US008986,963B2 (12) United States Patent (10) Patent No.: US 8,986,963 B2 Lee (45) Date of Patent: Mar. 24, 2015 (54) DESIGNER CALVIN-CYCLE-CHANNELED FOREIGN PATENT DOCUMENTS PRODUCTION OF BUTANOL AND RELATED WO 2005100.582 A2 10, 2005 HIGHER ALCOHOLS WO 2006119066 11, 2006 WO 2007032837 A2 3, 2007 (76) Inventor: James Weifu Lee, Cockeysville, MD WO 2007047148 4/2007 (US) WO 2007065035 6, 2007 WO 2007134340 A2 11/2007 WO 2008OO6038 A2 1, 2008 (*) Notice: Subject to any disclaimer, the term of this WO 201OO68821 A1 6, 2010 patent is extended or adjusted under 35 U.S.C. 154(b) by 714 days. OTHER PUBLICATIONS Appl. No.: 13/075,153 Chen et al., Photo. Res., 2005, 86:165-173.* (21) Raines et al., 2003, Photosynthesis Res., 75:1-10.* Filed: Mar. 29, 2011 Pickett-Heaps et al., 1999, Am. J. of Botany, 86:153-172.* (22) Shen et al., 2008, Metabolic Engineering, 10:312-320.* Prior Publication Data Sanderson, 2006, Nature, 444:673-676.* (65) Keneko Takakazu et al., Complete Genomic Sequence of the Fila US 2011/O177571 A1 Jul. 21, 2011 mentous Nitrogen-fixing Cyanobacterium Anabaena sp. Strain PCC 7120, DNA Research 8,205-213 (2001). Ramesh V. Nair et al., Regulation of the Sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC824 by Related U.S. Application Data a Putative Transcriptional Repressor, Journal of Bacteriology, Jan. (63) Continuation-in-part of application No. 12/918,784, 1999, vol. 181, No. 1, pp. 319-330. filed as application No. PCT/US2009/034801 on Feb. (Gfeller and Gibbs (1984) “Fermentative metabolism of Chlamydomonas reinhardtii, Plant Physiol. 75:212-218). 21, 2009, now Pat. No. 8,735,651. (Lee, Blankinship and Greenbaum (1995), “Temperature effect on (60) Provisional application No. 61/066,845, filed on Feb. production of hydrogen and oxygen by Chlamydomonas cold strain 23, 2008, provisional application No. 61/066,835, CCMP1619 and wild type 137c. Applied Biochemistry and filed on Feb. 23, 2008, provisional application No. Biotechnology 51/52:379-386). Lee et al., "Discovery of an Alternative Oxygen Sensitivity in Algal 61/426,147, filed on Dec. 22, 2010. Photosynthetic H2 Production”. Proceedings of the 2000 U.S. DOE Hydrogen Program Review, NREL/CP-570-28890. (51) Int. C. (Lee, Mets, and Greenbaum (2002). “Improvement of photosynthetic CI2P 7/16 (2006.01) efficiency at high light intensity through reduction of chlorophyll CI2P 7/04 (2006.01) antenna size.” Applied Biochemistry and Biotechnology, 98-100: CI2N IS/II3 (2010.01) 37-48). CI2O I/00 (2006.01) (Nakajima, Tsuzuki, and Ueda (1999) “Reduced photoinhibition of a (52) U.S. C. phycocyanin-deficient mutant of Synechocystis PCC 6714'. Journal CPC. CI2P 7/16 (2013.01); C12P 7/04 (2013.01); of Applied Phycology 10: 447-452). CI2Y 204/01021 (2013.01); C12N 15/I 137 (Continued) (2013.01): CI2O I/00 (2013.01); Y02E 50/10 (2013.01); C12N 23 10/11 (2013.01); C12N Primary Examiner — Ashwin Mehta 23 10/14 (2013.01) Assistant Examiner — Jason DeVeau Rosen USPC .......................................................... 435/160 (74) Attorney, Agent, or Firm — August Law, LLC; George (58) Field of Classification Search Willinghan None See application file for complete search history. (57) ABSTRACT (56) References Cited Designer Calvin-cycle-channeled and photosynthetic NADPH-enhanced pathways, the associated designer genes U.S. PATENT DOCUMENTS and designer transgenic photosynthetic organisms for photo biological production of butanol and related higher alcohols 6,699,696 B2 * 3/2004 Woods et al. ................. 435,161 7.682,821 B2 * 3/2010 Woods et al. .............. 435,292.1 from carbon dioxide and water are provided. The butanol and 2007/0037196 A1 2/2007 Gibson et al. related higher alcohols include 1-butanol, 2-methyl-1-bu 2007/0037197 A1 2/2007 Young et al. tanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 2007/O122826 A1 5, 2007 Glass et al. 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1- 2007.0128649 A1 6/2007 Young hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-me 2007,0264688 A1 11/2007 Venter et al. 2007,0269.862 A1 11, 2007 Glass et al. thyl-1-hexanol, and 6-methyl-1-heptanol. The designer pho 2009,0081746 A1 3/2009 Liao et al. tosynthetic organisms such as designer transgenic 2009/011 1154 A1 4/2009 Liao et al. oxyphotobacteria and algae comprise designer Calvin-cycle 2009,0176280 A1 7/2009 Hutchinson, III et al. channeled and photosynthetic NADPH-enhanced pathway 2009/0203070 A1 8, 2009 Devroe et al. gene(s) and biosafety-guarding technology for enhanced 2010/0105103 A1 4/2010 Juan et al. 2010, O151545 A1 6, 2010 Roessler et al. photobiological production of butanol and related higher 2010/0209986 A1 8, 2010 Liao et al. alcohols from carbon dioxide and water. 2010, 0221800 A1 9, 2010 Liao et al. 2010/0330637 A1* 12/2010 Lee ............................... 435,160 7 Claims, 12 Drawing Sheets US 8,986,963 B2 Page 2 (56) References Cited (Casey and Grossman (1994) “In vivo and in vitro characterization of the light-regulated cpcB2A2 promoter of Fremyella diplosiphont” OTHER PUBLICATIONS Journal of Bacteriology, 176(20):6362-6374). 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PCC 6803.” Current Microbiology 49:192-198). ethanol production with microalgal photosynthesis and intracellular (Nakamoto, Suzuki, and Roy (2000) “Constitutive expression of a anaerobic fermentation' Energy 22(2/3): 137-142). Small heat-shock protein confers cellular thermotolerance and ther The Eurasian Patent Office, Search Report, Apr. 15, 2011. mal protection to the photosynthetic apparatus in cyanobacteria.” FEBS Letters 483: 169-174). * cited by examiner U.S. Patent Mar. 24, 2015 Sheet 1 of 12 US 8,986,963 B2 sof*J af U.S. Patent US 8,986,963 B2 U.S. Patent Mar. 24, 2015 Sheet 4 of 12 US 8,986,963 B2 U.S. Patent Mar. 24, 2015 Sheet 6 of 12 US 8,986,963 B2 U.S. Patent Mar. 24, 2015 Sheet 7 of 12 US 8,986,963 B2 affs,~). U.S. Patent Mar. 24, 2015 Sheet 8 of 12 US 8,986,963 B2 ostaf U.S. Patent Mar. 24, 2015 Sheet 9 of 12 US 8,986,963 B2 of sof U.S. Patent Mar. 24, 2015 Sheet 10 of 12 US 8,986,963 B2 afsef U.S.