US 20160348O87A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0348087 A1 Behrendorff et al. (43) Pub. Date: Dec. 1, 2016

(54) GENETICALLY ENGINEERED Publication Classification MCROORGANISMS FOR THE PRODUCTION OF CHORISMATE-DERVED (51) Int. Cl. PRODUCTS CI2N 9/88 (2006.01) CI2P 7/62 (2006.01) (71) Applicant: LanzaTech New Zealand Limited, (52) U.S. Cl. Skokie, IL (US) CPC. CI2N 9/88 (2013.01); CI2P 7/62 (2013.01); CI2Y 401/0304 (2013.01) (72) Inventors: James Bruce Yarnton Haycock Behrendorff, Skokie, IL (US); Michael (57) ABSTRACT Koepke, Skokie, IL (US); Loan Phuong Tran, Skokie, IL (US); Wyatt The invention provides genetically engineered microorgan Eric Allen, Skokie, IL (US) isms and methods for producing chorismate-derived prod ucts, such as para-hydroxybenzoic acid, salicylate, 2-amin (21) Appl. No.: 15/166,224 obenzoate, 2,3-dihydroxybenzoate, and 4-hydroxycyclohexane carboxylic acid. Typically, the (22) Filed: May 26, 2016 microorganism comprises at least one of (a) an exogenous chorismate pyruvate lyase, (b) an exogenous isochorismate Related U.S. Application Data synthase, (c) an exogenous isochorismate pyruvate lyase, (60) Provisional application No. 62/167,101, filed on May and (d) a prephenate synthase comprising a disruptive 27, 2015. mutation.

erythrose-4-phosphate + phosphoenolpyruvate

aroG* arOG

DAHP

arokB 3-dehydroquinate aOD 3-dehydroshikimate aroE

shikimate arOKB shikimate 3-phosphate . arOA 5-O-(1-carboxyvinyl)-3-phosphoshikimate arOC chorismate Patent Application Publication Dec. 1, 2016 Sheet 1 of 32 US 2016/0348087 A1

erythrose-4-phosphate + phosphoenolpyruvate

aroG aroG

DAHP

arOKB 3-dehydroquinate aroD 3-dehydroshikimate arOE

shikimate aro KB shikimate 3-phosphate arOA 5-O-(1-carboxyvinyl)-3-phosphoshikimate aroC chorismate

Fig. 1 Patent Application Publication Dec. 1, 2016 Sheet 2 of 32 US 2016/0348087 A1

- erythrose-4-phosphate 38: ghosphoenogy wate , &* Siikiate pathway k. - Acetyl-CoA Pyravate sixxi-ji:gdahi* -- / Tryptoghai <-- *r Charisiiate' - beai 4 p-8A gathway - & ephenate8.- : as Y: Top-ABA CO, CC is /x \;Y. s: Foite ity site heyaarine Fig. 2

erythross-4-phosphate we ------: 3 phospice soigyravate f ; Shikimate gathway ', f -- Acetyl-CoA rivate oxi-jurgda: / ryptoghat: K-K-- Ctoristiate peria.A. sociatistate pct: saidylate pathway y - ’8. / Prephenate s p-ABA CR. O. his « ». *. w *. cate tyrosia Pterytsiarie Fig. 3 Patent Application Publication Dec. 1, 2016 Sheet 3 of 32 US 2016/0348087 A1

- erythrose-4-phosphate --> A phosphoeoisy rivate : : f Shikimate pathway * f yl - Acetyl-CoA fisci-3 agaiahi tryptophan K-x *r horismate patiway & Xphea x 8.& Pr:gh state {{, , i. & X, '. Foiate al Tytosine Pierryiaiaire 8 & 2-airobenzoate i 2,3-dihydroxybenzoate :3-hyxi oxycyclishexae carboxylicacic Fig. 4

gi:8A staircia's carya

Patent Application Publication Dec. 1, 2016 Sheet 4 of 32 US 2016/0348087 A1

&cc::::::: 488 s: 33-33- -

:::: s :33x :& 33: 333.38 38:33.** 1s: : 8 : 8

8 Patent Application Publication Dec. 1, 2016 Sheet 5 of 32 US 2016/0348087 A1

-- 8: - 38 S- 3.. (i)

3. s

3.

s:

s

(w)

