-upper U.S. Patent May 31, 2016 Sheet 56 of 66 US 9,353,375 B2 F.G. 37. xssex O.05 - O.04 BL21 pro-upper BL21 pg pro-upper U.S. Patent May 31, 2016 Sheet 57 of 66 US 9,353,375 B2 F.G. 38. 6.OE-06 5.OE-06 - 3.OE-06 - B21 pro-upporc-upper E BL21 pg pro-upper OE-06 - O.OE--OO &XSS & Time point 1 Time point 2 U.S. Patent May 31, 2016 Sheet 58 of 66 US 9,353,375 B2 F.G. 39. 7.OE-06 - 6.OE-06 --- A.OE-O6 - |OBL21 -pTrc-upper - BL21 pg pTrc-upper OE-06 O.OE--OO - Time point 1 Time point 2 U.S. Patent May 31, 2016 Sheet 60 of 66 US 9,353,375 B2 FIG. 40B. Amino acid sequence of PGL from P. aeruginosa (SEQ ID NO:12). MAISELKLPAGVGLQVWGSAAEQARGLAAEVAGRLRSALAEQGQALLVVSGGRSPVA FLEALSEEPLDWSRITVSLADERWVPESHADSNAGLVRRHLLRGEAAKARFIGLYQPAA SLEEAAELADHHLHELPLPIDVLVLGMGDDGHTASLFPNSPGLDLAMDPQGTRRCLPM WAPSVPHQRLTLPRAVLAAAKVOLLAIQGQSKLATLNAALAVEDERRMPVRAFLRAPL THWYP U.S. Patent May 31, 2016 Sheet 62 of 66 US 9,353,375 B2 FG, 4. l 5 O Ps aeruginosa pgll (1) -MAISEKLEAGVGCVWGSAAEOARGLAAEV------Ecoli MG1655 pgil (1) MKOTVY ASEESQCHVNNLNHECALTITOVVDVPCOVOPMVVSPDKRYL CC Sen SS (1) VW E A V 5. OO Ps aeruginosa pgll (32) ------AGRILRSALAEOGOALLWVSGGRSPVAEA Ecoli MG 1655 pgll (5) YWGVRPEFRVLAYRIAPDDGALTFAAESALPSPTHISDHQGOF VEVGS CCS enSUS (51) A. A. A. G S F. A 1 O1 15 O Ps aeruginosa pgll (62) LSEEP DWSRET----VSADERWVPESHADSNAGLVRRHILRGEAAKAR Ecoli MG1655 pgill (IOI) YNAGNSSVREDCLPSIGWVVVEGIGCHSAS ISPDNRTIWWPALKOD. CCS enSUS (101) SR W D D AN R R. 15 2OO Ps aeruginosa pgl (108) FIGLYOPAASLEEAAELADHHLHELPLEID GAGDSTASFNS Ecoli MC1655 pgi (151) ICLFTVSDDCHLVACPAEVTTVECACPRHRFHPNEQYAYCWNELNSSV CC Sen SS (151) A AD E. . A. S 2O1 25 O Ps aeruginosa pol (158) GDLAMCEOGTRREPMWAPSVPHORLILPRAVIAAAKVOLLAICGOSKI Ecoli MG1655 pgill (201) DSWELKIHGNIECSCTLDMMPENFSDTRWAADHITPDGRHLYACDRTA CCS enSUS (201) T. G C T A 251 3 O O Ps aeruginosa pgll (208) ATNAALAVE DERRPVRAFLRAPLTIHWYP------Ecoli MG1655. pg| (251) STITVFSVSEDGSVSKEGEOPTETOPRGENVDHSGKYLIAAGOKSHHIS CCS enSIS (251) A. I ED T. AF F 3 O 33 Ps aeruginosa pgl (239) ------ECOlli MG1655. pg| (301) VYEIWGEOGLTLHEKGRYAVGQGPMWVVVNAH CCS enSS (301) Identity 9.7%; Similarity 17.8% Vector Nti Align X E. coli MG 1655 pgl Pseudomonas aeruginosa pg| U.S. Patent May 31, 2016 Sheet 63 of 66 US 9,353,375 B2 F.G. 41A. O s EFT (h) U.S. Patent May 31, 2016 Sheet 64 of 66 US 9,353,375 B2 F.G. 4B. U.S. Patent May 31, 2016 Sheet 66 of 66 US 9,353,375 B2 F.G. 42B 8,O 6. so CMP234. OO 2. 3O.O aO.O SOO 6O.O 7OO Tire US 9,353,375 B2 1. 2 COMPOSITIONS AND METHODS OF PGL to unprecedented levels. Accordingly, there is a need to pro FOR THE INCREASED PRODUCTION OF cure a source ofisoprene from a low cost, renewable source ISOPRENE which is environmentally friendly. The improved methods and compositions described herein provide Such a source of CROSS-REFERENCE TO RELATED isoprene, capable of being derived at low cost and from APPLICATIONS renewable sources. Several recent advancements have been made in the pro This application is a divisional of U.S. patent application duction of isoprene from renewable sources (see, for Ser. No. 13/888,248 (U.S. Pat. No. 8,889,387), filed May 6, example, International Patent Application Publication No. 2013, which is a divisional of U.S. patent application Ser. No. 10 WO 2009/076676 A2). Such methods produce isoprene at 12/978,324 (U.S. Pat. No. 8,455,236), filed Dec. 23, 2010, rates, titers, and purity that may be sufficient to meet the which claims priority to U.S. Provisional Patent Application demands of a robust commercial process, however process No. 61/289.959, filed Dec. 23, 2009, the disclosures of which improvements to reduce the operational costs associated with are herein incorporated by reference in their entirety. the production of isoprene derived from biological sources 15 and to increase yields of isoprene are needed. Such as the INCORPORATION BY REFERENCE improved compositions and methods for the increased pro duction of isoprene and other heterologous polypeptides The Sequence Listing submitted in an ASCII text file, in capable of biological activity provided herein. accordance with 37 C.F.R.S1.821(c) and (e), is incorporated All patents, patent applications, documents, nucleotide and herein by reference. The text file name is protein sequence database accession numbers and articles 643842002211 SeqList.txt, the date of creation of the text file cited herein are incorporated herein by reference in their is Oct. 1, 2014, and the size of the ASCII text file in bytes is entirety. 83 KB. SUMMARY FIELD OF THE INVENTION 25 Disclosed herein are improved compositions and methods This disclosure relates to improved compositions and 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. 30 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, 35 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 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 40 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 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 in structure and properties to contain nucleic acid(s) encoding a PGL polypeptide, and natural rubber. Even though it is not identical to natural rub 45 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 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 50 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 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 55 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. 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 60 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 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 65 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 tose metabolism polypeptide is selected from the group con US 9,353,375 B2 3 4 sisting of galM, galK, galT and galE. In any of the aspects In other aspects, provided herein are cells of an Escherichia herein, nucleic acids encoding the PGL polypeptide, galac coli strain that does not encode a PGL polypeptide, wherein tose metabolism polypeptide, and molybdenum uptake the E. coli cells comprise one or more copies of a heterolo polypeptide are parta 17.257 base pair piece as shown in FIG. gous gene encoding a PGL polypeptide with one or more 20. In any of the aspects herein, the recombinant cell produces associated expression control sequences and a nucleic acid isoprene at a higher specific productivity than that of the same encoding a heterologous polypeptide capable of biological cells that do not contain (a) and (b). activity, and wherein the cells produce the heterologous In any of the aspects herein, the recombinant cell has a polypeptide capable of biological activity at a specific pro specific productivity of at least 15 mg/OD/hr. In any of the ductivity greater than that of the same cells lacking one or 10 more copies of a heterologous gene encoding a PGL polypep aspects herein, the nucleic acids encoding PGL polypeptide, tide with one or more associated expression control molybdenum uptake polypeptide, and/or galactose metabo sequences, when the cells are cultured in minimal medium. In lism polypeptide are from E. coli strain K12 MG 1655 or a Some aspects, the one or more copies of the heterologous gene 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 15 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 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 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 25 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 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. 30 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 35 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, 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 40 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 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 45 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 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. 50 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 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 55 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 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. 60 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) 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. 65 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 US 9,353,375 B2 5 6 cells are cultured in minimal medium. In some aspects, the In some aspects, the isoprene synthase polypeptide is a one or more copies of the heterologous gene encoding a PGL naturally-occurring polypeptide from the genus Pueraria. In polypeptide with one or more associated expression control Some aspects, the isoprene synthase polypeptide is a natu sequences is/are chromosomal copies (e.g., integrated into rally-occurring polypeptide from Pueraria montana. In some the E. coli chromosome). In some aspects, the E. coli cells are aspects, the isoprene synthase polypeptide is a naturally in culture. In some aspects, the cells further comprise a het occurring polypeptide from the genus Populus. In some erologous nucleic acid encoding an MVA pathway polypep aspects, the isoprene synthase polypeptide is a naturally tide. In some aspects, the MVA pathway polypeptide is an occurring polypeptide from Populus alba. upper MVA pathway polypeptide. In some aspects, the MVA In Some aspects, the cells comprise (i) an integrated nucleic pathway polypeptide is a lower MVA pathway polypeptide. 10 acid encoding the lower MVA pathway from S. cerevisiae In some aspects, the upper MVA pathway polypeptide is comprising a glucose isomerase promoter and a nucleic acid selected from the group consisting of: (i) an acetoacetyl encoding mevalonate kinase (MVK); a nucleic acid encoding Coenzyme A synthase (thiolase) polypeptide; (ii) a 3-hy phosphomevalonate kinase (PMK); a nucleic acid encoding droxy-3-methylglutaryl-Coenzyme A synthase polypeptide; diphosphomevalonate decarboxylase (MVD); and a nucleic and (iii) a 3-hydroxy-3-methylglutaryl-Coenzyme A reduc 15 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 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 25 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 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 30 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, 35 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 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. 40 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 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 45 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 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 50 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 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 55 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 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 60 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 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 65 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 US 9,353,375 B2 7 8 from the group consisting of hemiterpenoids, monoterpe polypeptide is from the genus Pseudomonas. In some aspects, noids, sesquiterpenoids, diterpenoids, Sesterterpenoids, trit the Pseudomonas is Pseudomonas aeruginosa. erpenoids, tetraterpenoids, and higher polyterpenoids. In In some aspects, the cells have a specific productivity Some aspects, the hemiterpenoid is prenol (i.e., 3-methyl-2- greater than about 20 mg/OD/hr ofisoprene. In some aspects, buten-1-ol), isoprenol (i.e., 3-methyl-3-buten-1-ol), 2-me the cells have a specific productivity greater than about 25 thyl-3-buten-2-ol, or isovaleric acid. In some aspects, the mg/OD/hr of isoprene. In some aspects, the heterologous monoterpenoid is geranyl pyrophosphate, eucalyptol, nucleic acid encoding an isoprene synthase polypeptide is limonene, or pinene. In some aspects, the sesquiterpenoid is operably linked to a promoter, and wherein the cells have a farnesyl pyrophosphate, artemisinin, or bisabolol. In some specific productivity greater than about 20 mg/OD/hr of iso aspects, the diterpenoid is geranylgeranyl pyrophosphate, ret 10 prene. In some aspects, the heterologous nucleic acid encod inol, retinal, phytol, taxol, forskolin, or aphidicolin. In some ing an isoprene synthase polypeptide is operably linked to a aspects, the triterpenoid is squalene or lanosterol. In some promoter, and wherein the cells have a specific productivity aspects, the tetraterpenoid is lycopene or carotene. In some greater than about 25 mg/L/hr ofisoprene. 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 15 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 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; 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 25 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 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 30 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 35 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 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 40 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; 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 45 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 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) 50 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 some aspects, the MVK polypeptide is from the genus Metha BRIEF DESCRIPTION OF THE DRAWINGS nosarcina. In some aspects, the MVK polypeptide is from 55 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 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, 60 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) 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 65 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 US 9,353,375 B2 9 10 2.7.1.36. Assay: Curr Genet 19:9-14, 1991. PMK; Phospho FIGS. 14A-F are graphs of isoprene production by E. coli mevalonate kinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol. strain expressing M. mazei mevalonate kinase, P alba iso 11:620-631, 1991; DPMDC; Diphosphomevalonate decar prene synthase, and pgl (RHM1 11608-2), and grown in fed boxylase, MVD1, EC 4.1.1.33. Assay: Biochemistry batch culture at the 15-L scale. FIG. 14A shows the time 33:13355-13362, 1994; IDI: Isopentenyl-diphosphate delta course of optical density within the 15-L bioreactor fed with isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem. 264: glucose. FIG. 14B shows the time course of isoprene titer 19169-19175, 1989. DXP Pathway: DXS: 1-Deoxyxylulose within the 15-L bioreactor fed with glucose. The titer is 5-phosphate synthase, dxs, EC 2.2.1.7. Assay: PNAS defined as the amount of isoprene produced per liter offer 94:12857-62, 1997: DXR: 1-Deoxy-D-xylulose 5-phosphate mentation broth. Method for calculating isoprene: cumulative reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem. 10 isoprene produced in 59 hrs, g/Fermentor volume at 59 hrs, 269:4446-4457, 2002: MCT: 4-Diphosphocytidyl-2C-me L =lg/L broth. FIG. 14C also shows the time course of thyl-D-erythritol synthase, IspD, EC 2.7.7.60. Assay: PNAS isoprene titer within the 15-L bioreactor fed with glucose. 97:6451-6456, 2000; CMK: 4-Diphosphocytidyl-2-C-me Method for calculating isoprene: f(Instantaneous isoprene thyl-D-erythritol kinase, IspE, EC 2.7.1.148. Assay: PNAS 15 production rate, g/L/hr)dt from t=0 to 59 hours =lg/L broth. 97: 1062-1067, 2000; MCS: 2C-Methyl-D-erythritol 2,4-cy FIG. 14D shows the time course of total isoprene produced clodiphosphate synthase, Isp, EC 4.6.1.12. Assay: PNAS from the 15-L bioreactor fed with glucose. FIG. 14E shows 96:11758-11763, 1999: HDS: 1-Hydroxy-2-methyl-2-(E)- volumetric productivity within the 15-L bioreactor fed with butenyl 4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. glucose. FIG. 14F shows carbon dioxide evolution rate Org. Chem. 70:9168-9174, 2005; HDR: 1-Hydroxy-2-me (CER), or metabolic activity profile, within the 15-L biore thyl-2-(E)-butenyl 4-diphosphate reductase, IspH. EC actor fed with glucose. 1.17.1.2. Assay: JACS, 126:12847-12855, 2004. FIG. 15 are graphs showing analysis of off-gas from fer FIG. 1B illustrates the classical and modified MVA path mentation in 15 L. bioreactors. Sample A is strain ways. 1, acetyl-CoA acetyltransferase (AACT); 2. HMG RM111608-2 sampled at 64.8 hours. Sample B is strain CoA synthase (HMGS);3, HMG-CoA reductase (HMGR);4, 25 EWL256 was E. coli BL21 (DE3), pCL upper, cmR-gi1.2- mevalonate kinase (MVK); 5, phosphomevalonate kinase yKKDyI, pTrcAlba-mMVK sampled at 34.5 hours. Hydro (PMK); 6, diphosphomevalonate decarboxylase (MVD or gen is detected above the baseline (0.95x10 torr) for both DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8. samples. phosphomevalonate decarboxylase (PMDC); 9, isopentenyl FIG. 16A shows an exemplary isoprene recovery unit. phosphate kinase (IPK). The classical MVA pathway pro 30 FIG. 16B shows an exemplary isoprene desorption/con ceeds from reaction 1 through reaction 7 via reactions 5 and 6. while a modified MVA pathway goes through reactions 8 and densation setup. 9. P and PP in the structural formula are phosphate and FIG. 17 shows a GC/FID chromatogram of an isoprene pyrophosphate, respectively. This figure was taken from product. The material was determined to be 99.7% pure. Koga and Morii, Microbiology and Mol. Biology Reviews 35 FIGS. 18A-C show the GC/FID chromatograms of an iso 71.97-120, 2007. The modified MVA pathway is present, for prene sample before (A) and after treatment with alumina (B) example, in some archaeal organisms, such as Methanosa or silica (C). The isoprene peak is not shown in these chro rcina mazei. matograms. FIG. 2 is a map of plasmid plT24 P. alba HGS. FIG. 19A shows a map of plasmid p)W34, encoding a FIG. 3A-B are the nucleotide sequence of plasmid pET24 40 truncated version of P alba isoprene synthase (MEA variant) P alba HGS (SEQID NO:1). under the control of the PTrc promoter and M. mazei MVK. FIG. 4 is a schematic diagram showing restriction sites FIG. 19B-D shows the complete nucleotide sequence of plas used for endonuclease digestion to construct plasmid mid plDW34 (SEQID NO:7). EWL230 and compatible cohesive ends between BspHI and FIG. 20 shows the chromosomal organization of E. coli NcoI sites. 45 K12 strain MG 1655 around the pgl locus (Graph imported FIG. 5 is a map of plasmid EWL230. from www.ecocyc.com). The region deleted in E. coli BL21 FIGS. 6A-B are the nucleotide sequence of plasmid (DE3) compared to E. coli K12 MG655 and restored instrains EWL230 (SEQ ID NO:2). CMP215 and CMP258 is shown in brackets. The predicted FIG. 7 is a schematic diagram showing restriction sites ORF of the ybgS gene is circled. A forward arrow (->) indi used for endonuclease digestion to construct plasmid 50 cates the annealing site of the galMF primer (SEQID NO:8). EWL244 and compatible cohesive ends between NsiI and A reverse arrow (e-) indicates the annealing site of the galMR Pst sites. primer (SEQ ID NO:9). FIG. 8 is a map of plasmid EWL244. FIG. 21 shows optical density (OD) plots from microfer FIGS. 9A-B are the nucleotide sequence of plasmid mentation experiments conducted with PGL+ (CMP312) and EWL244 (SEQ ID NO:3). 