1111111111111111111111111111111111111111111111111111111111111111111111 (12) United States Patent (1o) Patent No.: US 8,993,303 B2 Zhou et al. (45) Date of Patent: Mar. 31, 2015 (54) GENETICALLY ENGINEERED FOREIGN PATENT DOCUMENTS CYANOBACTERIA WO 2007084477 Al 7/2007 (75) Inventors: Ruanbao Zhou, Brookings, SD (US); OTHER PUBLICATIONS William Gibbons, Brookings, SD (US) A Tomar et al. The Effect of Acetate Pathway Mutations on the (73) Assignee: South Dakota State University, Production of Pyruvate in Escherichia coli, Applied Microbiology Brookings, SD (US) and Biotechnology, 2003, vol. 62, pp. 76. Mai Li et al. Effect of IpdA Gene Knockout on the Metabolism in Escherichia coli Based on Enzyme Activities, Intracellular Metabo- (*) Notice: Subject to any disclaimer, the term of this lite Concentrations and Metabolic Flux Analysis by 13C-labeling patent is extended or adjusted under 35 Experiments, Journal of Biotechnology, 2006, vol. 122, pp. 254. U.S.C. 154(b) by 158 days. Xiaojie Pan et al. Morphological Characteristics and Phylogenetic Relationship of Anabaena Species from Lakes Dianchi and Erhai, China, Hydrobiologia, 2008, vol. 614, pp. 353. (21) Appl. No.: 13/405,208 Ruanbao Zhou and C. Peter Wolk, A Two-component System Medi- ates Developmental Regulation of Biosynthesis of a Heterocyst (22) Filed: Feb. 24, 2012 Polysaccharide, Journal of Biological Chemistry, 2003, vol. 278 (22), pp. 19939. Yoshiko Miyagawa et al. Overexpression of a Cyanobacterial Fruc- (65) Prior Publication Data tose-1,6-/Sedoheptulose-1,7-Bisphosphatase in Tobacco Enhances US 2012/0276637 Al Nov. 1, 2012 Photosynthesis and Growth, Nature Biotechnology, 2001, vol. 19, pp. 965. T. Iwaki et al. Expression of Foreign Type I Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase (EC 4.1.1.39) Stimulates Related U.S. Application Data Photosynthesis in Cyanobacterium Synechococcus PCC7942 Cells, Photosynthesis Research, 2006, vol. 88, pp. 287. (60) Provisional application No. 61/446,366, filed on Feb. Masahiro Tamoi et al. Contribution of Fructose-1,6-Bisphosphate 24, 2011, provisional application No. 61/522,685, and Sedoheptulose-1,7-Bisphosphatase to the Photosynthetic Rate filed on Aug. 11, 2011. and Carbon Flow in the Calvin Cycle in Transgenic Plants, Plant Cell Physiology, 2006, vol. 47 (3), pp. 380. (51) Int. Cl. * cited by examiner C12N 1121 (2006.01) C12N 15100 (2006.01) Primary Examiner Oluwatosin Ogunbiyi (52) U.S. Cl. (74) Attorney, Agent, or Firm MDIP LLC USPC ..................... 435/252.3; 435/243; 435/252.1; 435/320.1 (57) ABSTRACT (58) Field of Classification Search The disclosed embodiments provide cyanobacteria spp. that None have been genetically engineered to have increased produc- See application file for complete search history. tion of carbon-based products of interest. These genetically engineered hosts efficiently convert carbon dioxide and light (56) References Cited into carbon-based products of interest such as long chained hydrocarbons. Several constructs containing polynucleotides U.S. PATENT DOCUMENTS encoding enzymes active in the metabolic pathways of cyano- bacteria are disclosed. In many instances, the cyanobacteria 6,699,696 B2 3/2004 Woods et al. strains have been further genetically modified to optimize 7,531,333 B2 * 5/2009 Miyake et al . ................ 