------xx-xx-as-s-s------

Fig. 6b Patent Application Publication Dec. 1, 2016 Sheet 6 of 32 US 2016/0348087 A1

Fig. 7 Patent Application Publication Dec. 1, 2016 Sheet 7 of 32 US 2016/0348087 A1

Patent Application Publication Dec. 1, 2016 Sheet 8 of 32 US 2016/0348087 A1

Oasis & i. i. Agai:3.33 Patent Application Publication Dec. 1, 2016 Sheet 9 of 32 US 2016/0348087 A1

2::::::::::::::::: 3:38

3.3-8:hy8roxybenzoic 3c: :

Fig. 10 Patent Application Publication Dec. 1, 2016 Sheet 10 of 32 US 2016/0348087 A1

::::::::::::::::::: aki: 3:2:3:23 *::::::::ce: 33 3:3:g: {3} :::::

ii: ::::

Fig. 1 a

*:::::::::::::c ::

:E: :::::::::::::::: 3.3 ::::::::::::33 a ::::::::::::3: 8. Patent Application Publication Dec. 1, 2016 Sheet 11 of 32 US 2016/0348087 A1

20 -: 1,000 : ...'...'...', * Siomass :

8 cis-a-hydroxy cyclohexanecarboxylic acid - 900

6 a 2-aminobenzoic acid 800 S. w8x3,4-dihydroxybenzoic acid E. 14 700 U 12 600 3. CO TO -: - 500 9 : W & s 8 : 400

6 -: 's 300

4 -:

2 -: 100

O ------C8------O O 2 3 4 6 7 8 9 O Patent Application Publication Dec. 1, 2016 Sheet 12 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession WP 0013266441 Bartonea carridgeiae WP 01354.4696.1 Escherichia Co WP 032343343.1 Providencia acaifaciens WP 036955412.1 WP 001295693.1 Providencia rustigianii WP OO6816067.1 WP 005071384.1 8artonella rattimassiliensis WP 0.0734.7862.1 Shigella sp. SF-2015 WP OO5002785.1 Edwardsiella anguiarum WP 034163029.1 Escherichia WP 000019214.1 Edwardsiella WP 045427790.1 Shigella sonnel WP 05299.0089.1 Phaseolibacter flectens WP 028684858.1 Escherichia alberti WP 000019228.1 Providencia Stuartii WP 004925912.1 Citrobacter youngae WP OO6688307.1 *dwardsiella ictauri WP 015869674.1 WP 054527959.1 Edwardsiella piscicida WP 015460726.1 Citrobacter WP 043213934.1 Sodalis praecaptivus WP 0514401951 Citrobacter pasteurii WP 0.05132668.1 Sodalis gossinidius WP 011411956.1 Citrobacter freundii complex WPO3294.2095.1 Edwardsiella tarda WP 035597793.1 WP 063411731.1 Providencia Sneebia WP 008916956.1 Providencia Citrobacter amatonaticus WP 061075585.1 burhodogranariea WP_008913736.1 WP 042999.031.1 Edwardsiela hoshinae WP 024524687.1 Candidatus Sodalis Enterobacter WP 014882105.1 pierantonius WP 025246620.1 Enterobacter cloacae Complex WP 045355219.1 Photobacterium aquae WP 047877737.1 Enterobacter sp. BEDMC 29 WP 041911565.1 Photobacterium marinum WP 007469524.1 Photobacterium Enterobacter sp., 35730 WP 045268808.1 Sanguinicanci WP 062688249.1 Enterobacter sp. T1-1 WP O29882656.1 Photobacterium Swingsii WP 048893785.1 Enterobacter cloacae Complex 'Hoffmann cluster IV WP 008500083.1 Photobacterium damseiae WP 044173922.1 Enterobacter asburiae WP 023617246.1 Photobacterium sanctipauli WP 036818650,1 Enterobacter sp. BEDMC92 WP 047957525.1 AgarivoranS albus WPO16399749.1 Enterobacter sp. 638 WP 011915505.1 Photobacterium profundum WP 0.06233275.1 Citrobacter farmer WP 042321063.1 Vibrio maritimus WP 042478790.1 Citrobacter koser WP 012134646.1 Vibrio metoecus WP O55052109.1 Enterobacter hormaechei WP 023299.087.1 Grimonitia celer WP 062666858.1 Escherichia fergusonii WP 000019211.1 Viorio WP 001072883.1 Enterobacter cancerogenus WP 042321179.1 Agarivoransgivus WP 055733842.1 Enterobacter sp. GNO24.54 WP 047742368.1 Vibrio aginolyticus WP 053311436.1 WP 000019223.1 Grimonitia indica WP 0025354O7.1 Leliottia amnigena WP 059179835.1 Photobacterium ganghwense WP 047887262.1 Enterobacter sp. Bisph1 WP 039055748.1 Grimontia sp. AD028 WP 0463.03386.1 Salmonella bongori WP 020845807.1 Vibrio neptunius WP 045975676.1 Fig. 13 Patent Application Publication Dec. 1, 2016 Sheet 13 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession Enterobacter sp. FY-07 WP 061.498857.1 Vibrio Corailiilyticus WP 0430.06692.1 Escherichia vulneris WP O42388891.1 Photobacterium aphoticum WP 047873744.1 Yokeneia regensburgei WP 04O902665,1. Photobacterium leiognathi WP O53987423.1 Trabusiella odontotermitis WP 054179777.1 Plesiomonas WP 010862816.1 Trabulsiella guamensis WP 038158396.1 Vibrio Xui WP 053441,696.1 Enterobacter sp. MT20 WP 061706855.1 Vibrio sp. VPAP30 WP O49845305. Kosakonia radicincitans WP 043955.711.1 Vibrio tubiashii WP 038.197373.1 Kluyvera intermedia WP 047372194.1 Sainvibrio Socompensis WP O256.73764.1 Enterobacter sp. Bisph2 WP 039077918.1 Grinnota marina WP 062709804.1