55 PGL- (CMP323) cultures. Black triangles along the X-axis FIG.10A is a map of the M. mazei archaeal Lower Pathway indicate when offline samples were taken. Other OD values operon. are interpolated. FIGS. 10B-C are the nucleotide sequence of the M. mazei FIG. 22 shows isoprene specific productivity plots from archaeal Lower Pathway operon (SEQID NO:4). microfermentation experiments conducted with PGL+ FIG. 11A is a map of MCM376-MVK from M. mazei 60 (CMP312) and PGL- (CMP323) cultures. Black triangles archaeal Lower in pET200D. along the X-axis indicate when offline samples were taken. FIGS. 11B-C are the nucleotide sequence of MCM376 Other OD values are interpolated. MVK from M. mazei archaeal Lower in pET200D (SEQ ID FIG. 23 shows a time course of optical density in a 15-L NO:5). bioreactor fed with glucose. FIG. 12 is a map of plasmid pBBRCMPGI1.5-pgl. 65 FIG. 24 shows a time course of isoprene titer in a 15-L FIGS. 13 A-B are the nucleotide sequence of plasmid bioreactor fed with glucose. Isoprene titer is defined as the pBBRCMPGI1.5-pgl (SEQID NO:6). amount of isoprene produced per liter of fermentation broth. US 9,353,375 B2 11 12 The equation for calculating isoprenetiter is: (Instantaneous FIG.38 shows the concentration of MvaS protein per OD isoprene production rate, g/L/hr)dt from t=0 to thrs =lg/L in E. coli strains MCM872 (BL21 pTrc-Upper) and MCM876 broth. (BL21 pglpTrc-Upper) in TM3 minimal medium with 0.02% FIG. 25 shows the time course of total isoprene produced yeast extract, taken at two different timepoints. from the 15-L bioreactors fed with glucose. FIG.39 shows the concentration of MvaE per OD in E. coli FIG. 26 shows isoprene specific productivity within the strains MCM872 (BL21 pTrc-Upper) and MCM876 (BL21 15-L bioreactors fed with glucose. Equation for calculating pglpTrc-Upper) grown in TM3 minimal medium with 0.02% Specific Productivity levels: (mg isoprene-mg isoprene)/ yeast extract. (OD550, L broth-OD550*L broth)/(2.7 OD*L/g cell)/ FIG. 40A shows the amino acid sequence of 6-phospho (t-to) = mg isoprene/g cell/hr. 10 gluconolactonase (PGL) from E. coli K12 MG1655 (SEQID FIG. 27 shows a time course of optical density within the NO:11). FIG. 40B shows the amino acid sequence of PGL 15-L bioreactor fed with glucose. The pgl+ sample was a from P. aeruginosa (SEQ ID NO:12). FIG. 40C shows the culture of strain CMP312. The pgl-sample was a culture of amino acid sequence of PGL from S. cerevisiae (SEQ ID Strain CMP323. NO:13). FIG. 40D shows an alignment of the amino acid 15 sequences of E. coli PGL and P. aeruginosa PGL. Identical FIG. 28 shows a time course of isoprene titer within the amino acids are shown highlighted in grey. Conservative 15-L bioreactor fed with glucose. The titer is defined as the amino acid Substitutions are shown highlighted in black. 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 =lg/L broth. MgSO4) containing 0.4% (w/v) glucose. Growth was mea FIG. 29 shows isoprene specific productivity within the sured at OD600. 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 25 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 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 30 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. isoprene production rate, g/L/hr)dt from t=0 to thrs =lg/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 35 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 =lg/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 40 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 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 45 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. 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 50 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 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 55 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 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. 60 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 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 65 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. 15, 2009). Consequently, E. coli BL21 and BL21 (DE3) lack US 9,353,375 B2 13 14 both PGL activity and the ability to utilize galactose as a Accordingly in one aspect, the invention encompasses carbon Source. (Aon et al., “Suppressing posttranslational recombinant cell(s) of an Escherichia coli (E. coli) strain gluconoylation of heterologous proteins by metabolic engi capable of producing isoprene, wherein the cell(s) comprise: neering of Escherichia coli,' Appl. Environ. Microbiol. (a) one or more copies of a heterologous nucleic acid(s) 74:950-958 (2008)). encoding a PGL polypeptide wherein the nucleic acid(s) Additionally, the deletion also included genes required for is/are integrated in the E. coli chromosome; and (b) one or high affinity transport of molybdate. While required in only more heterologous nucleic acid(s) encoding isoprene Syn trace amounts, molybdenum plays an important role in sev thase; wherein prior to the integration, the E. coli cell does not eral metabolic pathways in all organisms. Molybdate is used contain (a) nucleic acid(s) that encodes a encoding a PGL 10 polypeptide, and wherein the resulting recombinant cell pro as an enzymatic cofactor by bacteria in a number of oxidation/ duces isoprene at a greater titer than that of the same cell(s) reduction reactions, plays a critical role in nitrogen metabo that do not comprise (a) and (b). In some cases, the recombi lism, and, particularly in the case of anaerobic respiration, nant E. coli cell can use its own endogenous promoter(s) contributes to energy production. (See, e.g., Self et al., Res and/or its other regulatory systems to regulate the transcrip Microbiol. 152:311-321 (2001); Grunden & Shanmugam, 15 tion and Subsequent expression of the integrated PGL nucleic Arch Microbiol. 168:345-354 (1997)). acid. In Such cases, the expression of the heterologous nucleic The pentose phosphate pathway (PPP) is used during acids (e.g., PGL or isoprene) is not constitutive expression growth to provide NADPH and pentoses (5-carbon sugars) driven by a plasmid or elements on a plasmid. In other cases, (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 25 (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 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 30 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 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 35 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, 40 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 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 45 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 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 50 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 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 55 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 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 60 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 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 65 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 US 9,353,375 B2 15 16 polypeptide with one or more associated expression control vector, into an autonomously replicating plasmid or virus, or sequences and a heterologous nucleic acid encoding an upper into the genomic DNA of a prokaryote or eukaryote, or which mevalonate (MVA) pathway polypeptide, a lower MVA path exists as a separate molecule (e.g., a cDNA, a genomic DNA way polypeptide, or an isoprene synthase polypeptide, and fragment, or a cDNA fragment produced by PCR or restric producing isoprene. In some aspects, the cells have a specific 5 tion endonuclease digestion) independent of other sequences. productivity of isoprene greater than that of the same cells In some aspects, a recombinant nucleic acid is a nucleic acid lacking one or more copies of a heterologous gene encoding that encodes a non-naturally occurring polypeptide. a PGL polypeptide with one or more associated expression By "heterologous nucleic acid' is meant a nucleic acid control sequences, when the cells are cultured in minimal sequence derived from a different organism, species or strain medium. 10 General Techniques than the host cell. In some aspects, the heterologous nucleic The practice of the present invention will employ, unless acid is not identical to a wild-type nucleic acid that is found in otherwise indicated, conventional techniques of molecular the same host cell in nature. For example, a nucleic acid biology (including recombinant techniques), microbiology, encoding a PGL polypeptide isolated from E. coli K12 strain cell biology, biochemistry, and immunology, which are 15 MG 1655 or a derivative thereof, integrated into the chromo within the skill of the art. Such techniques are explained fully some of E. coli BL21 (DE3) by P1 transduction and expressed in the literature, “Molecular Cloning: A Laboratory Manual', in the cell is a heterologous nucleic acid. In one aspect, a second edition (Sambrook et al., 1989); “Oligonucleotide "heterologous nucleic acid can mean the introduction of a Synthesis” (M. J. Gait, ed., 1984): “Animal Cell Culture' (R. nucleic acid into a host cell that does not have that nucleic I. Freshney, ed., 1987); "Methods in Enzymology” (Aca acid. In some cases, a heterologous nucleic acid can be a demic Press, Inc.); "Current Protocols in Molecular Biology’ heterologous gene. One of skill in the art would appreciate the (F.M. Ausubel et al., eds., 1987, and periodic updates); "PCR: differences and also be able to use the context of the teaching The Polymerase Chain Reaction', (Mullis et al., eds., 1994). accordingly. Singleton et al., Dictionary of Microbiology and Molecular As used herein, an “expression control sequence” means a Biology 2nded. J. Wiley & Sons (New York, N.Y. 1994), and 25 nucleic acid sequence that directs transcription of a nucleic March, Advanced Organic Chemistry Reactions, Mecha acid of interest. An expression control sequence can be a nisms and Structure 4th ed., John Wiley & Sons (New York, promoter. Such as a constitutive or an inducible promoter, or N.Y. 1992), provide one skilled in the art with a general guide an enhancer. An expression control sequence can be "native' to many of the terms used in the present application. or heterologous. A native expression control sequence is Definitions 30 derived from the same organism, species, or strain as the gene The term "isoprene’ refers to 2-methyl-1,3-butadiene being expressed. A heterologous expression control sequence (CASH78-79-5). It can be the direct and final volatile C5 hydrocarbon product from the elimination of pyrophosphate is derived from a different organism, species, or strain as the from 3.3-dimethylallyl pyrophosphate (DMAPP). It may not gene being expressed. An "inducible promoter” is a promoter involve the linking or polymerization of IPP molecules to 35 that is active under environmental or developmental regula DMAPP molecules. The term "isoprene' is not generally tion. The expression control sequence is operably linked to intended to be limited to its method of production unless the nucleic acid segment to be transcribed. indicated otherwise herein. As used herein, the terms “minimal medium' or “minimal As used herein, the term “6-phosphogluconolactone' media' refer to growth medium containing the minimum refers to 6-phospho-D-glucono-1,5-lactone (CASH2641-81 - 40 nutrients possible for cell growth, generally without the pres 8). As used herein, the term “6-phosphogluconate” refers to ence of amino acids. Minimal medium typically contains: (1) 6-phospho-D-gluconate (CASH921-62-0). a carbon Source for bacterial growth; (2) various salts, which As used herein, the term “polypeptides includes polypep may vary among bacterial species and growing conditions; tides, proteins, peptides, fragments of polypeptides, and and (3) water. The carbon Source can vary significantly, from fusion polypeptides. 45 simple Sugars like glucose to more complex hydrolysates of As used herein, an "isolated polypeptide' is not part of a other biomass. Such as yeast extract, as discussed in more library of polypeptides, such as a library of 2, 5, 10, 20, 50 or detail below. The salts generally provide essential elements more different polypeptides and is separated from at least one Such as magnesium, nitrogen, phosphorus, and Sulfur to allow component with which it occurs in nature. An isolated the cells to synthesize proteins and nucleic acids. Minimal polypeptide can be obtained, for example, by expression of a 50 medium can also be supplemented with selective agents. Such recombinant nucleic acid encoding the polypeptide. An iso as antibiotics, to select for the maintenance of certain plas lated polypeptide can be a non-naturally occurring polypep mids and the like. For example, if a microorganism is resistant tide. to a certain antibiotic, such as amplicillin or tetracycline, then By "heterologous polypeptide' is meant a polypeptide that antibiotic can be added to the medium in order to prevent encoded by a nucleic acid sequence derived from a different 55 cells lacking the resistance from growing. Medium can be organism, species, or strain than the host cell. In some Supplemented with other compounds as necessary to select aspects, a heterologous polypeptide is not identical to a wild for desired physiological or biochemical characteristics. Such type polypeptide that is found in the same host cell in nature. as particular amino acids and the like. As used herein, a “nucleic acid refers to two or more As used herein, the term “terpenoid' or "isoprenoid refers deoxyribonucleotides and/or ribonucleotides covalently 60 to a large and diverse class of naturally-occurring organic joined together in either single or double-stranded form. chemicals similar to terpenes. Terpenoids are derived from By “recombinant nucleic acid' is meant a nucleic acid of five-carbon isoprene units assembled and modified in a vari interest that is free of one or more nucleic acids (e.g., genes) ety of ways, and are classified in groups based on the number which, in the genome occurring in nature of the organism of isoprene units used in group members. Hemiterpenoids from which the nucleic acid of interest is derived, flank the 65 have one isoprene unit. Monoterpenoids have two isoprene nucleic acid of interest. The term therefore includes, for units. Sesquiterpenoids have three isoprene units. Diterpe example, a recombinant DNA which is incorporated into a noids have four isoprene units. Sesterterpenoids have five US 9,353,375 B2 17 18 isoprene units. Triterpenoids have six isoprene units. Tetrater genome are PGL and galT. In some aspects, the genes penoids have eight isoprene units. Polyterpenoids have more restored to the BL21 or BL21 (DE3) genome are PGL and than eight isoprene units. galE. In some aspects, the genes restored to the BL21 or As used herein, the term “carotenoid refers to a group of BL21 (DE3) genome are PGL, galM, and galK. In some naturally occurring organic pigments produced in the chloro 5 aspects, the genes restored to the BL21 or BL21 (DE3) plasts and chromoplasts of plants, of Some other photosyn genome are PGL, galM, and galT. In some aspects, the genes thetic organisms, such as algae, in some types of fungus, and restored to the BL21 or BL21 (DE3) genome are PGL, galM, in Some bacteria. Carotenoids include the oxygen-containing and galE. In some aspects, the genes restored to the BL21 or Xanthophylls and the non-oxygen-containing carotenes. BL21 (DE3) genome are PGL, galK, and galT. In some Unless defined otherwise herein, all technical and scien 10 aspects, the genes restored to the BL21 or BL21 (DE3) tific terms used herein have the same meaning as commonly genome are PGL, galK, and galE. In some aspects, the genes understood by one of ordinary skill in the art to which this restored to the BL21 or BL21 (DE3) genome are PGL, galT. invention pertains. and galE. In some aspects, the genes restored to the BL21 or As used herein, the singular terms “a,” “an and “the BL21 (DE3) genome are PGL, galM, galK, and galT. In some include the plural reference unless the context clearly indi 15 aspects, genes restored to the BL21 or BL21 (DE3) genome cates otherwise. are PGL, galM, galK, and galE. In some aspects, genes It is intended that every maximum numerical limitation restored to the BL21 or BL21 (DE3) genome are PGL, galK, given throughout this specification includes every lower galT, and galE. numerical limitation, as if Such lower numerical limitations In some aspects, genes restored to the BL21 or BL21 (DE3) were expressly written herein. Every minimum numerical genome are PGL and modF. In some aspects, the genes 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 25 (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) 30 restored to the BL21 or BL21 (DE3) genome are PGL, modF. genomes includes the yghE gene (PGL), genes encoding modE, modA, modB, and modC. proteins involved in the utilization of galactose as a carbon In some aspects, the genes restored to the BL21 or BL21 Source, genes encoding proteins involved in molybdenum (DE3) genome are PGL, galM, and modF. In some aspects, transport, as well as several other genes of unknown function the genes restored to the BL21 or BL21 (DE3) genome are ality. See, for example, FIG. 20. The genes involved in the 35 PGL, galM, and modE. In some aspects, genes restored to the utilization of galactose are galM which encodes galactose-1- BL21 or BL21 (DE3) genome are PGL, galM, and modA, epimerase, galK, which encodes galactokinase, galT, which modB, and modC. In some aspects, the genes restored to the encodes galactose-1-phosphate uridylyltransferase, and BL21 or BL21 (DE3) genome are PGL, galK, and modF. In galE, which encodes UDP-glucose 4-epimerase. The genes some aspects, the genes restored to the BL21 or BL21 (DE3) encoding proteins involved in molybdenum transport are 40 genome are PGL, galK, and modE. In some aspects, the genes modF, which encodes the fused molybdate transporter sub restored to the BL21 or BL21 (DE3) genome are PGL, galK, units of the ABC Superfamily, modE, which encodes the modA, modB, and modC. In some aspects, the genes restored repressor of the modABC operon for molybdenum transport, to the BL21 or BL21 (DE3) genome are PGL, galT, and modF. and modA, modB, and modC, which each encode a molyb In some aspects, the genes restored to the BL21 or BL21 date transporter subunit protein. 