435/166 production of the carbon-based products of interest. The opti- 7,659,097 B2 * 2/2010 Renninger et al ............. 435/157 mization includes both up-regulation and down-regulation of 7,794,969 B1 9/2010 Reppas et al. 2009/0203070 Al* 8/2009 Devroe et al ................. 435/69.1 particular genes. 2010/0003739 Al* 1/2010 Duhring et al . ............ 435/252.3 2011/0039323 Al * 2/2011 Singsaas et al . .............. 435/167 10 Claims, 13 Drawing Sheets U.S. Patent Mar. 31, 2015 Sheet I of 13 US 8,993,303 B2 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- CO, H2.0 Light We will add copies of critical Calvin cycle genes to increase photosynthesis Pyruvate We will imidify MEPMEPua c P pathway to increase carbonca rbon Ethanol flow to end products Alkanes Methylbutenof Hydrocarbonarbor, myrcene farnescene FIG. I U.S. Patent Mar. 31, 2015 Sheet 2 of 13 US 8,993,303 B2 :... It E 3-phosphoglycerat e co Calvin CyCle PEA }y p r-0 a 4.t rp.¢ xa an GIP r-#5tzy A;.7. z d klw.-+ aivr kS'23 OH Ethanol ;.? g; t~ [ z ~, z€;,.c3 r . ~..: ~:#:. sz .:.:a:,~ .z; ~ s'~r•.3vcstt>: €~?rE's:?e;~t.~8sl t.?xie#<ssc~z`1isrt ~~ e >t .. ... Y:. •,.f,. fir.:£...: "' ... ..'cr; .. _,:. ,::•. ;yE 3 c5"•.c? €,§ s.3.:<? C phc pho, #Ct fi3y`E'<3vate catboxyline :.? <ItN }0 ,y ; S.:E 33t ptsc5 ipfEd>s €iE€:o ie €sioty}<?<# x` 3sCtS7s€s#t„':CSih' mum ii ~} €~Yruwatti~ #sar {a~~stsa €yeas? t~;,3) ;~~s~••;~€~.ELnsr €~Yr~€~€~E~s~,t~ srsr'ytr6.~c t;3 tsrtc3?c c€~,r src#rEs ~ E.?l3f~-s€s ErSsr>c 3syrs:5,~ 'r. ~ E}4g>£}<ary€tss ~: Tho heavy grE:es and red 3rfo fit indiuste iris. %:ar sisr flow from CO2 to ot:€samA in tees i ngirEeer€- d gyarie>bactc,..r. FI.G. 2 U.S. Patent Mar. 31, 2015 Sheet 3 of 13 US 8,993,303 B2 FIG. 3 U.S. Patent Mar. 31, 2015 Sheet 4 of 13 US 8,993,303 B2 A B Carbohydrates & arnin(: u't:IdS EtC_ CO2+H20+ light NADPH ATP 99 ycexa€dehyde 3-phosphate yr at € XS sugars & glycogen DXP MEP ACT 4 xx. xxxxxxxxxxx CDP-M, B Y to ice' a k a l €4A" 4 CDP- Pc EP S 4 ME-APP HMBPP ` .. (C H 0, rnethylbutenol) IPA (LAPP ~xa•r €€ €PP GFFJ (cjoHT.g)o Itnatoo€) GPP SQS jPP IFFPS (ClOH16,'rriyrcene) squa€ene FPP €PP farnesene (Cl SH24) te_'r q;tiods _&eWther L ~ - GGPP Engineering cyanobacteria to produce long-chain hydrocarbons A. Known MEP pathway exists in plants, algae, and some bacteria; B. Proposed a photosynthetic MEP pathway in cyanobacteria. *NADPH as cofactor. **ATP dependent G3P D-gtyceraldehyde-3-phosphate Rl:5P ribuloss-5-phosphate DXP 9-deoxy-D-xylulose 5-phosphate DXS DXP synthase MEP methylerythritol-4-phosphate DXR DXP reductoisomerase CDP-ME diphosphocytidylyl rnethylei-fihritol PACT CDP-ME synthase CDP-MEP CDP-methyierythritol-2-phosphate CMK CDP-ME kinase PIE-cPP methylerythritol-2,4-cyciodiphosphate MDS ME-cPP synthase