Kiebsiella oxytoca WP 004109561.1 Vibrio galatheae WP 045956983.1 Enterobacter xiangfangensis WP O58715018.1 Grimontia hoisae WP O055O1667.1 Leclercia adecarboxylata WPO39031929.1 Photobacterium sp. SKA34 WPOO6647642.1 Enterobacter aerogenes WP 045362420.1 WP 032480537.1 Kebsiela WP 01422.7537.1 Vibrio Caribbeanicus WP 0096.02602.1 Vibrionales bacterium SWAT Enterobacter massiiensis WP 044180994.1 3 WP O08224249.1 Citrobacter rodentium WP 012907701.1 Enterovibrio Caviensis WP 017016798.1 Raouitella terrigena WP045853463.1 Vibrio bivavicida WP 054963396.1 Klebsiella sp. OBRC7 WP 009654674.1 Vibrio Orientalis WP 004.409565.1 Klebsiella sp. RT-P-d WP 049838501.1 Vibrio sp. HOOD65 WPO63524665.1 Raouitella ornithinolytica WP 041143590.1 Wibrio ordaii WP 038198194.1 WP 023342419.1 Vibrio splendidus WP 032554291. Kebsiella sp. 10982 WP O25713803.1 Vibrio nigripulchritudo WP 045967092.1 Enterobacteriaceae bacterium Photobacterium strain FG 57 WP 015966334.1 phosphoreum WP 045031601.1 Pluralibacter gergoviae WP 04828491.1 Saliniwibrio sp. KP-1 WP 046074636.1 Kluyvera cryocrescens WP 061283371.1 Photobacterium gaetbulicola WP 044622288.1 Franconibacter helveticus WP 024553577.1 Photobacterium WP 045083799.1 Kluyvera aScorbata WP 035896877.1 Enterovibrio norvegicus WP 01696.1855.1 Franconibacter pulveris WP O29593165.1 Photobacterium angustum WP 005372020.1 Shimweia battae WP 002445222.1 Wibrio brasiliensis WP 040895.525.1 Enterobacteriaceae bacterium LSCA WP 017373629.1 Aiivibrio fischer WP 012534390.1 Cronobacter WP 007796820.1 Vibrio pacinii WP 038175692.1 Erwinia sp. SCU-B244 WP 058912420.1 Vibrio titoralis WP 051241116.1 Cronobacter sakazaki WP 054624,115.1 Vibrio sp. hENC-03 WP O09705152.1 Cronobacter turicensis WP 007764634.1 Vibrio genomosp, F6 WP 017051810.1 Cronobacter maionaticus WP 032994815.1 Photobacterium aquimaris WP 060997736.1 Cronobacter muytjensii WPO38867328.1 WP (246998.58.1 Enterobacter sp. Ag1 WP 0084.55844.1 Vibrio harveyi WPO33.007672. Cronobacter condimenti WP 0.07667577.1 Vibrio fortis WP 032553387.1 Fig. 13 cont'd Patent Application Publication Dec. 1, 2016 Sheet 14 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession Cronobacter universalis WP 007702330.1 Vibrio campbellii WP 051118327.1 Cedecea neteri WP 0392994.65.1 Vibrio sp. CAM 1540 WP 047049487.1 Cronobacter dubinensis WP 007752848.1 Photobacterium iliopiscarium WP 045035868.1 Klebsiella michiganensis WP 045780974.1 Morite a dasanensis WP 017222441.1 Buttiauxella agrestis WPO34456982.1 Vibrio harveyi group WP 0453723.02.1 Siccibacter Colletis WP 031521381.1 