45 (DE3) genome are PGL, galT, and modE. In some aspects, the Accordingly, bacterial (e.g., E. coli) cells can be engi genes restored to the BL21 or BL21 (DE3) genome are PGL, neered to integrate nucleic acids encoding a PGL polypeptide galT, modA, modB, and modC. In some aspects, the genes in the E. coli chromosome. Introduction of heterologous restored to the BL21 or BL21 (DE3) genome are PGL, galK, nucleic acids encoding for isoprene synthase (e.g., P alba and modF. In some aspects, the genes restored to the BL21 or isoprene synthase) can increase the total titer and/or specific 50 BL21 (DE3) genome are PGL, galE, and modE. In some activity for isoprene production. Furthermore, in addition to aspects, the genes restored to the BL21 or BL21 (DE3) the PGL integration, one or more genes encoding proteins genome are PGL, galE, modA, modB, and modC. involved in the utilization of galactose as a carbon source or In some aspects, the genes restored to the BL21 or BL21 proteins involved in molybdenum transport can also be intro (DE3) genome are PGL, galM, galK, and modF. In some duced into the E. coli cell to increase the overall fitness of the 55 aspects, the genes restored to the BL21 or BL21 (DE3) recombinant cell, which, in turn, can lead to increased pro genome are PGL, galM, galK, and modE. In some aspects, the duction of isoprene. genes restored to the BL21 or BL21 (DE3) genome are PGL, Various options of integrated PGL alone or integrated PGL galM, galK, and modA, modB, and modC. In some aspects, in combination with one or more genes encoding proteins the genes restored to the BL21 or BL21 (DE3) genome are involved in the utilization of galactose as a carbon source or 60 PGL, galM, galT, and modF. In some aspects, the genes proteins involved in molybdenum transport are contemplated restored to the BL21 or BL21 (DE3) genome are PGL, galM, within the scope of the invention. Thus, in Some aspects, the galT, and modE. In some aspects, the genes restored to the gene restored to the BL21 or BL21 (DE3) genome is PGL. In BL21 or BL21 (DE3) genome are PGL, galM, galT, and some aspects, the genes restored to the BL21 or BL21 (DE3) modA, modB, and modC. In some aspects, the genes restored genome are PGL and galM. In some aspects, genes restored to 65 to the BL21 or BL21 (DE3) genome are PGL, galM, galE, and the BL21 or BL21 (DE3) genome are PGL and galK. In some modF. In some aspects, the genes restored to the BL21 or aspects, the genes restored to the BL21 or BL21 (DE3) BL21 (DE3) genome are PGL, galM, galE, and modE. In US 9,353,375 B2 19 20 some aspects, the genes restored to the BL21 or BL21 (DE3) Mutant PGL polypeptides include those in which one or genome are PGL, galM, galE, and modA, modB, and modC. more amino acid residues have undergone an amino acid In some aspects, the genes restored to the BL21 or BL21 substitution while retaining PGL activity (i.e., the ability to (DE3) genome are PGL, galK, galT, and modF. In some convert 6-phosphogluconolactone to 6-phosphogluconate). aspects, the genes restored to the BL21 or BL21 (DE3) The amino acid Substitutions may be conservative or non genome are PGL, galK, galT, and modE. In some aspects, the conservative and Such substituted amino acid residues may or genes restored to the BL21 or BL21 (DE3) genome are PGL, may not be one encoded by the genetic code. The standard galK, galT, and modA, modB, and modC. In some aspects, twenty amino acid “alphabet” has been divided into chemical the genes restored to the BL21 or BL21 (DE3) genome are families based on similarity of their side chains. Those fami 10 lies include amino acids with basic side chains (e.g., lysine, PGL, galK, galE, and modF. In some aspects, the genes arginine, histidine), acidic side chains (e.g., aspartic acid, restored to the BL21 or BL21 (DE3) genome are PGL, galK, glutamic acid), uncharged polar side chains (e.g., glycine, galE, and modE. In some aspects, the genes restored to the asparagine, glutamine, serine, threonine, tyrosine, cysteine), BL21 or BL21 (DE3) genome are PGL, galK, galE, and nonpolar side chains (e.g., alanine, Valine, leucine, isoleu modA, modB, and modC. In some aspects, the genes restored 15 cine, proline, phenylalanine, methionine, tryptophan), beta to the BL21 or BL21 (DE3) genome are PGL, galE, galT, and branched side chains (e.g., threonine, Valine, isoleucine) and modF. In some aspects, the genes restored to the BL21 or aromatic side chains (e.g., tyrosine, phenylalanine, tryp BL21 (DE3) genome are PGL, galE, galT, and modE. In some tophan, histidine). A “conservative amino acid substitution aspects, the genes restored to the BL21 or BL21 (DE3) is one in which the amino acid residue is replaced with an genome are PGL, galE, galT, and modA, modB, and modC. amino acid residue having a chemically similar side chain 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) 25 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 30 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) 35 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 40 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 45 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 50 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 55 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 60 “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 65 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 US 9,353,375 B2 21 22 nucleic acids include naturally-occurring polypeptides and plary UDP-galactose-4-epimerase polypeptides include nucleic acids from any of the source organisms described polypeptides, fragments of polypeptides, peptides, and fusion herein as well as mutant polypeptides and nucleic acids polypeptides that have at least one activity of a UDP-galac derived from any of the Source organisms described herein. tose-4-epimerase polypeptide. Exemplary UDP-galactose-4- Exemplary PGL nucleic acids include, for example, PGL 5 epimerase polypeptides and nucleic acids include naturally isolated from E. coli K12 MG 1655 or derivatives thereof occurring polypeptides and nucleic acids from any of the (EcoGene Accession No. EG13231; part of E. coli K12 Source organisms described hereinas well as mutant polypep MG 1655 genomic sequence referenced by GenBank Acces tides and nucleic acids derived from any of the Source organ sion No. U0096; see also UniProtKB/Swiss-Prot Accession isms described herein that have at least one activity of a No. P52697 (PGL polypeptide))(see FIG. 40A and SEQ ID 10 UDP-galactose-4-epimerase polypeptide. NO:11); PGL isolated from Pseudomonas aeruginosa strain Exemplary galactose metabolic nucleic acids include PAO1 (Locus Tag PA3182 of GenBank Accession No. nucleic acids that encode a polypeptide, fragment of a AE004.091); see also GenBank Accession No. AAG06570.1 polypeptide, peptide, or fusion polypeptide that has at least (PGL polypeptide))(see FIG. 40B and SEQID NO:12); and one activity of a galactose metabolic polypeptide. Exemplary PGL isolated from Saccharomyces cerevisiae (Locus Tag 15 galactose metabolic nucleic acids include, for example, YHR163W of GenBank Accession No. NC 001140; see also galactose metabolic genes isolated from E. coli K12 MG 1655 UNIProtKB/Swiss-Prot Accession No. P38858 (PGL or derivatives thereof galactose metabolic genes isolated polypeptide))(see FIG. 40C and SEQ ID NO:13). Other from Pseudomonas aeruginosa strain PAO1; and galactose exemplary PGL nucleic acids can be isolated from any genus metabolic genes isolated from Saccharomyces cerevisie. in the family Enterobacteriaceae including, for example, Other exemplary galactose metabolic nucleic acids can be 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 25 (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 30 The polypeptide encoded by the modF gene is an unchar merase polypeptides include polypeptides, fragments of acterized member of the fused molybdate transporter sub polypeptides, peptides, and fusion polypeptides that have at units of ABC superfamily. Exemplary modF encoded least one activity of a galactose-1-epimerase polypeptide. polypeptides include polypeptides, fragments of polypep Exemplary galactose-1-epimerase polypeptides and nucleic tides, peptides, and fusion polypeptides that have at least one acids include naturally-occurring polypeptides and nucleic 35 activity of a modF encoded polypeptide. Exemplary modF acids from any of the source organisms described herein as encoded polypeptides and nucleic acids include naturally well as mutant polypeptides and nucleic acids derived from occurring polypeptides and nucleic acids from any of the any of the source organisms described herein that have at least Source organisms described hereinas well as mutant polypep one activity of a galactose-1-epimerase polypeptide. tides and nucleic acids derived from any of the Source organ Galactokinase (galK) catalyzes the phosphorylation of 40 isms described herein that have at least one activity of a modF D-galactose to D-galactose-1-phosphate. Exemplary galac encoded polypeptide. tokinase polypeptides include polypeptides, fragments of Repressor of the modABC operon for molybdenum trans polypeptides, peptides, and fusion polypeptides that have at port (modE) polypeptide is a regulatory protein that is least one activity of a galactokinase polypeptide. Exemplary believed to feedback inhibit the transcription of the modABC galactokinase polypeptides and nucleic acids include natu 45 operon in the presence of molybdate. Exemplary modE rally-occurring polypeptides and nucleic acids from any of encoded polypeptides include polypeptides, fragments of the Source organisms described herein as well as mutant polypeptides, peptides, and fusion polypeptides that have at polypeptides and nucleic acids derived from any of the Source least one activity of a modE encoded polypeptide. Exemplary organisms described herein that have at least one activity of a modE encoded polypeptides and nucleic acids include natu galactokinase polypeptide. 50 rally-occurring polypeptides and nucleic acids from any of Galactose-1-phosphate uridylyltransferase (galT) cata the source organisms described herein as well as mutant lyzes the second step of the Leloir pathway of galactose polypeptides and nucleic acids derived from any of the source metabolism by converting UDP-glucose and galactose organisms described herein that have at least one activity of a 1-phosphate to glucose 1-phosphate and UDP-galactose. modE encoded polypeptide. Exemplary galactose-1-phosphate uridylyltransferase 55 The high affinity trimeric molybdenum transporter protein polypeptides include polypeptides, fragments of polypep encoded by modA, modB, and modC is a membrane-associ tides, peptides, and fusion polypeptides that have at least one ated ABC-type transporter system for the uptake of molyb activity of a galactose-1-phosphate uridylyltransferase denum into the cell. When any one of the modABC genes are polypeptide. Exemplary galactose-1-phosphate uridylyl mutated or absent, molybdate transport is accomplished by transferase polypeptides and nucleic acids include naturally 60 the ABC-type Sulfate transport system or by a non-specific occurring polypeptides and nucleic acids from any of the anion transporter, but with about 100 times less efficiency. Source organisms described hereinas well as mutant polypep (Self et al., 2001, Res. Microbiol. 152:311-321). Exemplary tides and nucleic acids derived from any of the Source organ modABC encoded polypeptides include polypeptides, frag isms described herein that have at least one activity of a ments of polypeptides, peptides, and fusion polypeptides that galactose-1-phosphate uridylyltransferase polypeptide. 65 have at least one activity of a modABC encoded polypeptide. UDP-galactose-4-epimerase (galE) catalyzes the revers Exemplary modABC encoded polypeptides and nucleic acids ible conversion of UDP-galactose to UDP-glucose. Exem include naturally-occurring polypeptides and nucleic acids US 9,353,375 B2 23 24 from any of the Source organisms described herein as well as sequences and a heterologous nucleic acid encoding an iso mutant polypeptides and nucleic acids derived from any of prene synthase polypeptide. In some aspects, the bacterial the Source organisms described herein that have at least one cells produce isoprene at a specific productivity greater than activity of one of the modABC encoded polypeptides. that of the same cells lacking one or more copies of a heter Exemplary molybdenum transport nucleic acids include ologous gene encoding a PGL polypeptide with one or more nucleic acids that encode a polypeptide, fragment of a associated expression control sequences, when the cells are polypeptide, peptide, or fusion polypeptide that has at least cultured in minimal medium. one activity of a molybdenum transport polypeptide. Exem In some aspects, the cells further comprise an MVA path plary molybdenum transport nucleic acids include, for way polypeptide. In some aspects, the MVA pathway example, molybdenum transport genes isolated from E. coli 10 polypeptide is an upper MVA pathway polypeptide. In some K12 MG 1655 or derivatives thereof molybdenum transport aspects, the MVA pathway polypeptide is a lower MVA path genes isolated from Pseudomonas aeruginosa strain PAO1; way polypeptide. In some aspects, the upper MVA pathway and galactose metabolic genes isolated from Saccharomyces polypeptide is selected from the group consisting of: (i) an cerevisie. Other exemplary molybdenum transport nucleic acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) acids can be isolated from any genus in the family Enterobac 15 a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase teriaceae including, for example, Alishewanella, Alterococ polypeptide; and (iii) a 3-hydroxy-3-methylglutaryl-Coen cus, Aquamonas, Citrobacter, Cronobacter; Edwardsiella, Zyme A reductase polypeptide. In some aspects, the upper Enterobacter, Klebsiella (e.g., Klebsiella pneumoniae), Pan MVA pathway polypeptide is from the genus Enterococcus. toea (e.g., Pantoea citroea), Proteus (e.g., Proteus vulgaris), In some aspects, the upper MVA pathway polypeptide is from 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 25 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 In some aspects, the one or more copies of a heterologous wherein the nucleic acid is integrated in the E. coli chromo 30 gene encoding a PGL polypeptide with one or more associ Some; and (b) one or more heterologous nucleic acid(s) ated expression control sequences are chromosomal copies encoding isoprene synthase; wherein prior to the integration, (e.g., integrated into the E. coli chromosome). In some the E. coli cell does not contain nucleic acid(s) encoding a aspects, the E. coli cells are in culture. In some aspects, the PGL polypeptide, and wherein the resulting recombinant cell bacterial cells are of E. coli strain B. In some aspects, the produces isoprene at a greater titer than that of the same cells 35 bacterial strains are of E. coli Strain BL21. In some aspects, that do not comprise (a) and (b). In some aspects, the host the bacterial cells are of E. coli strain BL21 (DE3). cells are bacterial cells of an Escherichia coli strain that does Exemplary Cell Culture Media not encode a 6-phosphogluconolactonase (PGL) polypeptide, As used herein, the terms “minimal medium' or “minimal further comprising one or more copies of a heterologous gene media' refer to growth medium containing the minimum encoding a PGL polypeptide with one or more associated 40 nutrients possible for cell growth, generally, but not always, expression control sequences and a nucleic acid encoding a without the presence of one or more amino acids (e.g., 1,2,3, heterologous polypeptide capable of biological activity. In 4, 5, 6, 7, 8, 9, 10, or more amino acids). Minimal medium Some aspects, the bacterial cells produce the heterologous typically contains: (1) a carbon Source for bacterial growth; polypeptide at a specific productivity greater than that of the (2) various salts, which may vary among bacterial species and same cells lacking one or more copies of a heterologous gene 45 growing conditions; and (3) water. The carbon Source can encoding a PGL polypeptide with one or more associated vary significantly, from simple Sugars like glucose to more expression control sequences when the cells are cultured in complex hydrolysates of other biomass, such as yeast extract, minimal medium. In some aspects, the one or more copies of as discussed in more detail below. The salts generally provide a heterologous gene encoding a PGL polypeptide with one or essential elements such as magnesium, nitrogen, phosphorus, more associated expression control sequences are chromo 50 and sulfur to allow the cells to synthesize proteins and nucleic Somal copies (e.g., integrated into the E. coli chromosome). acids. Minimal medium can also be Supplemented with selec In some aspects, the E. coli cells are in culture. tive agents, such as antibiotics, to select for the maintenance In some aspects, the heterologous polypeptide capable of of certain plasmids and the like. For example, if a microor biological activity comprises one or more polypeptides ganism is resistant to a certain antibiotic, Such as amplicillin or involved in the biosynthesis ofterpenoid (isoprenoid) or caro 55 tetracycline, then that antibiotic can be added to the medium tenoid compounds, and the cells produce a terpenoid or caro in order to prevent cells lacking the resistance from growing. tenoid at a higher specific productivity than that of the same Medium can be supplemented with other compounds as nec cells lacking one or more copies of a heterologous gene essary to select for desired physiological or biochemical char encoding a PGL polypeptide with one or more associated acteristics, such as particular amino acids and the like. expression control sequences when cultured in minimal 60 Any minimal medium formulation can be used to cultivate medium. In some aspects, the method further comprises a the host cells. Exemplary minimal medium formulations step of recovering the terpenoid or carotenoid. include, for example, M9 minimal medium and TM3 minimal In some aspects, the host cells are bacterial cells of an medium. Each liter of M9 minimal medium contains (1) 200 Escherichia coli strain that does not encode a 6-phosphoglu ml sterile M9 salts (64 g NaHPO-7H2O, 15 g KHPO, 2.5 conolactonase (PGL) polypeptide, further comprising one or 65 g NaCl, and 5.0 g NHCl per liter); (2) 2 ml of 1 M MgSO more copies of a heterologous gene encoding a PGL polypep (sterile); (3) 20 ml of 20% (w/v) glucose (or other carbon tide with one or more associated expression control source); and (4) 100 ul of 1 M CaCl (sterile). Each liter of US 9,353,375 B2 25 26 TM3 minimal medium contains (1) 13.6 g. KHPO, (2) 13.6 Brock, Biotechnology: A Textbook of Industrial Microbiol g KHPO; (3)2g MgSO.7HO; (4) 2 g Citric Acid Mono ogy, Second Edition (1989) Sinauer Associates, Inc. hydrate; (5) 0.3 g Ferric Ammonium Citrate; (6) 3.2 g In some aspects, the cells are cultured under limited glu (NH4)2SO; (7) 0.2 g yeast extract; and (8) 1 ml of 1000x cose conditions. By “limited glucose conditions' is meant Trace Elements solution; pH is adjusted to ~6.8 and the solu that the amount of glucose that is added is less than or about tion is filter sterilized. Each liter of 1000x Trace Elements 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, contains: (1) 40 g Citric Acid Monohydrate; (2) 30 g 40%, 30%, 20%, or 10%) of the amount of glucose that is MnSOHO; (3) 10 g NaCl; (4) 1 g FeSO.7HO; (4) 1 g consumed by the cells. In particular aspects, the amount of CoCl*6HO; (5) 1 g ZnSO*7HO; (6) 100 mg glucose that is added to the culture medium is approximately 10 the same as the amount of glucose that is consumed by the CuSOSHO; (7) 100 mg HBO; and (8) 100 mg cells during a specific period of time. In some aspects, the rate NaMoO2H.O; pH is adjusted to -3.0. of cell growth is controlled by limiting the amount of added Any carbon Source can be used to cultivate the host cells. glucose such that the cells grow at the rate that can be Sup The term "carbon source” refers to one or more carbon ported by the amount of glucose in the cell medium. In some containing compounds capable of being metabolized by a 15 aspects, glucose does not accumulate during the time the cells host cell or organism. For example, the cell medium used to are cultured. In various aspects, the cells are cultured under cultivate the host cells may include any carbon Source Suitable limited glucose conditions for greater than or about 1, 2, 3, 5, for maintaining the viability or growing the host cells. In 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In various Some aspects, the carbon Source is a carbohydrate (such as aspects, the cells are cultured under limited glucose condi 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. 25 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 (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast Materials and methods suitable for the maintenance and 30 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 In some aspects, the E. coli cells are grown in fed-batch be found in International Publication No. WO 2009/076676, 35 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. 