HMBPP hydroxymethylbutenyl diphosphate HDS HR.IBPP synthase IPP isopentenyl diphosphate HDR HMBPP reductase DMAPP dimethylallyl-diphosphate EDE IPP:DMAPP isomerase GPP geranyl-diphosphate GPPS GPP synthase APP 6nesyl-diphosphate FPPS Fr P synthase GGPP geratiylgerar:yl-diphosphate GGPPS GGPP synttase XY5P Yylulose-5-phosphate SQS squalene synthase FIG. 4 U.S. Patent Mar. 31, 2015 Sheet 5 of 13 US 8,993,303 B2 W CO2 ^ li )U 8 q8 ° G6p F1,6 P ` G1 P ADP-GI c ~ "PISN UDPGic+ F6P ---* %c6P+ UDP H2O UPS' SAS R UDPGic+ F Sucrose ,-D phosphoglucose isomerase nvert am phosphoglucose mutase (~-Z ADP-glucose pyrophosphorylase ~~ + F UDRglucose pyrophosphorylase f Phosphofructokinase FIG. 5 U.S. Patent Mar. 31, 2015 Sheet 6 of 13 US 8,993,303 B2 `Y R lll i..zfl 2 1-11, <4 r. x'i i3R } GC m h inalo l standard } 'F h5-wG"if } .s8~ ....s47 :e:cJGY ate. 'ar±•. ;3.3^.ci .i•c}.cL:. •s [s.Car•~ xs > c T ` ~ e a BpXD~B }R linalool ki q `, ;Z 3 A:k xx:t 3t~ i. ryr .L- t.a,(x) umx)ryzk-_3;x ~;5,xtc 110000 Wil vz- xx~ d -type A ab en a w , s f3F #+.C32 ]fX3~ r32 4C r~X~€ '5 [' LXe :°_f'7:C, FAG. 6 U.S. Patent Mar. 31, 2015 Sheet 7 of 13 US 8,993,303 B2 S man B1O From .:'—to cai2O CIO .40970_s— Sped Y SP 81 (22 T545Y = 1 ❑ C34e} 2.' 0.448TO.sms 10.044 mi.. Scan: 5Y0 Chan: Y Ion: E& us RIC: Y 1558845 5C 8Y Y444 F 54: 544 t 3T 254 1 21 43 185 24Y 21T 254 2T5 2^v1 4 4' 5 Y Y 2 2 m f2 FIG. 7. Mass spectra for linalool standard U.S. Patent Mar. 31, 2015 Sheet 8 of 13 US 8,993,303 B2 Scan 599 fr®m c=Esa cu rnws4da ~~ 12666_A6672_sms sn—t 1 BP 81 (45133T=14 %) 2i .44BT2. sms 4.854 min. a—n: 584 Chan: 1 Ion: 110 us RIC: 31BTT4 BC 81 1434 T59 549: A3 121 259 1 3T 1 ° } 1 T1 215 2234 254 243 b9 S i 1 2 2 m rx FIG. 8. Mass spectra for linalool produced by engineered Anabaena (see FIG. bB) U.S. Patent Mar. 31, 2015 Sheet 9 of 13 US 8,993,303 B2 FIG. 9. Hydrocarbons produced by A imbaena Q14hidrica 29414 U.S. Patent Mar. 31, 2015 Sheet 10 of 13 US 8,993,303 B2 Or. s xr:Yff£ Tea Nth ~ne .r M.:I € £# Ytc 011 : m, et m CO2 OG ~:yt d~tf~1:331~#Et,`x~%i..IF ~~~~i'#.~~~? ~~ i ~[sY~~ E'•i~iS ArgkF€i i o docaL'hox)*a c p;*t~'YL4dayY >`,':<E„s ~~bt£oArtY4~.r#~+.c,~ '}+t'#t'i~ t;o4: ~fi3 ±.:~ ~'~.x~~#.#:tMkrYfa`}~fi"~:~a~jf`•rkt~`io"3 FIG. 10. Engineering Anabaena to synthesize urea using solar energy U.S. Patent Mar. 31, 2015 Sheet 11 of 13 US 8,993,303 B2 FIG.11. Sucrose produced by Anabaena sp PCC7120 U.S. Patent Mar. 31, 2015 Sheet 12 of 13 US 8,993,303 B2 Lind Kna pLinS C 2 -e Ll Double cto.ssover All,, FIG. 12. is integrated toy A nc-d-wim cliromv,~,(xtne, at lod, A <iud 11 U.S. Patent Mar. 31, 2015 Sheet 13 of 13 US 8,993,303 B2 ......... ........ ........ ......... _ Hosoolcgous Eije11es fie ni MEP patllway found ill five cyallobactelial stl in , td~~7~~~~ 3•ia~.~r~y~~ nwsrQ ~ Arr6aear 1st; 5? r~echa~ ystrs Arabidopsiv tlaivr a saiabdbATt C ch~ orac prariftame p ; C7120 ~ YCC803 29413 efonat'V P.1 AMC29133 aI10519 Av.a 41432 tR062 :1 x€015-#5 Hpull—F5366 AT 3G21100MXSTE 1E-i3' 1E-13"c 1E•143 1E ,139 1E.1s5 Drs AT4C11566(DXSA 1t 14 t.