Psychromonas arctica WP 028869261.1 Cedecea davisae WPO16517422.1 Vibrio rotiferianus WP 029560889.1 Mangrovibacter sp. MFB070 WP 036102985.1 Bermanea marisrubri WP 050758020.1 Enterobacter udwigii WP 061.718382.1 Vibrio nereis WP 053396837.1 Pantoea sp. RT-P-b WP O49851273.1 Vibrio sp. OY15 WP 033907265.1 Pantoea sp., GMO1 WPO09128583.1 Alivibrio Wodanis WP 060993987.1 Pantoea sp. 3.5.1. WP 031375526.1 Vibrio renipiscarius WP 040988992.1 Pantoea sp. YR343 WP 008109935.1 Vibrio sp. AND4 WP 04399.1786.1 Erwinia billingiae WP 013200342.1 Vibrio Shionii WP 050798660.1 Pantoea sp. AS-PWVM4 WP 021184075.1 Vibrio ichthyoenteri WP 006713584.1 Pantoea WP 038643645.1 Vibrio azureus WP 033004368.1 Pantoea sp. BL1 WP 045832390.1 Vibrio sp. 3062 WP 063605216.1 Erwinia typographi WPO348982.91.1 Vibrio diazotrophicus WPO42489735.1 Erwinia WP 0145396.19.1 Wibrio crassostreae WP 048662880.1 Erwinia iniecta WP 052902020.1 Vibrio sp., MED222 WP 009848243.1 Pantoea sp. PSNEH2 WP 038629825.1 Wibrio tasmaniensis WP 032500146.1 Erwinia piriflorinigrans WP 023656527.1 Vibrio hyugaensis WP 0454.66146.1 Pantoea agglomerans WP 033780412.1 Moritela Wiscosa WP 045112351.1 Erwinia toletana WP 017801681.1 Vibrio sp. 32-17 WP 050654326.1 Erwinia maotivora WPO34935.024.1 Vibrio navarrensis WP 039422096.1 Pantoea sp. MH WP 024966000.1 Psychromonas ingrahamii WP 011771703.1 Erwinia amylovora WP 004160504.1 idiomarina Xiamenensis WP 008489429.1 Type-F symbiont of Plautia stati WP 058956993.1 Vibrio Sagamiensis WP 039980960.1 Pantoea sp. Sc1 WP 009092618.1 Vibrio owensii WP 04298O126.1 Pantoea anthophila WP 046101010.1 Vibrio sp. 32-4 WP 050649045.1 Erwinia persicina WP 062748343.1 Aliiwibrio Salmonicida WP 012551314.1 Pantoea sp., At-9b WP 013507482.1 Vibrio rhizosphaerae WP 038.185523.1 Symbiont BFo1 of Frankiniella occidentatis WP 048917577.1 Alivibrio logei WP 01702.0963.1 Erwinia tasmaniensis WP 012442801.1 Vibrio genomosp, F10 WP 017036869.1 Erwinia sp. Leaf S3 WP 056235041.1 Vibrio breogani WP 017243149.1 Pantoea sp. PSNEH1 WPO39381957.1 Afteromonas nacleodii WP 041693341.1 Pantoea ananatis WPO29569357.1 Wibrio hepatarius WP 05341.0630.1 Type-D symbiont of Plautia stati WP 058972255.1 Vibrio mytili WP 041155288.1 Pantoea stewartii WP 006121550.1 Vibrio Scophthalmi WP 005599707.1 Pantoea dispersa WP O58757568.1 Psychromonas sp. SPO41 WP 025564742.1 Fig. 13 cont’d Patent Application Publication Dec. 1, 2016 Sheet 15 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession Type-B symbiont of Plautia stati WP O59028282.1 Vibrio sp. S234-5 WP 045569648.1 Pantoea