40 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 (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 45 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 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 50 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 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 55 (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. polypeptide is from E. coli strain K12 MG 1655. In some Standard culture conditions and modEs of fermentation, 60 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. Pseudomonas is Pseudomonas aeruginosa. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/ 65 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 US 9,353,375 B2 27 28 heterologous nucleic acid(s) encoding a PGL polypeptide medium. In some aspects, the heterologous gene encoding a wherein the nucleic acid(s) is/are integrated in the E. coli PGL polypeptide is from E. coli strain K12 MG 1655. In some chromosome; and (b) one or more heterologous nucleic aspects, the heterologous gene encoding a PGL polypeptide is acid(s) encoding isoprene synthase; wherein prior to the inte from a derivative of E. coli strain K12 MG 1655. In some gration, the E. coli cell does not contain nucleic acid(s) that aspects, the heterologous gene encoding a PGL polypeptide is encode(s) a encoding a PGL polypeptide, and wherein the from the genus Pseudomonas. In some aspects, the resulting recombinant cell(s) produce(s) isoprene at a greater Pseudomonas is Pseudomonas aeruginosa. titer than that of the same cell(s) that does/do not comprise (a) In some aspects, the cells further comprise an MVA path and (b). way polypeptide. In some aspects, the MVA pathway In some aspects, the host cells are bacterial cells of an 10 polypeptide is an upper MVA pathway polypeptide. In some Escherichia coli strain that do not encode a 6-phosphoglu aspects, the MVA pathway polypeptide is a lower MVA path conolactonase (PGL) polypeptide, further comprising one or way polypeptide. In some aspects, the upper MVA pathway more copies of a heterologous gene encoding a PGL polypep polypeptide is selected from the group consisting of: (i) an tide with one or more associated expression control acetoacetyl-Coenzyme A synthase (thiolase) polypeptide; (ii) sequences and a heterologous nucleic acid encoding an iso 15 a 3-hydroxy-3-methylglutaryl-Coenzyme A synthase prene synthase polypeptide. In some aspects, the host cells polypeptide; and (iii) a 3-hydroxy-3-methylglutaryl-Coen are bacterial cells of an Escherichia coli strain that does not Zyme A reductase polypeptide. In some aspects, the upper encode a 6-phosphogluconolactonase (PGL) polypeptide, MVA pathway polypeptide is from the genus Enterococcus. polypeptides transcribed from genes for galactose metabo In some aspects, the upper MVA pathway polypeptide is from 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 25 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 The recombinant bacterial cells described herein have the porter polypeptides. In some aspects, the one or more copies 30 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 in minimal medium. In some cases, the heterologous gene ologous gene encoding a PGL polypeptide, the one or more 35 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). 40 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 gene encoding one or more molybdate transport polypeptides mented with 0.1% (w/v) yeast extractor less. In some aspects, 45 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), a specific productivity greater than about 15 mg/OD/hr of or 0.1% (w/v) glucose. In certain aspects, the minimal 50 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), aspects, the E. coli cells have a specific productivity greater or 0.01% (w/v) yeast extract. In some aspects, the minimal 55 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% 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% 60 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 23 mg/OD/hr of isoprene. In some aspects, the E. coli cells medium. In some aspects, the minimal medium is TM3 65 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. US 9,353,375 B2 29 30 In other aspects, the E. coli cells have an upper limit of In some aspects, the E. coli cells further comprise a heter specific productivity of about 25, 24, 23, 22, 21, 20, 19, 18, ologous nucleic acid encoding an isoprene synthase polypep 17, 16, 15, 14, 13, 12, 11, 10,9,8,7,6, or 5 mg/OD/hr of tide. In some cases, the isoprene synthase polypeptide can be isoprene. In other aspects, the E. coli cells have a lower limit one or more copies of an endogenous isoprene synthase. In of specific productivity of about 5, 6,7,8,9, 10, 11, 12, 13, 14, 5 Some aspects, the isoprene synthase polypeptide is a plant 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg/OD/hr of isoprene synthase polypeptide. In some aspects, the isoprene isoprene. synthase polypeptide is a naturally-occurring polypeptide In some aspects, the heterologous nucleic acid encoding an from the genus Pueraria. In some aspects, the isoprene Syn isoprene synthase polypeptide is operably linked to a pro thase polypeptide is a naturally-occurring polypeptide from moter, and the cells have a specific productivity greater than 10 Pueraria montana. In some aspects, the isoprene synthase about 15 mg/OD/hr of isoprene. In some aspects, the heter polypeptide is a naturally-occurring polypeptide from the ologous nucleic acid encoding an isoprene synthase polypep genus Populus. In some aspects, the isoprene synthase tide is operably linked to a promoter, and the cells have a polypeptide is a naturally-occurring polypeptide from Popu specific productivity greater than about 16 mg/OD/hr of iso 15 lus alba. Other isoprene synthase polypeptides or isoprene prene. In some aspects, the heterologous nucleic acid encod synthase variants that can be used to practice the invention ing an isoprene synthase polypeptide is operably linked to a include, but is not limited to, the isoprene synthases, variants promoter, and the cells have a specific productivity greater thereof and/or isoprene synthase mutants as described in WO than about 17 mg/OD/hr of isoprene. In some aspects, the 2009/132220 or WO 2010/124146 (the contents of which are heterologous nucleic acid encoding an isoprene synthase incorporated by reference in their entirety, especially with polypeptide is operably linked to a promoter, and the cells respect to isoprene synthases, variants thereof and/or iso have a specific productivity greater than about 18 mg/OD/hr prene synthase mutants). of isoprene. In some aspects, the heterologous nucleic acid Methods for the Increased Production of Isoprene encoding an isoprene synthase polypeptide is operably linked Genetically engineered cell cultures in bioreactors have to a promoter, and the cells have a specific productivity 25 produced isoprene more efficiently, in larger quantities, in greater than about 19 mg/OD/hr ofisoprene. In some aspects, higher purities and/or with unique impurity profiles, and the heterologous nucleic acid encoding an isoprene synthase methods of producing commercially useful quantities of iso polypeptide is operably linked to a promoter, and the cells prene from renewable resources are described and exempli have a specific productivity greater than about 20 mg/OD/hr fied, for example, in International Patent Application Publi of isoprene. In some aspects, the heterologous nucleic acid 30 cation No. WO2009/076676 A2, U.S. Patent Application encoding an isoprene synthase polypeptide is operably linked 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 US2010/0167371 A1, US2010/0196977 A1, US2010/ polypeptide is operably linked to a promoter, and the cells 35 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, 40 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 the nucleic acid is integrated in the E. coli chromosome; and encoding an isoprene synthase polypeptide is operably linked 45 (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 ologous nucleic acid encoding an IDI polypeptide. In some duces isoprene at a greater titer than that of the same cells that aspects, the E. coli cells further comprise a chromosomal 50 do not comprise (i) and (ii) and (b) producing the isoprene. In copy of an endogenous nucleic acid encoding an IDI polypep Some aspects, the improved method of producing isoprene tide. In some aspects, the E. coli cells further comprise a comprises the steps of: (a) culturing bacterial cells of an heterologous nucleic acid encoding a DXS polypeptide or Escherichia coli strain that does not encode a 6-phosphoglu other DXP pathway polypeptides. In some aspects, the E. coli conolactonase (PGL) polypeptide in minimal medium, cells further comprise a chromosomal copy of an endogenous 55 wherein the E. coli cells comprise one or more copies of a nucleic acid encoding a DXS polypeptide or other DXP path heterologous gene encoding a PGL polypeptide with one or way polypeptides. In some aspects, the E. coli cells further more associated expression control sequences and a heterolo comprise one or more nucleic acids encoding an IDI polypep gous nucleic acid encoding an isoprene synthase polypeptide; tide and a DXS polypeptide or other DXP pathway polypep and (b) producing isoprene, wherein the E. coli cells have a tides. In some aspects, one nucleic acid encodes the isoprene 60 specific productivity ofisoprene greater than that of the same synthase polypeptide, IDI polypeptide, and DXS polypeptide cells lacking one or more copies of a heterologous gene or other DXP pathway polypeptides. In some aspects, one encoding a PGL polypeptide with one or more associated plasmid encodes the isoprene synthase polypeptide, IDI expression control sequences, when the cells are cultured in polypeptide, and DXS polypeptide or other DXP pathway minimal medium. In some aspects, the one or more copies of polypeptides. In some aspects, multiple plasmids encode the 65 a heterologous gene encoding a PGL polypeptide with one or isoprene synthase polypeptide, IDI polypeptide, and DXS more associated expression control sequences are chromo polypeptide or other DXP pathway polypeptides. Somal copies (e.g., integrated into the E. coli chromosome). US 9,353,375 B2 31 32 In some aspects, the improved method of producing isoprene bacterium. In some aspects, the bacterium is from the genus further comprises a step of recovering the isoprene. Enterococcus. In some aspects, bacterium is from Enterococ In some aspects, the improved method of producing iso cus faecalis. prene comprises the steps of culturing the recombinant cells In some aspects, the lower MVA pathway polypeptide is described herein under conditions suitable for the production 5 selected from the group consisting of: (i) mevalonate kinase of isoprene and allowing the recombinant cells to produce (MVK); (ii) phosphomevalonate kinase (PMK); (iii) diphos isoprene. In some aspects, the improved method of producing phomevalonate decarboxylase (MVD); and (iv) isopentenyl isoprene further comprises a step of recovering the isoprene. diphosphate isomerase (IDI). In some aspects, the lower Without being bound by theory, recombinant cells having MVA pathway polypeptide is MVK. In some aspects, the chromosomally integrated heterologous nucleic acids encod 10 MVK is from the genus Methanosarcina. In some aspects, the Methanosarcina is Methanosarcina mazei. In some aspects, ing PGL polypeptide produce isoprene at a higher titer and a the lower MVA pathway polypeptide is PMK, MVD, or IDI. higher specific productivity than cells where a heterologous In some aspects, the PMK. MVD, or IDI is from the genus PGL nucleic acid is on a plasmid. Surprisingly, recombinant Saccharomyces. In some aspects, the Saccharomyces is Sac cells comprising one or more copies of chromosomally inte 15 charomyces cerevisiae. In some aspects, the lower MVA path grated PGL polypeptide, and optionally with one or more way polypeptide is PMK. In some aspects, the PMK is from copies of one or more polypeptides encoded by chromosoma the genus Saccharomyces. In some aspects, the Saccharomy lly integrated galactose metabolism genes (for example, ces is Saccharomyces cerevisiae. In some aspects, the lower galM, galK, galT and galE), and/or one or more copies of one MVA pathway polypeptide is MVD. or more polypeptides encoded by chromosomally integrated In some aspects, the MVD is from the genus Saccharomy molybdenum transport genes (for example, modF, modE. ces. In some aspects, the Saccharomyces is Saccharomyces modA, modB, and modC) conveya substantial growth benefit cerevisiae. In some aspects, the lower MVA pathway to the cells, a higher titer of isoprene production, and/or a polypeptide is IDI. In some aspects, the lower MVA pathway higher specific production of isoprene versus cells compris polypeptide is from the genus Saccharomyces. In some ing a heterologous PGL nucleic acid on a plasmid. 25 aspects, the Saccharomyces is Saccharomyces cerevisiae. Therefore, in one aspect the improved method of produc In some aspects, the isoprene synthase polypeptide is from ing isoprene comprises the steps of: (a) culturing bacterial a plant. In some aspects, the plant is kudzu. In some aspects, cells of an Escherichia coli strain that does not encode a the plant is poplar (Populus albaxtremula CAC35696). In 6-phosphogluconolactonase (PGL) polypeptide, one or more Some aspects, the plant is aspen (Populus tremuloides). In polypeptides encoded by genes for galactose metabolism (for 30 Some aspects, the plantis English oak (Quercus robur). In one example, galM, galK, galT and galE), and/or one or more aspect, the plant is Populus alba. Other isoprene synthase polypeptides encoded by genes for molybdenum transport polypeptides or isoprene synthase variants that can be used to 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 35 mutants as described in WO 2009/132220 or WO 2010/ chromosomally integrated heterologous gene encoding a 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 40 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 45 polypeptide. In some aspects, the E. coli cells further com heterologous gene encoding PGL is located on a plasmid. 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 50 other DXP pathway polypeptides. In some aspects, the E. coli lly integrated PGL host cell system can optionally include 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. 55 polypeptide. In some aspects, one plasmid encodes the iso In some aspects, the upper MVA pathway polypeptide is 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 60 DXP pathway polypeptides. tase polypeptide. In some aspects, the upper MVA pathway 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 65 aspects, the E. coli K12 strain MG 1655 polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. In 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 US 9,353,375 B2 33 34 prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or TM3 medium. In some aspects, the minimal medium is M9 19, 20, or more amino acid substitutions compared to SEQID medium. In some aspects, the minimal medium is TM3 NO:11. In some aspects, the amino acid substitutions are medium. conservative. In some aspects, the amino acid Substitutions In some aspects, the E. coli cells have a specific productiv are non-conservative. In some aspects, the E. coli K12 strain ity greater than about 15 mg/L/hr of isoprene. In some MG1655 polypeptide having PGL activity has 99%, 98%, aspects, the E. coli cells have a specific productivity greater 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, than about 16 mg/OD/hr of isoprene. In some aspects, the E. 87%. 86%, or 85% amino acid sequence identity to SEQID coli cells have a specific productivity greater than about 17 NO:11. mg/OD/hr of isoprene. In some aspects, the E. coli cells have 10 a specific productivity greater than about 18 mg/OD/hr of In some aspects, the heterologous gene encoding a PGL isoprene. In some aspects, the E. coli cells have a specific polypeptide is from the genus Pseudomonas. In some aspects, productivity greater than about 19 mg/OD/hr of isoprene. In the Pseudomonas is Pseudomonas aeruginosa. In some Some aspects, the E. coli cells have a specific productivity aspects, the Paeruginosa polypeptide having PGL activity is greater than about 20 mg/OD/hr ofisoprene. In some aspects, SEQID NO:12. In some aspects, the P. aeruginosa polypep 15 the E. coli cells have a specific productivity greater than about tide having PGL activity comprises 1, 2, 3, 4, 5, 6,7,8,9, 10, 21 mg/OD/hr of isoprene. In some aspects, the E. coli cells 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid have a specific productivity greater than about 22 mg/OD/hr substitutions compared to SEQID NO:12. In some aspects, of isoprene. In some aspects, the E. coli cells have a specific the amino acid substitutions are conservative. In some productivity greater than about 23 mg/OD/hr of isoprene. In aspects, the amino acid Substitutions are non-conservative. In Some aspects, the E. coli cells have a specific productivity some aspects, the E. coli K12 strain MG 1655 polypeptide greater than about 24 mg/OD/hr ofisoprene. In some aspects, having PGL activity has 99%, 98%, 97%, 96%. 95%, 95%, the E. coli cells have a specific productivity greater than about 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino 25 mg/OD/hr of isoprene. acid sequence identity to SEQID NO:12. In Some aspects, the heterologous nucleic acid encoding an In some aspects, the heterologous gene encoding a PGL 25 isoprene synthase polypeptide is operably linked to a pro polypeptide is from the genus Saccharomyces. In some moter and the E. coli cells have a specific productivity greater aspects, the Saccharomyces is Saccharomyces cerevisiae. In than about 15 mg/OD/hr of isoprene. In some aspects, the Some aspects, the S. cerevisiae polypeptidehaving PGL activ heterologous nucleic acid encoding an isoprene synthase ity is SEQ ID NO:13. In some aspects, the S. cerevisiae polypeptide is operably linked to a promoter and the E. coli polypeptide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, 30 cells have a specific productivity greater than about 16 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino mg/OD/hr of isoprene. In some aspects, the heterologous acid substitutions compared to SEQ ID NO:13. In some nucleic acid encoding an isoprene synthase polypeptide is aspects, the amino acid substitutions are conservative. In operably linked to a promoter and the E. coli cells have a Some aspects, the amino acid Substitutions are non-conserva specific productivity greater than about 17 mg/OD/hr of iso tive. In some aspects, the E. coli K12 strain MG 1655 polypep 35 prene. In some aspects, the heterologous nucleic acid encod tide having PGL activity has 99%, 98%, 97%, 96%. 