sp. OXWO6B1 WP O63877979.1 Vibrio sp. 2423-01 WP 061893561.1 WP 019212311.1 Marinobacter lipolyticus WP 036190774.1 Erwinia tracheiphila WP 016191385.1 Vibrio sp. MEBiC08052 WP O59123026.1 Yersinia frederiksenii WP 004711220.1 Alteromonas mediterranea WP 012516591.1 Pantoea sp. A4 WP O26042541.1 Vibrio toranzoniae WP 060468829.1 Yersinia rodei WP 032817534.1 Vibrio Cycitrophicus WPO16798923.1 Yersinia aldovae WP 049689167.1 Wibrio rumoiensis WPO17024312.1 Rouxiela chamberiensis WP 045048258.1 Vibrio sinatoensis WP 039481766.1 WP 057648693.1 Vibrio sp., N418 WP O09384140.1 Candidatus Hamiltone:a Marinobacterium defensa WP 016857191.1 rhizophilum WP 020679626.1 Yersinia moa retii WP 050536989.1 Psychromonas hadais WP 022942336.1 Yersinia kristensenii WP 004391858.1 Vibrio kanaloae WP 017055514.1 Yersinia intermedia WP 005191439.1 Marinobacterium titorale WP O27854.294.1 Serratia odorifera WP 004957855.1 Vibrio sp. ECSMB14106 WP 046224925.1 Pseudomonas sp. NBRC Yersinia bercovieri WP 005271235.1 111130 WP O54884750.1 Serratia fonticola WP O59199031.1 Galibacterium genomosp. 2 WPO39136020.1 Yersinia pekkanenii WP 04.9613832.1 Pseudomonas sp. 2-92 (2010) WP 028618504.1 Serratia narcescens WP 015962093.1 Moriteia sp. PE36 WP OO6030970.1 Serratia rubidaea WPO54305351.1 Vibrio mimicus WP 032467641.1 Serratia protearmaculans WP 012147158.1 Shewanella waksmanii WP 028772807.1 Serratia ficaria WP 061799.193.1 Wibrio fluvialis WP 032081097.1 Serratia Eduefaciens WP 044553804.1 Pseudomonas Vranovensis WP 028942480.1 Serratia sp. S4 WP 017894211.1 idiomarina atlantica WP 034733921.1 Serratia grimesii WP 037416107.1 Gallibacterium genomosp. 1 WP 039174,494.1 Serratia WP 020837172.1 Shewanella frigidimarina WP_059745295.1 Serratia plymuthica WP 062868878.1 Pseudomonas putida WPO43209917.1 Yersinia rucker WP 004719425.1 Vibrio haioticoi WP 023405283.1 Yersinia pseudotuberculosis WP 050117587.1 Simiduia agarivorans WP O15047857.1 WPO54104465.1 Alteromonas austratica WP 052806549.1 Serratia sp. YD25 WP 06391.8667.1 Afteromonas marina WP 039222473.1 Yersinia pseudotuberculosis complex WP 033848617.1 Pseudomonas fluorescens WP 034101515.1 Serratia symbiotica WP 061770918.1 Pseudomonas sp. FeS53a WP 044,401466.1 gamma proteobacterium Serratia sp. FS14 WP 044030326.1 MCC1989 WP 009670102.1 Serratia sp. LeafSO WP 055774138.1 Spiribacter salinus WP 016352700,1 Fig. 13 cont’d Patent Application Publication Dec. 1, 2016 Sheet 16 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession Enterobacteriaceae bacterium B14. WP 051014381.1 Galibacterium anatis WPO39166724.1 Aggregatibacter Serratia sp. M2413 WP 009638599.1 actinomycetemcomitans WP 005538626.1 Yersinia nurnii WP 049598056.1 Cellvibrio sp., BR WP 007638851.1 aturnelia Saanichensis WP O29686.453.1 Aggregatibacter aphrophilus WP 050694113.1 Photorhabdus luminescens WP 040154039.1 idiomarina sediminum WP 051207005.1 Xenorhabdus poinarii WPO4595.9602.1 Shewanea sediminis WP 012144760.1 Bartonelia Senegadensis WP 019221445.1 Wibrio furnissi WP 055466655.1 Xenorhabdus Szentirmaii WP 038235621.1 Vibrio eZurae WP 021715061.1 ?seudomonas sp. TU2014 Pectobacterium carotovorum WP 039355229, 1 185ASC WP 058063592.1 Serratia sp., DD3 WP 023490517.1 Vibrio proteolyticus WP 021707164.1 Chania multitudinisentens WP 024913341.1 Baneatrix apica WP 051527455.1 Pectobacterium betavasculorum WP 039303019.1 Pseudomonas parafulva WP 039582433.1 Pectobacterium atro septicum WP 011092244.1 Shewanea oihica WP 011867537.1 Photorhabdus heterorhabditis WP054478023.1 Vibrio Vunificus WPO39541844.1 Pectobacterium wasabiae WP 012.822481.1 Nitrococcus mobilis WP 04.0661924.1 Xenorhabdus WP 047769935.1 Pseudomonas sp. 5 WP 0451861.99.1 Xenorhabdus nematophia WPO38219455.1 Pseudomonas trivia is WP 049710900.1 Bartonea henselae WP 011181,000.1 Pseudomonas Stutzeri WP 038663939.1 Bartonelia koehlerae WP 034459798.1 Pseudomonas tuomuerensis WP 039606725.1 atumea morbirosei WPO38023855.1 Pseudomonas rhodesiae WP 0402.66714.1 Serratia sp. ATCC 39006 WP 021014322.1 Pseudomonas sp. ARP3 WP 047881785.1 Ewingelia americana WP 034793486.1 Afteromonas sp., Ati 199 WP 025257082.1 Hafnia awei WP 043490453.1 Celvibrio sp. peatriver WP O49631176.1 Rahneia WP 013577374.1 Psychromonas acquimarina WP 028862328.1 Rahnella aquatilis WP 047612327.1 Pseudomonas WP 043314995.1 Serratia sp. LeafS1 WP O56776024.1 :Marinobacter daepoensis WP 0296.54624.1 Sedimenticola Pectobacterium sp. SCC3193 WP 01469870.2.1 selenatireducerns WP 037375012.1 Bartonea baciliformis WP 041849739.1 ?seudomonas sp. TKP WP 024078144.1 Dickeya sp. DW 0440 WP 035339654.1 Pseudomonas corrugata WP 024.777876. Budvicia aquatica WP 036017158.1 ?seudomonas sp. AAC WP 043268085.1 Photorhabdus asymbiotica WP 036770256.1 Colwelia psychrerythraea WP 033093585.1 Brenneria sp., EniD312 WP 009114521.1 Shewanella sp., P1-14-1 WP O550.24157.1 iPseudomonas fluorescens Xenorhabdus doucetiae WP 045972447.1 grOup WP 033897276.1 WPO36938822.1 Pseudomonas mediterranea WP 047704174.1 Bartonea bovis WP 010702680.1 :Marinobacter subterrani WP 048497131.1 Fig. 3 cont’d Patent Application Publication Dec. 1, 2016 Sheet 17 of 32 US 2016/0348087 A1