95%, ing an isoprene synthase polypeptide is operably linked to a 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% promoter and the E. coli cells have a specific productivity amino acid sequence identity to SEQID NO:13. greater than about 18 mg/OD/hr ofisoprene. In some aspects, In some aspects, the bacterial cells of an Escherichia coli the heterologous nucleic acid encoding an isoprene synthase strain that does not encode a PGL polypeptide are of E. coli 40 polypeptide is operably linked to a promoter and the E. coli strain B. In some aspects, the bacterial cells are of E. coli cells have a specific productivity greater than about 19 strain BL21. In some aspects, the bacterial cells are of E. coli mg/OD/hr of isoprene. In some aspects, the heterologous strain BL21 (DE3). nucleic acid encoding an isoprene synthase polypeptide is In some aspects, the E. coli cells are cultured in minimal operably linked to a promoter and the E. coli cells have a medium. In some aspects, the E. coli cells of E. coli strain B 45 specific productivity greater than about 20 mg/OD/hr of iso are cultured in minimal medium. In some aspects, the E. coli prene. In some aspects, the heterologous nucleic acid encod cells of E. coli strain BL21 are cultured in minimal medium. ing an isoprene synthase polypeptide is operably linked to a In some aspects, the E. coli cells of E. coli strain BL21 (DE3) promoter and the E. coli cells have a specific productivity are cultured in minimal medium. In some aspects, the mini greater than about 21 mg/OD/hr ofisoprene. In some aspects, mal medium is Supplemented with 1% (w/v) or less glucose. 50 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 55 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 60 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), 65 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 US 9,353,375 B2 35 36 prene to about 100 mg/OD/hr of isoprene. In some aspects, In some aspects, the bacterial cells of an Escherichia coli the heterologous nucleic acid encoding an isoprene synthase strain that does not encode a PGL polypeptide are of E. coli polypeptide is operably linked to a promoter and the E. coli strain B. In some aspects, the bacterial cells are of E. coli cells have a specific productivity greater than about 15 strain BL21. In some aspects, the bacterial cells are of E. coli mg/OD/hr of isoprene to about 100 mg/OD/hr of isoprene. strain BL21 (DE3). The invention also provides for recombinant E. coli cells In some aspects, the heterologous gene encoding a PGL with PGL integration that have been engineered to produce polypeptide is from E. coli strain K12 MG 1655. In some isoprene that also have better growth due to their increased aspects, the heterologous gene encoding a PGL polypeptide is overall fitness. One of skill in the art can appreciate that from a derivative of E. coli strain K12 MG 1655. In some increased growth rate can lead to enhanced production of 10 aspects, the E. coli K12 strain MG 1655 polypeptide having isoprene, Such as higher specific activity, more isoprene pro PGL activity is SEQID NO:11. In some aspects, the E. coli duced over a period of time, or higher isoprene titers. In one K12 strain MG 1655 polypeptide having PGL activity com aspect, the recombinant E. coli cells with PGL integration that prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, have been engineered to produce isoprene has at least 10% 19, 20, or more amino acid substitutions compared to SEQID increased growth as compared to those cells without PGL 15 NO:11. In some aspects, the amino acid Substitutions are integration and/or the restoration of the 17,257 base pair piece conservative. In some aspects, the amino acid Substitutions as described herein (see, for example, FIG. 20). In other are non-conservative. In some aspects, the E. coli K12 strain aspects, the recombinant E. coli cells with PGL integration MG1655 polypeptide having PGL activity has 99%, 98%, that have been engineered to produce isoprene has at least 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, about 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 87%. 86%, or 85% amino acid sequence identity to SEQID 20%, 21%. 22%, 23%, 24%, or 25% growth as compared to NO:11. those cells without PGL integration and/or the restoration of In some aspects, the heterologous gene encoding a PGL the 17,257 base pair piece as described herein. polypeptide is from the genus Pseudomonas. In some aspects, Methods for the Increased Production of Other Heterologous the Pseudomonas is Pseudomonas aeruginosa. In some Polypeptides Capable of Biological Activity 25 aspects, the Paeruginosa polypeptide having PGL activity is Also provided herein are improved methods for the pro SEQID NO:12. In some aspects, the Paeruginosa polypep duction of other heterologous polypeptides capable of bio tide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, logical activity or other products. One non-limiting example 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid of a product is mevalonate. One of skill in the art can produce substitutions compared to SEQID NO:12. In some aspects, mevalonate by: (a) culturing a composition comprising the 30 the amino acid substitutions are conservative. In some recombinant cell of an Escherichia coli (E. coli) strain, or aspects, the amino acid Substitutions are non-conservative. In progeny thereof, capable of producing isoprene, the cellcom some aspects, the P. aeruginosa polypeptide having PGL prising: (i) one or more copies of a heterologous nucleic activity has 99%, 98%, 97%, 96%. 95%, 95%, 93%, 92%, acid(s) encoding a PGL polypeptide wherein the nucleic acid 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino acid is integrated in the E. coli chromosome; (ii) one or more 35 sequence identity to SEQID NO:12. heterologous nucleic acid(s) encoding isoprene synthase; and In some aspects, the heterologous gene encoding a PGL (iii) (c) a heterologous nucleic acid encoding an upper meva polypeptide is from the genus Saccharomyces. In some lonate (MVA) pathway polypeptide and/or a lower MVA aspects, the Saccharomyces is Saccharomyces cerevisiae. In pathway polypeptide; wherein prior to the integration, the E. Some aspects, the S. cerevisiae polypeptide having PGL activ coli cell does not contain nucleic acid(s) encoding a PGL 40 ity is SEQ ID NO:13. In some aspects, the S. cerevisiae polypeptide, and wherein the resulting recombinant cell pro polypeptide having PGL activity comprises 1, 2, 3, 4, 5, 6, 7, duces isoprene at a greater titer than that of the same cells that 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino do not comprise (i) and (ii) under Suitable culture conditions acid substitutions compared to SEQ ID NO:13. In some for the production of mevalonate and (b) producing meva aspects, the amino acid Substitutions are conservative. In lonate. 45 Some aspects, the amino acid Substitutions are non-conserva In some aspects, the improved method of producing heter tive. In some aspects, the E. coli K12 strain MG 1655 polypep ologous polypeptides capable of biological activity com tide having PGL activity has 99%, 98%, 97%, 96%. 95%, prises the steps of: (a) culturing cells of an Escherichia coli 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% strain that does not encode a 6-phosphogluconolactonase amino acid sequence identity to SEQID NO:13. (PGL) polypeptide, further comprising one or more copies of 50 In some aspects, the bacterial cells of an Escherichia coli a heterologous gene encoding a PGL polypeptide with one or strain that does not encode a 6-phosphogluconolactonase more associated expression control sequences and a nucleic (PGL) polypeptide are cultured in minimal medium. In some acid encoding a heterologous polypeptide capable of biologi 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 55 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 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 60 (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 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 65 (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 US 9,353,375 B2 37 38 mented with 1% (w/v) glucose or less and 0.1% (w/v) or less. tutions compared to the native E. coli K12 strain MG 1655 In some aspects, the minimal medium is Supplemented with polypeptide. In some aspects, the amino acid substitutions are 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), conservative. In some aspects, the amino acid Substitutions 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% are non-conservative. In some aspects, the E. coli K12 strain (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% MG 1655 polypeptide having PGL activity, galactose meta (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), bolic activity, and/or molybdenum transport activity has 99%, 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. In 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, some aspects, the minimal medium is M9 medium or TM3 88%, 87%. 86%, or 85% amino acid sequence identity to the medium. In some aspects, the minimal medium is M9 native E. coli K12 strain MG 1655 polypeptide. medium. In some aspects, the minimal medium is TM3 10 In some aspects, the heterologous gene encoding a PGL medium. polypeptide, one or more galactose metabolism polypeptides, Also provided herein are improved methods for the pro and/or one or more molybdenum transport polypeptides is duction of other heterologous polypeptides capable of bio from the genus Pseudomonas. In some aspects, the logical activity. In some aspects, the improved method of Pseudomonas is Pseudomonas aeruginosa. In some aspects, producing heterologous polypeptides capable of biological 15 the Paeruginosa polypeptide having PGL activity, galactose activity comprises the steps of: (a) culturing cells of an metabolic activity, and/or molybdenum transport activity Escherichia coli strain that does not encode a 6-phosphoglu comprises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17. conolactonase (PGL) polypeptide, a gene that encodes one or 18, 19, 20, or more amino acid substitutions compared to the 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 25 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 polypeptide, one or more galactose metabolism polypeptides, heterologous gene encoding one or more molybdenum trans and/or one or more molybdenum transport polypeptides is port polypeptides; and (b) producing the heterologous 30 from the genus Saccharomyces. In some aspects, the Saccha polypeptide, wherein the E. coli cells have a specific produc romyces is Saccharomyces cerevisiae. In some aspects, the S. tivity of the heterologous polypeptide greater than that of the cerevisiae polypeptide having PGL activity, galactose meta same cells lacking one or more copies of a heterologous gene bolic activity, and/or molybdenum transport activity com encoding a PGL polypeptide with one or more associated prises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, expression control sequences, one or more copies of a heter 35 19, 20, or more amino acid substitutions compared to the ologous gene encoding one or more galactose metabolism native Saccharomyces cerevisiae. In some aspects, the amino polypeptides, and/or one or more copies of a heterologous acid substitutions are conservative. In some aspects, the gene encoding one or more molybdenum transport polypep amino acid substitutions are non-conservative. In some tides when the cells are cultured in minimal medium. In some aspects, the Saccharomyces cerevisiae polypeptide having aspects, the one or more copies of a heterologous gene encod 40 PGL activity has 99%, 98%, 97%, 96%. 95%, 95%, 93%, ing a PGL polypeptide with one or more associated expres 92%, 91%, 90%, 89%, 88%, 87%. 86%, or 85% amino acid sion control sequences, the one or more copies of a heterolo sequence identity to the native Saccharomyces cerevisiae gous gene encoding one or more galactose metabolism polypeptide. polypeptides, and/or the one or more copies of a heterologous In some aspects, the bacterial cells of an Escherichia coli gene encoding one or more molybdenum transporter 45 strain that does not encode a 6-phosphogluconolactonase polypeptides are chromosomal copies (e.g., integrated into (PGL) polypeptide, one or more galactose metabolism the E. coli chromosome). In some aspects, the E. coli cells are polypeptides, and/or one or more molybdenum transport in culture. In some aspects, the improved method of produc polypeptides are cultured in minimal medium. In some ing heterologous polypeptides capable of biological activity aspects, the bacterial cells of E. coli strain B are cultured in further comprises a step of recovering the polypeptide. 50 minimal medium. In some aspects, the bacterial cells of E. In some aspects, the bacterial cells of an Escherichia coli coli strain BL21 are cultured in minimal medium. In some strain that does not encode a PGL polypeptide, one or more aspects, the bacterial cells of E. coli strain BL21 (DE3) are galactose metabolic genes, and/or one or more molybdenum cultured in minimal medium. In some aspects, the minimal transport genes are of E. coli Strain B. In some aspects, the medium is supplemented with 1% (w/v) or less glucose. In bacterial cells are of E. coli strain BL21. In some aspects, the 55 Some aspects, the minimal medium is Supplemented with 1% bacterial cells are of E. coli strain BL21 (DE3). (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% In some aspects, the heterologous gene encoding a PGL (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) polypeptide, one or more galactose metabolic genes, and/or glucose. In certain aspects, the minimal medium is Supple one or more molybdenum transport genes is from E. coli mented 0.1% (w/v) or less yeast extract. In some aspects, the strain K12 MG 1655. In some aspects, the heterologous gene 60 minimal medium is supplemented with 0.1% (w/v), 0.09% encoding a PGL polypeptide, one or more galactose meta (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), bolic genes, and/or one or more molybdenum transport genes 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast is from a derivative of E. coli strain K12 MG 1655. In some extract. In some aspects, the minimal medium is Supple aspects, the E. coli K12 strain MG 1655 polypeptide having mented with 1% (w/v) glucose or less and 0.1% (w/v) or less. PGL activity, galactose metabolic activity, and/or molybde 65 In some aspects, the minimal medium is Supplemented with num transportactivity comprises 1,2,3,4,5,6,7,8,9, 10, 11, 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substi 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% US 9,353,375 B2 39 40 (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), Chrysosporium sp., C. lucknowense, Gliocladium sp., 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. In Aspergillus sp., Such as A. Oryzae, A. niger; A Sojae, A. japoni some aspects, the minimal medium is M9 medium or TM3 cus, A. nidulans, or A. awamori, Fusarium sp., Such as F. medium. In some aspects, the minimal medium is M9 5 roseum, F. graminum F. cerealis, F. Oxysporuin, or F. venena medium. In some aspects, the minimal medium is TM3 tum, Neurospora sp., Such as N. crassa, Hypocrea sp., Mucor medium. sp., Such as M. miehei, Rhizopus sp. or Emericella sp. In some In some aspects, the heterologous polypeptide capable of aspects, the fungus is A. nidulans, A. awamori, A. Oryzae, A. biological activity comprises one or more polypeptides aculeatus, A. niger; A. japonicus, T. reesei, T. viride, F. involved in the biosynthesis ofterpenoid (isoprenoid) or caro 10 Oxysporum, or F. Solani. In some aspects, the Source organism tenoid compound(s), and the cells produce a terpenoid or is a yeast, such as Saccharomyces sp., Schizosaccharomyces carotenoid at a higher specific productivity than that of the sp., Pichia sp., or Candida sp. In some aspects, the Saccha same cells lacking one or more copies of a heterologous gene romyces sp. is Saccharomyces cerevisiae. encoding a PGL polypeptide with one or more associated In some aspects, the source organism for the heterologous expression control sequences when cultured in minimal 15 polypeptide capable of biological activity is a bacterium. In medium. In some aspects, the method further comprises a Some aspects, the bacterium is of the genus Bacillus, Such as step of recovering the terpenoid or carotenoid. B. lichenformis or B. subtilis, the genus Pantoea, such as P 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 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 25 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 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 30 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 (such as Populus alba or Populus albaxtremula 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 35 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 40 ales, Nostocales, or Stigonematales. consisting of hemiterpenoids, monoterpenoids, sesquiterpe Isoprene Compositions Produced from Renewable Resources noids, diterpenoids, sesterterpenoids, triterpenoids, tetrater 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 45 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 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. 50 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 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 55 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. 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. 60 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. 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 65 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, isoprene dimer or a linear isoprene trimer derived from the US 9,353,375 B2 41 42 polymerization of multiple isoprene units). Products derived Exemplary DXS polypeptides and nucleic acids and methods from isoprene produced from renewable resources contain of measuring DXS activity are described in more detail in one or more of the biological byproducts or compounds International Publication No. WO 2009/076676, U.S. patent derived from any of the by-products. In addition, products application Ser. No. 12/335,071 (US Publ. No. 2009/ derived from isoprene produced from renewable resources 0203102), WO 2010/003007, US Publ. No. 2010/0048964, may contain compounds formed from these by-products dur WO 2009/132220, and US Publ. No. 2010/0003716. ing Subsequent chemical conversion. Examples of such com Exemplary DXP Pathway Polypeptides and Nucleic Acids pounds include those derived from Diels-Alder cycloaddition Exemplary DXP pathways polypeptides include, but are of dienophiles to isoprene, or the oxidation of isoprene. not limited to any of the following polypeptides: DXS Isoprene compositions produced from renewable 10 polypeptides, DXR polypeptides, MCT polypeptides, CMK resources, including particular byproducts or impurities, are polypeptides, MCS polypeptides, HDS polypeptides, HDR described in more detail in U.S. Provisional Patent Applica polypeptides, IDI polypeptides, and polypeptides (e.g., tion No. 61/187,959 and WO 2010/14825. fusion polypeptides) having an activity of one, two, or more The amount of isoprene produced by cells can be greatly of the DXP pathway polypeptides. In particular, DXP path increased by introducing a heterologous nucleic acid encod 15 way polypeptides include polypeptides, fragments of ing an isoprene synthase polypeptide (e.g., a plant isoprene polypeptides, peptides, and fusions polypeptides that have at synthase polypeptide), a DXS polypeptide, other DXP path least one activity of a DXP pathway polypeptide. Exemplary 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. 25 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 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 30 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. 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 35 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 40 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 5-phosphate (DXP). Standard methods can be used to deter lus alba, Populus migra, Populus trichocarpa, or Populus mine whether a polypeptide has DXS polypeptide activity by albaxtremula (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 45 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 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 50 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 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. 55 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 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 60 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 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 65 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. 5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into US 9,353,375 B2 43 44 2-C-methyl-D-erythritol 2.4-cyclodiphosphate (ME-CPP or PMK, MVD, and IDI nucleic acids. In some aspects, the cells cMEPP). Standard methods can be used to determine whether contain an entire MVA pathway that includes AA-CoA thio a polypeptide has MCS polypeptides activity by measuring lase, HMG-CoA synthase, HMG-CoA reductase, MVK, the ability of the polypeptide to convert CDP-MEP in vitro, in PMK, MVD, and IDI nucleic acids. In some aspects, the cells a cell extract, or in vivo. 5 contain an entire MVA pathway that includes AA-CoA thio HDS polypeptides convert 2-C-methyl-D-erythritol 2,4- lase, HMG-CoA synthase, HMG-CoA reductase, MVK, cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl PMDC, IPK, and IDI nucleic acids. diphosphate (HMBPP or HDMAPP). Standard methods can The E. coli cells described herein can also be used for be used to determine whether a polypeptide has HDS improved methods of producing isoprene and a co-product, polypeptides activity by measuring the ability of the polypep 10 Such as hydrogen, ethanol, or propanediol (e.g., 1,2-pro tide to convert ME-CPP in vitro, in a cell extract, or in vivo. panediol or 1,3-propanediol). Exemplary hydrogenase HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2- polypeptides and nucleic acids, polypeptides and nucleic en-1-yl diphosphate into isopentenyl diphosphate (IPP) and acids for genes related to production of fermentation side dimethylallyl diphosphate (DMAPP). Standard methods can products, and polypeptides and nucleic acids for genes relat be used to determine whether a polypeptide has HDR 15 ing to hydrogen reuptake can also be used with the composi polypeptides activity by measuring the ability of the polypep tions and methods described in. Such polypeptides and tide to convert HMBPP in vitro, in a cell extract, or in vivo. nucleic acids are described in U.S. Publ. No. 2010/0196977 IDI polypeptides convert isopentenyl diphosphate into and WO 2010/078457. dimethylallyl diphosphate. Standard methods can be used to Exemplary Methods for Isolating Nucleic Acids determine whether a polypeptide has IDI polypeptides activ Isoprene synthase, DXS. IDI, DXP pathway, MVA path ity by measuring the ability of the polypeptide to convert way, PGL, hydrogenase, hydrogenase maturation and/or tran isopentenyl diphosphate in vitro, in a cell extract, or in vivo. Scription factor nucleic acids can be isolated using standard Exemplary IDI Polypeptides and Nucleic Acids methods. Methods of obtaining desired nucleic acids from a Isopentenyl diphosphate isomerase polypeptides (isopen Source organism of interest (such as a bacterial genome) are tenyl-diphosphate delta-isomerase or IDI) catalyses the inter 25 common and well known in the art of molecular biology (see, conversion of isopentenyl diphosphate (IPP) and dimethyl for example, WO 2004/033646 and references cited therein). allyl diphosphate (DMAPP) (e.g., converting IPP into Standard methods of isolating nucleic acids, including PCR DMAPP and/or converting DMAPP into IPP). Exemplary amplification of known sequences, synthesis of nucleic acids, IDI polypeptides include polypeptides, fragments of screening of genomic libraries, screening of cosmid libraries polypeptides, peptides, and fusions polypeptides that have at 30 are described in International Publication No. WO 2009/ least one activity of an IDI polypeptide. Standard methods 076676, U.S. patent application Ser. No. 12/335,071 (US (such as those described herein) can be used to determine Publ. No. 2009/0203102), WO 2010/003007, US Publ. No. whether a polypeptide has IDI polypeptide activity by mea 2010/0048964, WO 2009/132220, and US Publ. No. 2010/ suring the ability of the polypeptide to interconvert IPP and OOO3716. DMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI 35 Exemplary Promoters and Vectors polypeptides and nucleic acids and methods of measuring IDI Any of the isoprene synthase, DXS. DXP pathway, IDI, activity are described in more detail in International Publica MVA pathway, PGL, hydrogenase, hydrogenase maturation, tion No. WO 2009/076676, U.S. patent application Ser. No. transcription factor, galactose metabolic, and/or molybde 12/335,071 (US Publ. No. 2009/0203102), WO 2010/ num transport nucleic acids described herein can be included 003007, US Publ. No. 2010/0048964, WO 2009/132220, and 40 in one or more vectors. Accordingly, also described hereinare US Pub1. No. 201O/OOO3716. vectors with one more nucleic acids encoding any of the Exemplary MVA Pathway Polypeptides and Nucleic Acids isoprene synthase, DXS. IDI, DXP pathway, MVA pathway, Exemplary MVA pathway polypeptides include acetyl PGL, hydrogenase, hydrogenase maturation, transcription CoA acetyltransferase (AA-CoA thiolase) polypeptides, factor polypeptides, galactose metabolic polypeptides, and/ 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA syn 45 or molybdenum transport polypeptides that are described thase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA herein. In some aspects, the vector contains a nucleic acid reductase (HMG-CoA reductase) polypeptides, mevalonate under the control of an expression control sequence. In some kinase (MVK) polypeptides, phosphomevalonate kinase aspects, the expression control sequence is a native expres (PMK) polypeptides, diphosphomevalonate decarboxylase sion control sequence. In some aspects, the expression con (MVD) polypeptides, phosphomevalonate decarboxylase 50 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 55 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. 60 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 65 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 US 9,353,375 B2 45 46 using Such vectors are known to those in the art (see, for Exemplary Source Organisms example, Sambrook et al., Molecular Cloning: A Laboratory Isoprene synthase. DXP pathway, IDI, MVA pathway, Manual. 2" ed., Cold Spring Harbor, 1989). Suitable vectors PGL, hydrogenase, hydrogenase maturation, transcription compatible with the cells and methods described herein are factor, galactose metabolic and/or molybdenum transport described in International Publication No. WO 2009/076676, nucleic acids (and their encoded polypeptides) can be U.S. patent application Ser. No. 12/335,071 (US Publ. No. obtained from any organism that naturally contains isoprene 2009/0203102), WO 2010/003007, US Publ. No. 2010/ synthase, DXP pathway, IDI, MVA pathway, PGL, hydroge 0048964, WO 2009/132220, and US Publ. No. 2010/ nase, hydrogenase maturation, transcription factor, galactose OOO3716. metabolic and/or molybdenum transport nucleic acids. As Promoters are well known in the art. Any promoter that 10 noted above, isoprene is formed naturally by a variety of functions in the host cell can be used for expression of an organisms. Such as bacteria, yeast, plants, and animals. isoprene synthase, DXS. IDI, DXP pathway, MVA pathway, Organisms contain the MVA pathway, DXP pathway, or both PGL, hydrogenase, hydrogenase maturation, transcription the MVA and DXP pathways for producing isoprene (FIGS. factor, galactose metabolic and/or molybdenum transport 1A and 1B). Thus, DXP pathway nucleic acids can be nucleic acid in the host cell. Initiation control regions or 15 obtained, e.g., from any organism that contains the DXP 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 25 obtained or identified, e.g., from any organism that undergoes patible with the cells and methods described herein are oxygen-limited or anaerobic respiration, such as glycolysis. described in International Publication No. WO 2009/076676 The nucleic acid sequence of the isoprene synthase, DXP A2 and U.S. Patent Application Publication No. US2009/ pathway, IDI, MVA pathway, PGL, hydrogenase, hydroge O2O31 O2A1. nase maturation, transcription factor, galactose metabolic In some aspects, the expression vector also includes a 30 and/or molybdenum transport nucleic acids can be isolated termination sequence. Termination control regions may also from a bacterium, fungus, plant, algae, or cyanobacterium. be derived from various genes native to the host cell. In some Exemplary source organisms include, for example, yeasts, aspects, the termination sequence and the promoter sequence Such as species of Saccharomyces (e.g., S. cerevisiae), bacte are derived from the same source. Suitable termination ria, Such as species of Escherichia (e.g., E. coli), or species of sequences compatible with the cells and methods described 35 Methanosarcina (e.g., Methanosarcina mazei), plants, such herein are described in International Publication No. WO as kudzu or poplar (e.g., Populus alba or Populus albax 2009/076676A2 and U.S. Patent Application Publication No. tremula CAC35696) or aspen (e.g., Populus tremuloides). US2O09/O2O31 O2 A1 Exemplary host organisms are described in International An isoprene synthase, DXS. IDI, DXP pathway, MVA Publication No. WO 2009/076676, U.S. patent application pathway, PGL, hydrogenase, hydrogenase maturation, tran 40 Ser. No. 12/335,071 (US Publ. No. 2009/0203102), WO Scription factor, galactose metabolic and/or molybdenum 2010/003007, US Publ. No. 2010/0048964, WO 2009/ nucleic acid can be incorporated into a vector, Such as an 132220, and US Publ. No. 2010/0003716. expression vector, using standard techniques (Sambrook et Exemplary Transformation Methods al., Molecular Cloning: A Laboratory Manual, Cold Spring Isoprene synthase. DXP pathway, IDI, MVA pathway, Harbor, 1982). Suitable techniques compatible with the cells 45 PGL, hydrogenase, hydrogenase maturation, transcription and methods described herein are described in International factor, galactose metabolic and/or molybdenum transport Publication No. WO 2009/076676A2 and U.S. Patent Appli nucleic acids or vectors containing them can be inserted into cation Publication No. US2009/0203102 A1. a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a In some aspects, it may be desirable to over-express iso bacterial cell described herein) using standard techniques for prene synthase, DXP pathway, IDI, MVA pathway, PGL, 50 introduction of a DNA construct or vector into a host cell, hydrogenase, hydrogenase maturation, transcription factor, Such as transformation, electroporation, nuclear microinjec galactose metabolic and/or molybdenum transport nucleic tion, transduction, transfection (e.g., lipofection mediated or acids at levels far higher than currently found in naturally DEAE-Dextrin mediated transfection or transfection using a occurring cells. In some aspects, it may be desirable to under recombinant phage virus), incubation with calcium phos express (e.g., mutate, inactivate, or delete) isoprene synthase, 55 phate DNA precipitate, high velocity bombardment with DXP pathway, IDI, MVA pathway, PGL, hydrogenase, DNA-coated microprojectiles, and protoplast fusion. General hydrogenase maturation, transcription factor polypeptide, transformation techniques are known in the art (see, e.g., galactose metabolic polypeptide and/or molybdenum trans Current Protocols in Molecular Biology (F. M. Ausubel et al. port polypeptide-encoding nucleic acids at levels far below (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A that those currently found in naturally-occurring cells. Suit 60 Laboratory Manual, 2" ed., Cold Spring Harbor, 1989; and able methods for over- or under-expressing the isoprene Syn Campbell et al., Curr. Genet. 16:53-56, 1989). The intro thase, DXP pathway, IDI, MVA pathway, PGL, hydrogenase, duced nucleic acids may be integrated into chromosomal hydrogenase maturation, transcription factor, galactose meta DNA or maintained as extrachromosomal replicating bolic and/or molybdenum transport nucleic acids compatible sequences. Transformants can be selected by any method with cells and methods described herein are described in 65 known in the art. Suitable methods for selecting transfor International Publication No. WO 2009/076676 A2 and U.S. mants are described in International Publication No. WO Patent Application Publication No. US2009/0203102A1. 2009/076676, U.S. patent application Ser. No. 12/335,071 US 9,353,375 B2 47 48 (US Publ. No. 2009/0203102), WO 2010/003007, US Publ. plates (water+cells control plate for reversion and water and No. 2010/0048964, WO 2009/132220, and US Publ. No. P1 lysate control plate for lysate contamination. 201O/OOO3716. Four transductants were picked and used to inoculate 5 mL. Exemplary Purification Methods L. Broth and 20 g/ul chloramphenicol. The cultures were In some aspects, any of the methods described herein fur grown overnight at 30°C. with shaking at 200 rpm. To make ther include a step of recovering the compounds produced. In genomic DNA preparations of each transductant for PCR some aspects, any of the methods described herein further analysis, 1.5 mL of overnight cell culture were centrifuged. include a step of recovering the isoprene. In some aspects, the The cell pellet was resuspended with 400 ul Resuspension isoprene is recovered by absorption stripping (see, e.g., U.S. Buffer (20 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 7.5) and 10 4 ul RNase, DNase-free (Roche) was added. The tubes were Prov. 61/288,142 or U.S. application Ser. No. 12/969,440). In incubated at 37°C. for 30 minutes followed by the addition of some aspects, any of the methods described herein further 4 Jul 10% SDS and 4 ul of 10 mg/ml Proteinase K stock include a step of recovering the heterologous polypeptide. In solution (Sigma-Aldrich). The tubes were incubated at 37°C. some aspects, any of the methods described herein further for 1 hour. The cell lysate was transferred into 2 ml Phase include a step of recovering the terpenoid or carotenoid. 15 Lock Light Gel tubes (Eppendorf) and 200 ul each of satu 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 25 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 30 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 35 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. 40 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) 45 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, 50 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., 55 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 60 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 65 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, US 9,353,375 B2 49 50 but not LA and 20 ugful chloramphenicol plates, were the correct size and shipped to Quintara Biosciences (Berke deemed to have the chloramphenicol marker looped out ley, Calif.) for sequencing with primers MCM65, MCM66, (picked one and designated as strain EWL204). EL1000 (Table 1). DNA sequencing results showed all 6 (iii) Construction of Plasmid pEWL230 (pTrc P alba) plasmids were correct. One plasmid was picked designated as Generation of a synthetic gene encoding Populus alba plasmid EWL230 (FIGS. 5, 6A-B: SEQID NO:2). isoprene synthase (P alba HGS) was outsourced to DNA2.0 iv) Construction of PlasmidpEWL244 (pTrcP alba-mMVK) Inc. (Menlo Park, Calif.) based on their codon optimization A PCR reaction was performed to amplify the Methanosa method for E. coli expression. The synthetic gene was custom rcina mazei (M. mazei) MVK gene using MCM376 as the cloned into plasmid pET24a (Novagen brand, EMD Bio template, primers MCM165 and MCM177 (see Table 1), and sciences, Inc.) and delivered lyophilized (FIGS. 2, 3A-B; 10 Pfu Ultra II Fusion DNA polymerase (Stratagene) according SEQID NO:1). A PCR reaction was performed to amplify the P alba to manufacturer's protocol. PCR conditions were as follows: isoprene synthase (P alba HGS) gene using pET24 P alba 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, HGS as the template, primers MCM182 and MCM192, and 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28 Herculase II Fusion DNA polymerase (Stratagene) according 15 cycles, with final extension at 72° C. for 1 minute. The M. 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 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 was incubated for 2 hours at 37°C. The digested PCR frag 25 containing 2 ul NsiI endonuclease (Roche), 4.7 ul 10x Buffer ment was then purified using the QIAquick PCR Purification H, and 40 ul of Pme digested M. mazei MVK fragment. The Kit. A secondary restriction digest was performed in a 20 ul reaction containing 1 Jul PstI endonuclease (Roche) with 2 ul reaction was incubated for 3 hours at 37°C. The digested PCR 10x Buffer H. The reaction was incubated for 2 hours at 37° fragment was then gel purified using a 1.2% E-gel and C. The digested PCR fragment was then purified using the 30 extracted using the QIAquick Gel Extraction Kit. Plasmid QIAquick PCR Purification Kit. Plasmid pTrcHis2B (Invit EWL230 was digested in a 40 ul reaction containing 10 ul rogen Corp.) was digested in a 20 Jul reaction containing 1 ul plasmid, 2 ul PmeI endonuclease, 4 Jul 10xNEB Buffer 4, 4 ul NcoI endonuclease (Roche), 1 ul PstI endonuclease, and 2 ul 10xNEB BSA, and 20 ul of ddHO. The reaction was incu 10x Buffer H. The reaction was incubated for 2 hours at 37° bated for 3 hours at 37°C. The digested PCR fragment was C. The digested pTrcHis2B vector was gel purified using a 35 then purified using the QIAquick PCR Purification Kit. A 1.2% E-gel (Invitrogen Corp.) and extracted using the secondary restriction digest was performed in a 47 ul reaction QIAquick Gel Extraction Kit (Qiagen) (FIG. 4). Using the containing 2 ulPstI endonuclease, 4.7 ul 10x Buffer H, and 40 compatible cohesive ends of BspHI and NcoI sites, a 20 ul ul of PmeI digested EWL230 linear fragment. The reaction ligation reaction was prepared containing 5 ul P alba iso was incubated for 3 hours at 37°C. The digested PCR frag prene synthase insert, 2 ul pTrc vector, 1 ul T4 DNA ligase 40 (New England Biolabs), 2 Jul 10x ligase buffer, and 10 ul ment was then gel purified using a 1.2% E-gel and extracted ddHO. The ligation mixture was incubated at room tempera using the QIAquick Gel Extraction Kit (FIG. 7). Using the ture for 40 minutes. The ligation mixture was desalted by compatible cohesive ends of NsiI and PstI sites, a 20 ul floating a 0.025 um nitrocellulose membrane filter (Milli ligation reaction was prepared containing 8 ul M. mazei MVK pore) in a petri dish of ddH2O and applying the ligation 45 insert, 3 ul. EWL230 plasmid, 1 ul T4 DNA ligase, 2 Jul 10x mixture gently on top of the nitrocellulose membrane filter ligase buffer, and 6 ul ddHO. The ligation mixture was for 30 minutes at room temperature. MCM446 cells were incubated overnight at 16°C. The next day, the ligation mix grown in LB to midlog phase and then washed three times in ture was desalted by floating a 0.025 um nitrocellulose mem ice-cold, sterile water. An aliquot of 50 ul of cell suspension brane filter in a petridish of ddHO and applying the ligation was mixed with 5 ul of desalted pTrc P alba HGS ligation 50 mix. The cell Suspension mixture was electroporated in a 2 mixture gently on top of the nitrocellulose membrane filter mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser for 30 minutes at room temperature. MCM446 cells were Electroporator. 