Genbank Microorganism ACCession Microorganism Genbank Accession Proteus mirabis WPO36971463.1 Pseudohongiella Spirulinae WPO58022208.1 Proteus hauser WP 023583078.1 Pseudomonas sp. URHB0015 WP 027616796.1 Bartonea birties Pseudomonadaceae WP O27588895.1 Bartonea quintana WP 042995.424.1 Marinobacter Santoriniensis WP 040886922.1 Bartonea winsonii WP O15399.203.1 Vibrio metSchnikovii WP 040903602.1 Xenorhabdus sp. NBA Pseudomonas sp. NBRC XenSa O4 WP 047683929.1 1123 WP 060483806.1 Pseudomonas sp. XenorhabduS bowieni WP 038187361.1 URMO17WK2:3 WP 027917043.1 sp. DB5-6 WP 007553455.1 Marinobacter sp. ClS70 WP 022993199.1 Bartonella taylorii WP 004859565.1 Pseudomonas fuscovaginae WP 054.057810.1 Xenorhabdus khoisanae WP 047963672.1 Pseudomonas sp., M1 WP 024128089.1 Xenorhabdus cabaniasii WP 03826O250.1 Pseudomonas poae WP 015373328.1 Marinobacter Bartonea washoensis WP OO6924009.1 hydrocarbonoclasticus WP 014.422862.1 bacteria Symbiont BFo2 of Frankiniela occidentalis WP 0439.1280.1 Marinobacter sp. CP1 WP 053113189.1 Bartonelia florencae WP 019218918.1 Marinobacter similis WP 052471995.1 Bartonea elizabethae WP OO5773162.1 Morite a marina WP 019441181.1 Dickeya dadantii WP 038924493.1 Vibrio sp. RC586 WP 001072884.1 Lonsdalea quercina WP 026739591.1 Shewaneta Coweliana WP 028764686.1 Pseudomonas Photorhabdus temperata WP 046974225.1 Cremoricolorata WP 03841.1439.1 Dickeya dianthicola WP 024104487.1 Aestuariibacter Salexigens WP O51275567.1 Arsenophonus nasoniae WP 034249744.1 Pseudomonas simiae WP 047542440.1 Dickeya zeae WP 016943166.1 Pseudomonas sp., SHCS2 WP 041020540.1 Pragia fontium WP 047782060.1 Saccharospirium impatiens WP 051208090.1 Pseudomonas Bartonella meiophagi WP 007476822.1 chioritidismutans WP 042927861.1 Dickeya WP 038917764.1 Nitrincola sp. A-D6 WP 036522654.1 eminorelia grimontii WP 027275775.1 Shewanella sp. cp20 WP 041509787.1 gamma proteobacterium Bartonea alsatica WP O05864859.1 HCC22O7 WP 007231113.1 Dickeya sp. NCPPB 3274 WP 042858576.1 Pseudomonas sp. p.21 WP 06391224.5.1 Bartonella queenslandensis WP 039758997.1 Alteromonas WP 032094739.1 Arsenophonus endosymbiont of Niaparvata lugens WP 032116478.1 Pseudomonas batumici WP 040071885.1 Bartonea doshiae WP 004.855905.1 Pseudomonas sp. Ant30-3 WP 028620438.1 Bartonea Schoenbuchensis WP 010704040.1 Marinobacter sp., EVN1 WP 02300.9487.1 Providencia rettgeri WP 004261323.1 Pseudomonas monteii WP 060477249.1 Fig. 13 cont d Patent Application Publication Dec. 1, 2016 Sheet 18 of 32 US 2016/0348087 A1