1 ml of LB was immediately added to the grown in LB to midlog phase and then washed three times in cells, then transferred to a 14 ml polypropylene tube ice-cold, sterile water. An aliquot of 50 ul of cell suspension (Sarstedt) with a metal cap. Cells were allowed to recover by 55 was mixed with 5ul of desalted pTrc P alba-mMVK ligation growing for 2 hour at 30°C. Transformants were selected on mix. The cell Suspension mixture was electroporated in a 2 LAgar and 50 ug/ul carbenicillin and 10 mM mevalonic acid mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser and incubated at 30°C. The next day, 6 transformants were Electroporator. 1 ml of LB is immediately added to the cells, picked and grown in 5 ml L. Broth and 50 lug/ul carbenicillin then the cells are transferred to a 14 ml polypropylene tube tubes overnight at 30° C. Plasmid preps were performed on 60 the overnight cultures using QIAquick Spin Miniprep Kit with a metal cap. Cells were allowed to recover by growing (Qiagen). Due to the use of BL21 cells for propagating plas for 2 hour at 30°C. Transformants were selected on LA and mids, a modification of washing the spin columns with PB 50 ug/ul carbenicillin and 5 mM mevalonic acid plates and Buffer 5x and PE Buffer 3x was incorporated to the standard incubated at 30°C. The next day, 6 transformants were picked manufacturer's protocol for achieving high quality plasmid 65 and grown in 5 ml LB and 50 lug/ul carbenicillin tubes over DNA. Plasmids were digested with PstI in a 20 ul reaction to night at 30° C. Plasmid preps were performed on the over ensure the correct sized linear fragment. All 6 plasmids were night cultures using QIAquick Spin Miniprep Kit. Due to the US 9,353,375 B2 51 52 use of BL21 cells for propagating plasmids, a modification of TABLE 1. washing the spin columns with PB Buffer 5x and PE Buffer 3x was incorporated to the standard manufacturer's protocol Primer Sequences for achieving high quality plasmid DNA. Plasmids were Primer digested with PstI in a 20 ul reaction to ensure the correct ale Primer sequence sized linear fragment. Three of the 6 plasmids were the cor MCM13 O ACCAATTGCACCCGGCAGA (SEQ ID NO: 14) rect size and shipped to Quintara Biosciences for sequencing GB Cm GCTAAAGCGCATGCTCCAGAC (SEO ID NO: 15) with primers MCM65, MCM66, EL1000, EL1003, and Rew 10 EL 1006 (Table 1). DNA sequencing results showed all 3 WD GACTGGCCTCAGATGAAAGC (SEQ ID NO: 16) plasmids were correct. One was picked and designated as For plasmid EWL244 (FIGS. 8 and 9A-B; SEQID NO:3). WD CAAACATGTGGCATGGAAAG (SEQ ID NO: 17) v) Construction of Plasmid MCM376-MVK from M. mazei Rew Archaeal Lower in pET200D. 15 The MVK ORF from the M. mazei archaeal Lower Path CM182 GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTT way operon (FIGS. 10A-C: SEQID NO:4) was PCR ampli CAAACGGCAGAA (SEQ ID NO: 18) fied using primers MCM161 and MCM162 (Table 1) using CM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA the Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix (SEQ ID NO: 19) was combined with 1 uL template, 1 uL of each primer at 10 CM65 ACAATTTCACACAGGAAACAGC (SEQ ID NO: 2O) uM, and 2 LL water. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30 cycles of 94° C. for 0:30 minutes, 55° CM66 CCAGGCAAATTCTGTTTTATCAG (SEO ID NO: 21) C. for 0:30 minutes and 68°C. for 1:15 minutes; and then 72° EL1OOO GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 22) C. for 7:00 minutes, and 4° C. until cool. 3 uIl of this PCR reaction was ligated to Invitrogen pET200D plasmid accord 25 CM165 GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCT ing to the manufacturer's protocol. 3 uIl of this ligation was GTTCTGCGCCGGGTAAGATTTACCTG introduced into Invitrogen TOP10 cells, and transformants (SEQ ID NO: 23) were selected on LA/kans0. A plasmid from a transformant CM177 GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCA was isolated and the insert sequenced, resulting in MCM376 GACCTTGC (SEO ID NO. 24) 30 (FIGS. 11A-C). EL10O3 GATAGTAACGGCTGCGCTGCTACC (SEO ID NO: 25) vi) Construction of Strain EWL251 (BL21 (DE3), Cm-GI1.2- KKDyI, pTrc P. Alba-mMVK) EL1OO6 GACAGCTTATCATCGACTGCACG (SEO ID NO: 26) MCM331 cells (which contain chromosomal construct MCM161 CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 27) gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, 35 mevalonate phosphate kinase, mevalonate pyrophosphate MCM162 TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 28) decarboxylase, and IPP isomerase) were grown in LB to midlog phase and then washed three times in ice-cold, sterile viii) Construction of Strain RM111608-2 (Cm-GI1.2- water. Mixed 50 ul of cell suspension with 1 ul of plasmid KKDyI, pTrc P alba-mMVK, pCL Upper MVA, pBBRC EWL244. The cell suspension mixture was electroporated in 40 MPGI1.5-pgl) a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser The BL21 strain of E. coli producing isoprene (EWL256) Electroporator. 1 ml of LB is immediately added to the cells, was constructed with constitutive expression of the ybhE and then the cells were transferred to a 14 ml polypropylene gene (encoding E. coli 6-phosphogluconolactonase) on a rep tube with a metal cap. Cells were allowed to recover by licating plasmid pPBR1MCS5(Gentamycin) (obtained from growing for 2 hours at 30°C. Transformants were selected on 45 Dr. K. Peterson, Louisiana State University). LA and 50 g/ul carbenicillin and 5 mM mevalonic acid FRT-based recombination cassettes, and plasmids for Red/ plates and incubated at 37°C. One colony was selected and ET-mediated integration and antibiotic marker loopout were designated as strain EWL251. obtained from Gene Bridges GmbH (Germany). Procedures vii) Construction of Strain EWL256 (BL21 (DE3), using these materials were carried out according to Gene Cm-GI1.2-KKDyI, pTrc P Alba-mMVK, pCL Upper MVA) 50 Bridges protocols. Primers Pgl-F (SEQ ID NO:29) and EWL251 cells were grown in LB to midlog phase and then PglGI1.5-R (SEQID NO:30) were used to amplify the resis washed three times in ice-cold, sterile water. Mixed 50 ul of tance cassette from the FRT-gb2-Cm-FRT template using cell suspension with 1 ul of plasmid MCM82 (comprising Stratagene Herculase II Fusion kit according to the manufac pCL PtrcUpperPathway (also known as “pCLUpper MVA), turer's protocol. The PCR reaction (50 uL final volume) con encoding E. faecalis mvaE and mvaS). Plasmid pCL Ptrc 55 tained: 5uL buffer, 1 uL template DNA (FRT-gb2-Cm-F from Upper Pathway was constructed as described in Example 8 of Gene Bridges), 10 pmols of each primer, and 1.5 uL 25 mM International Publication No. WO 2009/076676 A2 and U.S. dNTP mix, made to 50 uL with dHO. The reaction was patent application Ser. No. 12/335,071 (US Publ. No. 2009/ cycled as follows: 1x2 minutes, 95°C. then 30 cycles of (30 0203102). The cell suspension mixture was electroporated in seconds at 95°C.; 30 seconds at 63°C.: 3 minutes at 72° C.). a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser 60 The resulting PCR product was purified using the Electroporator. 1 ml of LB was immediately added to the QIAquick R. PCR Purification Kit (Qiagen) and electropo cells. Cells were then transferred to a 14 ml polypropylene rated into electrocompetent MG 1655 cells harboring the tube with a metal cap. Cells were allowed to recover by pRed-ET recombinase-containing plasmid as follows. Cells growing for 2 hours at 30°C. Transformants were selected on were prepared by growing in 5 mLs of L broth to and LA and 50 ug/ul carbenicillin and 50 g/ul spectinomycin 65 OD600-0.6 at 30°C. The cells were induced for recombinase plates and incubated at 37° C. One colony was picked and expression by the addition of 4% arabinose and allowed to designated as strain EWL256. grow for 30 minutes at 30° C. followed by 30 minutes of US 9,353,375 B2 53 54 growth at 37° C. An aliquot of 1.5 mLs of the cells was - Continued washed 3-4 times in ice cold dHO. The final cell pellet was resuspended in 40 uL of ice cold dHO and 2-5 uL of the PCR Primers: product was added. The electroporation was carried out in Bottom Pgb2: 1-mm gap cuvettes, at 1.3 kV in a Gene Pulser Electroporator 5 (SEQ ID NO: 33) (Bio-Rad Inc.). Cells were recovered for 1-2 hours at 30°C. GGTTTAGTTCCT CACCTTGTC and plated on Lagar containing chloramphenicol (5 g/mL). Top GB's CMP (946) : Five transformants were analyzed by PCR and sequencing (SEQ ID NO: 34) using primers flanking the integration site (2 primer sets: pgl ACTGAAACGTTTTCATCGCTC and 49 rev and 3' EcoRV-pglstop; Bottom Pgb2 and Top GB’s 10 Pglconfirm-F CMP (946)). A correct transformant was selected and this (SEO ID NO : 35) strain was designated MG1655 GI1.5-pgl::CMP. 5'-ACCGCCAAAAGCGACTAATTTTAGCT-3' The chromosomal DNA of MG1655 GI1.5-pgl:CMP was used as template to generate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct. This construct was 15 cloned into pBBR1MCS5(Gentamycin) as follows. The frag Example 2 ment, here on referred to as CMP-GI1.5-pgl, was amplified using the 5' primer Pglconfirm-F (SEQ ID NO:31) and 3' Improvement of Isoprene Production by Constitutive primer 3' EcoRV-pglstop (SEQ ID NO:32). The resulting Expression of ybhE (Pgl) from a Plasmid in E. coli fragment was cloned using the Invitrogen TOPO-Blunt clon ing kit into the plasmid vector pCR-Blunt II-TOPO as sug This example shows production of isoprene in a strain gested from the manufacturer. The Nsil fragment harboring constitutively expressing E. coli ybhE (pgl) compared to a the CMP-GI1.5-pgl fragment was cloned into the PstI site of control strain expressing ybhE at wild-type levels (i.e., pBBR1MCS5 (Gentamycin). A 20 Jul ligation reaction was EWL256). The gene ybhE (pgl) encodes E. coli 6-phospho prepared containing 5 ul CMP-GI1.5-pgl insert, 2 ul 25 gluconolactonase that Suppresses posttranslational glucony pBBR1MCS5 (Gentamycin) vector, 1 ul T4 DNA ligase lation of heterologously expressed proteins and improves (New England Biolabs), 2 Jul 10x ligase buffer, and 10 ul product solubility and yield while also improving biomass ddH2O. The ligation mixture was incubated at room tempera yield and flux through the pentose phosphate pathway (Aonet ture for 40 minutes then 2-4 uL were electroporated into al., Applied and Environmental Microbiology 74(4): 950 electrocompetent Top10 cells (Invitrogen) using the param 30 958, 2008). eters disclosed above. Transformants were selected on Lagar i) Small Scale Analysis containing 10 g/ml chloramphenicol and 5 g/ml Gentamy Media Recipe (per liter fermentation media): KHPO 13.6 cin. The sequence of the selected clone was determined using g, KHPO, 13.6 g. MgSO.7HO 2 g, citric acid monohy a number of the primers described above as well as with the drate 2 g, ferric ammonium citrate 0.3 g. (NH4)2SO 3.2 g, in-house T3 and Reverse primers provided by Sequetech, 35 yeast extract 1 g, 1000x Trace Metals Solution 1 ml. All of the Calif. This plasmid was designated pBBRCMPGI1.5-pg| components were added together and dissolved in diH2O. (FIGS. 12, 13 A-B and SEQID NO:6). The pH was adjusted to 6.8 with ammonium hydroxide (30%) Plasmid pPBRCMPGI1.5-pgl was electroporated into and brought to volume. Media was filter-sterilized with a 0.22 EWL256, as described herein and transformants were plated micron filter. Glucose 5.0 g and antibiotics were added after on Lagar containing Chloramphenicol (10 ug/mL), Genta 40 sterilization and pH adjustment. mycin (5 g/mL), spectinomycin (50 ug/mL), and carbenicil 1000x Trace Metal Solution (per liter fermentation media): lin (50 ug/mL). One transformant was selected and desig Citric Acid'HO 40 g, MnSOHO 30 g, NaCl 10 g, nated Strain RM1 11608-2. FeSO.7HO 1 g, CoCl*6HO 1 g, ZnSO*7HO 1 g, CuSO4.5H2O 100 mg., HBO 100 mg. NaMoO2HO 100 45 mg. Each component was dissolved one at a time in diFIO. Primers: The pH was adjusted to 3.0 with HCl/NaOH, and then the Pgl-F solution was brought to volume and filter-sterilized with a (SEQ ID NO: 29) 0.22 micron filter. 5'-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGA 50 (a) Experimental Procedure ATTAACCCTCACTAAAGGGCGGCCGC-3' PglGI1.5-R Isoprene production was analyzed by growing the strains in (SEQ ID NO: 30) a CelleratorTM from MicroReactor Technologies, Inc. The s' - GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGG working volume in each of the 24 wells was 4.5 mL. The 55 temperature was maintained at 30° C., the pH setpoint was GTTAC CTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTA 7.0, the oxygen flow setpoint was 20 scem and the agitation TTTGCTCAGGATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAA rate was 800 rpm. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with TACGACT CACTATAGGGCTCGAG-3 antibiotics) and incubated at 30° C. A single colony was 3' EcoRV-pglstop: 60 inoculated into media with antibiotics and grown overnight. (SEQ ID NO: 31) The bacteria were diluted into 4.5 mL of media with antibi s' - CTT GAT ATC TTA. GTG TGC GTT AAC CAC CAC otics to reach an optical density of 0.05 measured at 550 nm. Off-gas analysis of isoprene was performed using a gas pgll + 49 rev: (SEQ ID NO: 32) chromatograph-mass spectrometer (GC-MS) (Agilent) head CGTGAATTTGCTGGCTCTCAG 65 space assay. Sample preparation was as follows: 100 L of whole broth was placed in a sealed GC vial and incubated at 30° C. for a fixed time of 30 minutes. Following a heat kill US 9,353,375 B2 55 56 step, consisting of incubation at 70° C. for 5 minutes, the fermentation to a maximum value of 40.0 g/L at 40 hours sample was loaded on the GC.. (60.5 g/L at 59 hours) (FIG. 14C). The total amount of iso Optical density (OD) at a wavelength of 550 nm was prene produced during the 40-hour (59-hour) fermentation obtained using a microplate reader (Spectramax) during the was 281.3 g (451.0 g at 59 hours) and the time course of course of the run. Specific productivity was obtained by production is shown in FIG. 14D. The time course of volu dividing the isoprene concentration (ug/L) by the ODreading metric productivity is shown in FIG. 14E and shows that an and the time (hour). average rate of 1.0 g/L/hr was maintained between 0 and 40 The two strains EWL256 and RM11608-2 were assessed at hours (1.4 g/L/hour between 19 and 59 hour). The metabolic 200 and 400 uM IPTG induction levels. Samples were ana activity profile, as measured by CER, is shown in FIG. 14F. lyzed for isoprene production and cell growth (OD550) at 1, 10 The molar yield of utilized carbon that went into producing 2.5, 4.75, and 8 hours post-induction. Samples were done in isoprene during fermentation was 19.6% at 40 hours (23.6% duplicate. at 59 hours). The weight percent yield of isoprene from glu cose was 8.9% at 40 hours (10.7% at 59 hours). (b) Results 15 Example 3 The experiment demonstrated that at 2 different concen trations of IPTG the strain expressing the ybhE (pgl) had a Recovery of Isoprene Produced from Renewable dramatic 2-3 fold increase in specific productivity ofisoprene Resources compared to the control strain. ii) Isoprene Fermentation from E. coli Expressing Cm-GI1.2- Isoprene was recovered from a set of four 14-L scale fer KKDyI. M. mazei Mevalonate Kinase, P alba Isoprene Syn mentations in a two-step operation involving stripping of thase, and ybhE (Pgl) (RM111608-2) and Grown in Fed isoprene from the fermentation off-gas stream by adsorption Batch Culture at the 15-L Scale to activated carbon, followed by off-line steam desorption Medium Recipe (per liter fermentation medium): KHPO and condensation to give liquid isoprene (FIGS. 16A and 7.5 g. MgSO47H2O2 g, citric acid monohydrate 2 g, ferric 25 16B). The total amount of isoprene produced by the four ammonium citrate 0.3 g yeast extract 0.5g, 1000x Modified fermentors was 1150 g (16.9 mol), of which 953 g (14 mol, 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 30 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, CuSO5HO 100 mg, 35 determine the nature and levels of impurities. The product HBO 100 mg. NaMoO2H2O 100 mg. Each component 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 40 rity profile was similar to other isoprene batches produced on BL21 (DE3) E. coli cells containing the upper mevalonic acid 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 45 Summary of the nature and levels of impurities seen in (pBBR-pgl). This experiment was carried out to monitor iso several 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 50 Ethanol 1.59 11.89 <50 ppm nm, 500 mL was used to inoculate a 15-L bioreactor bringing Acetone 1624 12.673 <100 ppm the initial volume to 5-L. Methacrolein 1851 15.369 <200 ppm Methyl vinyl ketone 1923 16.333 <20 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- 2.27 18.875 50 to 500 ppm decreased to meet metabolic demands. The total amount of 55 pentadiene glucose delivered to the bioreactor during the 40 hour (59 Methyl vinyl oxirane 2.548 19.931 <100 ppm Isoprenol 2.962 21.583 <500 ppm hour) fermentation was 3.1 kg (4.2 kg at 59 hour). Induction 3-methyl-1-butanol 2.99 21.783 <50 ppm was achieved by adding IPTG. The IPTG concentration was 3-hexen-1-ol 4.019 24.8.19 <100 ppm brought to 110 uM when the optical density at 550 nm Isopentenyl acetate 4.466 25.733 200 to 1000 ppm (ODsso) reached a value of 4. The IPTG concentration was 60 3-hexen-1-yl acetate 5.339 27.223 <400 ppm raised to 192M when ODsso reached 150. The ODsso profile limonene 5.715 27.971 <500 ppm within the bioreactor over time is shown in FIG. 14A. The Other cyclics S.SO-6.SO 27.5-28.0 <200 ppm isoprene level in the off gas from the bioreactor was deter mined using a Hiden mass spectrometer. The isoprene titer Purification of Isoprene Produced from Renewable increased over the course of the fermentation to a maximum 65 Resources by Treatment with Adsorbents value of 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. Adsorbents are widely used by industry for the removal of 14B). The isoprene titer increased over the course of the trace impurities from hydrocarbon feedstocks. Suitable US 9,353,375 B2 57 58 adsorbents include Zeolite, alumina and silica-based materi above, and then recovered in 500 uL SOC medium at 37° C. als. Isoprene produced from renewable resources can be Sub for 1.5-3 hours. Transformants were selected on LB+ 10 stantially purified by passage over silica gel, and to a lesser ug/mL, kanamycin (LB/kan10) plates at 37° C. extent with alumina. FIG. 18 shows the GC/FID chromato Recombination of the amplicon at the target locus was grams of an isoprene sample before (A) and after treatment confirmed by PCR with primers GB-DW (SEQ ID NO:38) with alumina (B) or silica (C). The SelexsorbTM adsorbent and MCM208 (SEQ ID NO:39). The resulting amplicons products from BASF is one of the adsorbents of choice for the were sequenced to identify four clones having the sequences removal of polar impurities from isoprene produced from listed below. Four carbenicillin-sensitive clones were frozen renewable resources. Specifically, the SelexsorbTM CD and as Strains MCM518-MCM521. CDX products are preferred given their proven utility for 10 Strains MCM518-MCM521 were re-streaked onto removal of polar impurities from isoprene and butadiene LB/kan 10 and grown overnight at 37° C. Colonies of strains feedstocks. MCM518-MCM521 were picked, cultured in LB/kan 10 at Example 4 37° C. and electrotransformed with plasmid pCP20, which 15 encodes the yeast Flp recombinase, chloramphenicol and Construction of Strains MCM518-521 and 528-531: ampicillin resistance genes and confers temperature sensitive Lambda Promoters Driving Integrated mKKDyI replication on host cells (Cherepanov, P. P. et al., Gene 158 (1):9-14 (1995)). Cells were recovered in 500 uL SOC P1 transduction enables movement of up to 100kb of DNA medium by shaking at 30°C. for 1 hour. Transformants were between bacterial strains (Thomason et al. 2007). A 17,257 bp selected on LB/carb50 plates at 30°C. overnight. The follow deletion in E. coli BL21 (DE3) (see FIG. 20) was replaced by ing morning a colony from each plate was grown at 30°C. in moving a piece of the bacterial chromosome from E. coli K12 LB/carb50 medium until visibly turbid. The culture was then MG1655 to E. coli BL21 (DE3) using P1 transduction. shifted to 37°C. for at least 3 hours. Cells were streaked from Two strategies were used employing different selectable that culture onto LB plates and grown overnight at 37° C. markers to identify colonies containing the recombined bac 25 The following day colonies were patched to LB, terial chromosome. First, an antibiotic marker in a gene close LB/carb50 and LB/kan10. Clones that were sensitive to both to the 17,257 bp sequence to be transferred, whose deletion carbenicillin and kanamycin (i.e., which could not grow on was not likely to be detrimental to the strain, was inserted. A carb50 and kan10) were cultured in liquid LB and frozen as strain containing that antibiotic marker would likely have the Strains MCM528-MCM531. 17.257 bp piece of bacterial chromosome transduced at the 30 same time as the marker. In this case, a gene encoding kana TABLE 3 mycin resistance (“kan”) was inserted into the ybgS gene, E. coli strains encoding a 126 amino acid protein of unknown function. Second, since it is known that a number of genes involved in Strain Description Parent utilization of galactose are close topgl in the 17,257 bp piece 35 MCMSO8 BL21 gi1.6-mKKDyI+ predet.-carb MCM446 to be transduced into E. coli BL21 (DE3), colonies transduced MCMS.18 BL21 neo-PL.6-mKKDyI, clone 10 MCMSO8 with a P1 lysate obtained from E. coli K12 MG1655 (which MCMS 19 BL21 neo-PL.0-mKKDyI, clone 11 MCMSO8 contains the 17,257 bp sequence deleted in E. coli BL21 MCMS2O BL21 neo-PL.0-mKKDyI MCMSO8 (bad RBS in front of mMVK), clone 13 (DE3)) and isolated in M9 medium (6 g/L NaHPO, 3 g/L MCMS21 BL21 neo-PL.2-mKKDyI, clone 15 MCMSO8 KHPO, 0.5g/L NaCl, 0.5 g/L NHCl, 0.1 mM CaCl2 mM 40 MCMS28 BL21 PL.6-mKKDyI, neo looped out MCMS18 MgSO4) containing 0.4% (w/v) galactose would likely con MCMS29 BL21 PL.0-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, neoR looped out MCMS21 tance ("Cm) cassette from the GeneBridges FRT-gb2-Cm 45 FRT template using the Stratagene HerculaseTM II Fusion kit (Agilent Technologies, Stratagene Products Division, La TABLE 4 Jolla, Calif.) according to the manufacturer's protocol. Four 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° 50 Primer C./1 minute; and 72° C./3 minutes. Reactions were then ale Sequence 5' -> 3' cooled to 4°C. The four reactions were pooled, loaded onto a MCM120 aaagtagcc.galagatgacggtttgtcacatggagaggcag Qiagen PCR column according to the manufacturer's proto gatgtagattaaaagcaattalacc ct cactaaagggcgg col and eluted with 60 uL elution buffer (“EB) at 55° C. (SEQ ID NO: 36) Plasmid pRedET-carbenicillin' (GeneBridges, Heidel 55 MCM224 taaatcttacccgg.cgcagaac aggataccatgtttittitt berg, Germany) was electroporated into E. coli BL21 (DE3) acctic ctittgcaccitt catggtggtcagtgcgt.cctgctg strain MCM446 (Cm', gi1.6mKKDyI A1-3) using standard atgtgct cagtat caccgc.cagtgg tatttalNgtcaacac procedures. Transformants were recovered by shaking for cgc.ca.gagataattitat caccc.ca.gatggittatctgtatg one hour in SOC medium at 30° C. and then selected on tatttatatgaatttaatacgact cactatagggctic g (SEQ ID NO: 37) (where N can be a t, c. LB+50 ug/mL carbenicillin (“LB/carb50) plates at 30° C. 60 overnight. A carbenicillin-resistant colony was frozen as or g) Strain MCM508. GB-DW aaagaccgaccaag.cgacgtctga (SEQ ID NO: 38) Strain MCM508 was grown from a fresh streak in 5 mL LB/carb50 at 30° C. to an ODoo of -0.5. At that point, 40 mM MCM2O8 gctctgaatagtgatagagt ca (SEQ ID NO: 39) L-arabinose was added, and the culture was incubated at 37° 65 C. for 1.5 hours. Cells were then harvested by centrifugation, The assemblies integrated into the chromosomes of strains electroporated with 3 uI of purified amplicons as described MCM518-MCM521 include new P. promoters derived from US 9,353,375 B2 59 60 bacteriophage lambda (W) and the very beginning of the Promoter/mMVK sequence integrated into MCM521 mMVK ORF, with sequences from the Gene Bridges FRT (SEQID NO:43): gb2-Cm-FRT cassette integrated upstream of the promoter/ mMVK assembly, as well as the remainder of the mMVK ORF followed by the rest of the lower MVA pathway integron 5 aaagaccgacca agcgacgtctgagagct Coctggcga attcggta from Strain MCM508. c caataaaagagctittattitt catgatctgtgtgttggitttttgtg Promoter/mMVK sequence integrated into MCM518 (SEQ ID NO:40): tgcggcgcggaagttcctatt ct ct agaaagtataggaactt CCtc 10 gag.ccctatagtgagt cqt attaaatt catataaaaaacatacaga aaagaccgacca agcgacgtctgagagct Coctggcga attcggta talaccatctg.cggtgataa attatct ctggcggtgttgacgtaaat c caataaaagagctittattitt catgatctgtgtgttggitttttgtg accactggcggtgatactgagcacat cagcaggacgcactgaccac tgcggcgcggaagttcct attct ct agaaagtataggaactt CCtc Catgaaggtgcaaaggaggtaaaaaaacatggitatic ctgttctgcg 15 gag.ccctatagtgagt cqtattaaatt catataaaaaacatacaga ccgggtaagatttacctgttcggtgaacacgc.cgtagttt atggcg taaccatctg.cggtgataaattatctotgg.cggtgttgacataaat aaactgcaattgcgtgtgcggtggaactg.cgt accc.gtgttcgc.gc accactggcggtgatactgagcacat cagcaggacgcactgaccac ggaact caatgactictatoactatt cagagc Catgaaggtgcaaaggaggtaaaaaaacatggitatic ctgttctg.cg Example 5 ccgggtaagatttacctgttcggtgaacacgc.cgtagttt atggcg aaactgcaattgcgtgtgcggtggaactg.cgtaccc.gtgttcgc.gc Construction of Strains DW199 and DW202 25 ggaact caatgactictat cactatt cagagc This example describes the construction of an isoprene producing E. coli strain harboring the truncated version of P Promoter/mMVK sequence integrated into MCM519 alba isoprene synthase (the MEA variant) under control of the (SEQ ID NO:41): PTrc promoter. 30 The plasmid harboring truncated P alba isoprene synthase (IspS) was constructed by QuikchangeTM (Agilent Technolo aaagaccgac Caag.cgacgtctgagagct Coctggcga attcggta gies, Stratagene Products Division, La Jolla, Calif.) PCR c caataaaagagctittattitt catgatctgtgtgttggitttttgtg mutagenesis from the template pEWL244 (also referred to as pTrc-P alba(MEA)-mMVK (described in Example 10 of tgcggcgcggaagttcct attct ct agaaagtataggaactt CCtc 35 U.S. patent application Ser. No. 12/335,071). The PCR reac gag.ccctatagtgagt cqtattaaatt catataaaaaacatacaga tion contained the following components: 1 ul pEWL244 (encoding pTrc P alba-mMVK), 5 Jul 10x Pful Jltra High taaccatctg.cggtgataaattatctotgg.cggtgttgacctaaat Fidelity buffer, 1 ul 100 mM dNTPs, 1 ul 50 uMQC EWL244 accactggcggtgatactgagcacat cagcaggacgcactgaccac MEAF primer (SEQ ID NO:44), 1 ul 50 uM QC EWL244 40 MEA R primer (SEQID NO:45), 2 ul DMSO, 1 ul Pful Jltra Catgaaggtgcaaaggaggtaaaaaaacatggitatic ctgttctg.cg High Fidelity polymerase (Agilent Technologies, Stratagene Products Division, La Jolla, Calif.), and 39 ul diHO. The ccgggtaagatttacctgttcggtgaacacgc.cgtagttt atggcg PCR reaction was cycled as follows: 95°C./1 minute; and 18 aaactgcaattgcgtgtgcggtggaactg.cgtaccc.gtgttcgc.gc cycles of 95° C./30 seconds, 55° C./1 minute, 68° C./7.3 45 minutes. The reaction was then cooled to 4°C. ggaact caatgactictat cactatt cagagc The PCR product was visualized by gel electrophoresis using an E-gel (Invitrogen, Carlsbad, Calif.), and then treated Promoter/mMVK sequence integrated into MCM520 with 1 ul DpnI restriction endonuclease (Roche, South San (SEQ ID NO:42): Francisco, Calif.) for three hours at 37° C. Tenul of the PCR 50 product were then de-salted using a microdialysis membrane (MilliPore, Billerica, Mass.) and transformed into electro aaagaccgacca agcgacgtctgagagct Coctggcga attcggta competent E. coli strain MCM531 (prepared as described c caataaaagagctittattitt catgatctgtgtgttggitttttgtg above) using standard molecular biology techniques. Cells were recovered in one ml of LB medium for 1.5 hours at 30° tgcggcgcggaagttcct attct ct agaaagtataggaactt CCtc 55 C., plated onto LB-agar plates containing 50 ug/ml carbeni cillin and 5 mM mevalonic acid, and then incubated overnight gag.ccctatagtgagt cqtattaaatt catataaaaaacatacaga at 37° C. The next day, positive colonies (of strain DW195, taaccatctg.cggtgataaattatctotgg.cggtgttgacctaaat see below) were selected for growth, plasmid purification (Qiagen, Valencia, Calif.), confirmed by DNA sequencing accactggcggtgatactgagcacat cagcaggacgcactgaccac 60 (Quintara Biosciences, Berkeley, Calif.) with the primers Catgaaggtgcaaaggtaaaaaaac atggitatic ctgttctgcgc.cg listed below. The final plasmid, pIDW34 (FIG. 19 A; SEQID NO:7), was confirmed to carry the open reading frame that ggtaagatttacctgttctggtgaac acgc.cgtagtt tatggcgaaa encodes the truncated version of P alba IspS. Ctgcaattgcgtgtgcggtggaactg.cgt accc.gtgttcgc.gcgga Strain DW199 was generated by transformation of pDW34 65 and pMCM82 (described in Example 10 of U.S. patent appli act caatgactictato act attcagagc cation Ser. No. 12/335,071) into electrocompetent MCM531 (prepared as described above). Cells were recovered in 1 ml US 9,353,375 B2 61 62 of LB medium for 1 hour at 37°C., plated on LBagar plates A P1 lysate was made of strain JW0736, in which theybgS containing 50 spectinomycin and 50 g/ml carbenicillin, and gene was replaced with a kanamycin resistance gene then incubated overnight at 37° C. The next day, antibiotic (“Kan')(i.e., ybgS:Kan mutation) from the Keio collec resistant colonies of strain DW199 were chosen for further study. 5 tion (Baba et al. 2006). That lysate was used to infect strain Strain DW202 was generated by transformation of MCM531 (described above), producing strain CMP215. The pBBRCMPGI1.5-pgl (described in example 1) into electro genotype of CMP215 was confirmed by PCR using primers competent DW199 (prepared as described above). Cells were galMR (5'-GTCAGGCTG GAA TACTCTTCG-3'; SEQID recovered in 1 ml of LB medium for 1 hour at 37°C., plated NO:8) and galM F (5'-GAC GCT TTC GCC AAG TCA on LB agar plates containing 50 ug/ml spectinomycin, 50 10 GG-3'; SEQ ID NO:9). Those primers anneal to the galM ug/ml carbenicillin and 5 ug/ml gentamycin, and then incu gene, as shown on FIG. 20, but only produce a PCR product bated overnight at 37° C. The next day, antibiotic resistant colonies of strain DW202 were chosen for further study. from E. coli BL21 (DE3) chromosomal DNA having the 17,257 bp deletion. TABLE 5 15 Integration of the 17,257 bp fragment following P1 trans duction was verified by PCR with the following protocol. One Primers bacterial colony was stirred in 30 ul HO and heated to 95°C. Primer Name Sequence 5' -> 3' for 5 minutes. The resulting Solution was spun down and 2 ul QC EWL,244 MEA F gaggaataaaccatggaagct cqtcgttct of the supernatant used as template in the following PCR (SEQ ID NO: 44) reaction: 2 ul colony in HO,5ul Herculaser Buffer, 1 Jul 100 mM dNTPs, 1 Jul 10 uM Forward primer, 1 ul 10 uM Reverse QC EWL,244 MEA R agaacgacgagctt coatggitttatt cotc primer, 0.5 ul of Herculase R. Enhanced DNA Polymerase (SEQ ID NO: 45) (Agilent Technologies, Stratagene Products Division, La EL-1OO6 gacagctitat catcgactgcacg Jolla, Calif.), and 39.5 ul diHO. The PCR reaction was (SEQ ID NO: 26) 25 cycled in a PCR Express Thermal Cycler (Thermo Hybaid, EL-1OOO gcactgtctitt.ccgtctgctgc Franklin, Mass.) as follows: 95°C./2 minutes; 30 cycles of (SEQ ID NO: 22) 95°C/30 seconds, 52°C/30 seconds, 72°C./60 seconds; and 72°C./7 minutes. The reaction was then cooled to 4°C. The A.-rew Ctcgtacaggct caggatag 30 annealing temperature of 52° C. was 3° C. lower than the (SEQ ID NO: 48) lower T" of the primer pair. The size of the resulting PCR A.-rew-2 ttacgt.cccaacgct Caact fragment was determined on a pre-cast 0.8% E-Gel(R) (Invit (SEQ ID NO: 49) rogen, Carlsbad, Calif.), using DNA Molecular Weight QB1493 Cttctgcaacgcatggaaat Marker X (75-12,216 bp) (Roche Diagnostics, Mannheim, (SEO ID NO : 50) 35 Germany) as size marker. Successful transduction was also MCM2O8 gctctgaatagtgatagagt ca confirmed by the ability of strain CMP215 to grow on galac (SEO ID NO. 39) tOSe. MCM66 (aka c caggcaaattctgttittat cag Alternatively, a lysate of E. coli MG 1655 was used to pTrc Reverse) (SEQ ID NO: 21) transduce strain BL21 (as described in Example 1 above). A 40 colony selected on M9 medium supplemented with 0.4% (w/v) galactose was named CMP258. Presence of the 17,257 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| Example 6 bp region containing pgl was confirmed by PCR using prim ers galM R (SEQ ID NO:9) and galM F (SEQ ID NO:8), essentially as described above. Construction of E. coli BL21 Strains CMP215, 60 Strain CMP215 was cotransformed by electroporation CMP258 and CMP234 with plasmids pCLPtrcUpperPathway expressing mvaE and mvaS (described in Example 8 of U.S. patent application Ser. This example describes the construction of E. coli strains No. 12/335,071) and pL)W34 (containing a truncated P alba derived from BL21 transduced with P1 phage containing E. isoprene synthase and M. mazei mevalonate kinase, as coli MG 1655 genomic DNA and selected for recombination 65 described above). Transformants were selected on LB agar of a 17.257 bp piece present in MG 1655 but absent in BL21 plates including 50 g/ml carbenicillin+50 ug/ml spectino and BL21 (DE3). mycin. One colony was picked and named CMP234. US 9,353,375 B2 63 64 Example 7 Diagnostics, Mannheim, Germany) as size marker. The PCR reaction was purified using the QIAquick R. PCR Purification Construction of E. coli BL21 Strains CMP269 and Kit (Qiagen, La Jolla, Calif.). CMP312 Plasmid pRedETAmp (GeneBridges Gmbh, Heidelberg, Germany) was electroporated into CMP269 to form This example describes the construction of E. coli strains derived from BL21 transduced with P1 phage containing E. CMP296. CMP296 was grown and induced with L-arabinose coli MG 1655 genomic DNA and selected for recombination according to the manufacturers instructions (GeneBridges) of a 17.257 bp piece present in MG 1655 but absent in BL21 and transformed with the pgl/ybhE::kan“ PCR product and BL21 (DE3). The marker used for selection has been 10 described in this example. Transformants were selected on looped out. LB agar including 20 ppm kanamycin. One colony was Strain CMP215 (described above) was transformed with picked, its genotype checked by PCR with Herculase(R) poly pCP20 (Cherepanov, P. P. et al., 1995, Gene 158(1):9-14: merase using pg|Ampf (5'-cagcaaatagcaggtgtaticcagc-3', Datsenko and Wanner, 2000, Proc. Nat'l Acad. Sci. USA, SEQ ID NO:54) and pg|RecCheck (5'-GGTTACAAA ATG 97(12):6645) and the kan' marker contained in theybgS gene 15 ATT GGC GTA CGC-3'; SEQ ID NO:56) and named was looped out according to a previously described procedure CMP298. The marker was removed as described above in (Datsenko and Wanner, Proc. Nat'l Acad. Sci. USA, 97(12): Example 2 to form strain CMP315. Plasmids pCLPtrcupper 6645 (2000)). Marker loopout was verified by PCR as Pathway and plW34 (see Example 1) were introduced in described above, but usingybgSAmp F primer (5'-CCTGGA CMP315 as described above in Examples 4-5 to form strain ATT AGC AAG AAA AAC GC-3'; SEQ ID NO:52) and CMP323. ybgSAmp R primer (5'-GTG AAA ATT GCA CGG CGA GTA GG-3'; SEQ ID NO:53). That strain was designated TABLE 7 CMP269. Strain CMP269 was cotransformed by electropo ration with plasmids pCLPtrcUpperPathway (expressing Description of strains mvaE and mvaS) and plW34 (see FIG. 19A) containing a 25 truncated P alba IspS and M. mazei MVK to produce strain Strain Description Parent CMP3.12. MCMS31 BL21 PL.2-mKKDy CMP215 BL21 PL.2-mKKDyI tybgS:Kan MCMS31 Example 8 CMP258 BL21 tpg| BL21 30 (Nowagen) Construction of E. coli BL21 Strains CMP296, CMP234 BL21 PL.2-mKKDyI tybgS:Kan, CMP215 CMP315 and CMP323 pCLPtrcUpperPathway, pDW34 CMP269 BL21 PL.2-mKKDyI tybgSML CMP215 CMP296 BL21 PL.2-mKKDyI tybgSML, pRedETAmp CMP269 This example describes the construction of strains derived CMP312 BL21 PL.2-mKKDyI tybgSML, CMP269 from E. coli BL21 transduced with P1 phage containing E. 35 pCLPtrcUpperPathway, pIDW34 coli MG 1655 genomic DNA and selected for recombination CMP315 BL21 PL.2-mKKDyI tybgSML rpg| ML CMP296 of a 17.257 bp piece present in MG 1655 but deleted in BL21 CMP323 BL21 PL.2-mKKDyI tybgSML rpg| ML, CMP315 and BL21 (DE3), thereby restoring a functional copy of pgl to pCLPtrcUpperPathway, pIDW34 the E. coli BL21 and BL21 (DE3) derived strains. A strain in which the restored pgl gene has been precisely knocked out 40 by inserting a kanamycin cassette which was Subsequently References cited: Aon et al., 2008, “Suppressing posttrans looped out was also constructed. lational gluconoylation of heterologous proteins by meta A PCR product containing a copy of pgl/ybhE in which a bolic engineering of Escherichia coli, 'Appl. Environ. Micro kan' gene has been inserted (pgl/ybhE::kan') was amplified biol. 74:950-958; Baba et al., 2006, “Construction of from E. coli strain JW0750 from the Keio collection using the 45 Escherichia coli K-12 in-frame, single-gene knockout primer pair pg|Ampf (5'-Cagcaaatagcaggtgtatccagc-3'; SEQ mutants: the Keio collection.” Mol. Syst. Biol. 2: 2006.0008: ID NO:54) and pg|AmpR (5'-GCA ACC GAC TGT TGA Cherepanov, P. P. et al., 1995, “Gene disruption in Escheri TAG AAC AAC-3'; SEQ ID NO:55). That primer pair pro chia coli: TcP and KmR cassettes with the option of Flp duces a fragment containing pgl/ybhE::kan plus ~350 bp of catalyzed excision of the antibiotic-resistance determinant.” flanking sequence from each side of the mutation. PCR tem 50 Gene 158(1): 9-14: Datsenko, K., and Wanner, B., 2000, plate was prepared as follows: one colony of E. coli JW0750 “One-step inactivation of chromosomal genes in Escherichia carrying pgl/ybhE::kan“ was stirred in 30 ul H2O and heated coli K-12 using PCR products, Proc. Nat. Acad. Sci. USA to 95°C. for 5 minutes. The resulting solution was spun down 97:6640-6645; Neidhart, F., Ingraham, J., and Schaechter, and 2 ul of the supernatant was used as the template in a PCR M., 1990, Physiology of the bacterial cell: a molecular reaction performed as follows: 2 ul colony in H2O, 5ul Pfu 55 Ultra II Buffer, 1 ul 100 mM dNTPs, 1 ul 10 uM Forward approach (Sinauer Associates, Inc. Sunderland, Mass.); Tho primer, 1 Jul 10 uM Reverse primer, 1 ul of Pfu Ultra II mason, L., Court, D., Datta, A., Khanna, R. and Rosner, J., polymerase (Agilent Technologies, Stratagene Products 2004, “Identification of the Escherichia coli K-12 ybhE gene Division, La Jolla, Calif.), and 39 ul H2O. The PCR reaction as pgl, encoding 6-phosphogluconolactonase. J. Bact. 186: was cycled in a PCR Express Thermal Cycler (Thermo 60 8248-8253; Thomason, L., Costantino, N., Court, D., 2007, Hybaid, Franklin, Mass.) as follows: 95°C./2 minutes; 30 “E. coli genome manipulation by P1 transduction. Curr. cycles of 95°C./20 seconds, 53.4° C./20 seconds, 72° C./40 Protocols Mol. Biol. Chapter 1, Unit 1.17; Studier F., Dae seconds; 72°C./3 minutes. The reactions were then cooled to gelen, P. Lenski, R., Maslov, S., Kim, J. F., 2009, “Under 40 C. standing the differences between genome sequences of The size of the resulting PCR fragments was determined on 65 Escherichia coli B strains REL606 and BL21 (DE3) and com a pre-cast 0.8% E-Gel R (Invitrogen, Carlsbad, Calif.), using parison of the E. coli B and K-12 genomes. J. Mol. Biol. DNA Molecular Weight Marker X (75-12,216 bp)(Roche 394(4):653-80, 2009).