Genbank Microorganism Accession Microorganism Genbank Accession Actinobacilus Dickeya sp. 2812 WP 033570629.1 WPOO5625006.1 Pseudomonas sp. Dickeya Solani WP 022632063.1 RMO17WK12:4. WP 027908969.1 atunnella WPO2590.0954.1 Pseudomonas sp. FG 182 WPO25341124.1 Morganella morganii WP 024475151.1 Pseudomonas agarici WP 017132350.1 Morganella WP 004.241531.1 Pseudomonas Veronii WP 017849993.1 Bartonea tribocorum WP 0384.73768.1 Dickeya chrysanthemi WP 040002537.1 Brenneria goodwinii WP 048636333.1 Dickeya paradisiaca WP O15855063.1 Candidatus Regiella insecticola WP O06705673.1 Dickeya sp. NCPPB 569 WPO42868420.1 Bartonella grahamii WP 034451706.1 Moellerella wisconsensis WP O539.07569.1 Bartonea rattalustraliani WP 01922.2387.1 Fig. 13 contid Patent Application Publication Dec. 1, 2016 Sheet 19 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession

Pseudomonas WP 003114686.1 Bacius sp. NH71 WP 060697735.1 WP 023089494.1 Bacius sp., WP8 WP 039180923.1 Pseudomonas sp. 2126 WP 009316330.1 Bacius Sp. Aph1. WP 034271361.1 Microvirgula aerodenitrificans WP 028498979.1 Azospiritum sp. B506 WP 04997.5975.1 Burkholderia contaminans WP O39366334.1 Virgibacilius pantothenticus WP 050350407.1 Burkhoideria cepacia WP 059.525390.1 Bacius sp. B-jedd WP 043826224.1 Burkhoideria Cepacia complex WP 027789658.1 Bacius simplex WP 061142094.1 Burkhoideria cenocepacia WP 043887 199.1 Virgibacilius sp. SK37 WP 040955898.1 Burkhoideria oklahomensis WP 010108306.1 Bacius thermotolerans WP 03923.8772.1 Burkholderia WPO48024784.1 unclassified Baciliaceae WP 040037859.1 Burkholderia anthina WP 05964.08.04.1 Bacius sp. Root020 WP O56766977, 1 Burkholderia ata WP 011354275.1 Anoxybacilius thermarum WP 043966577.1 Burkholderia sp., MSh1 WP 031398726.1 Bacius Coahuiensis WP 01.0174447.1 Burkholderia sp. MSh2 WP 0341.98724.1 8acius safensis WP 044332225.1 Burkhoideria sp. ABCPW 11 WP 059505050.1 Alkalibacillus haloalkaliphius WP 017187026.1 Burkhoideria Seminalis WP 059556868.1 Bacius sp. 333 WP 034263269.1 Burkholderia pseudomaiei WP 004.551415.1 Viridibacilius arenos WP 038188166.1 Burkhoideria thailandensis WP 009900942.1 Bacilius sp. FAT-14578 WP 028395275.1 Burkholderia gumae WP 052498364.1 Oceanobacilius kimchii WP 017797441.1 Burkhoideria plantarii WP O551394.95.1 Bacius decisifrondis WP 053592881.1 Virgibacilius Pseudomonas mandei WP 0504.82791.1 haodernitrificans WP 019379016.1 Pseudomonas fluorescens WP 047337084.1 Gracilibacilius sp. Awa-1 WP 058306868.1 Nitrococcus mobilis WP 005.004375.1 Bacius sp. MSP13 WP 039074850.1 Pseudomonas protegens WP 041752315.1 !ysinibacilus macroides WP 053996681.1 Pseudomonas sp. PH1b WP O25129888.1 Bacius sp. FSAE-27251 WP 0533644.01.1 Pseudomonas putida WP 023535698.1 iysinibacilius massiliensis WPO36180684.1 Pseudomonas sp. ABAC61 lysinibacilius sp. FJAR-14745 WP 0534.85081.1 Pseudomonas veronii WPO32804924.1 Sphaerobacter thermophilus WP 052295394.1 Pseudomonas sp. ARP3 WP 053060097.1 8acius sp., SA2-6 WP 046525629.1 Pseudomonas sp. PAMC 25886 WP 01.0169497.1 Bacius Selenatarsenatis WPO41968022.1 Fig ... 4 Patent Application Publication Dec. 1, 2016 Sheet 20 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Pseudomonas sp. MRSN221 WP 044,463386.1 Gracilimonas tropica WP 020401972.1 Pseudomonas rhodesiae WP 040269528.1 Oceanobacilus oncorhynchi WP 04253.0686.1 Pseudomonas sp. 2(2015) WP 045198122,1 Bacius mura is WP 057915654.1 Pseudomonas sp., BRG-100 WP 032876543.1 Bacius maiacitensis WP 059291772.1 Pseudomonas chlororaphis WP 052712847.1 Anoxybacillus sp. KU2-6(11) WP 03504.8665.1 Pseudomonas sp. CF 68 WP 018605339.1 Domibacilus enciensis WP 052698560.1 Pseudomonas heter WP 048388690.1 Bacius axarquiensis WP 059352147.1 Pseudomonas sp., RootS69 WP 056846015.1 Brevibacterium halotolerans WP 059335649.1 Pseudomonas sp. FH1. WP 033901147.1 lysinibacilius Xylani lyticus WP 0496674.04.1 Pseudomonas sp. 2 92 (2010) WP 050587840.1 | Bacius tecuiensis WP 024.714703.1 Bacius sp. Pseudomonas Ibanensis WP 059396815.1 | UNC322M FChirA.1 WP 035432514.1 Pseudomonas simiae WP 047543762.1 | Solibacilius WP 008408138.1 Alcanivorax dieseloe WP 014994344.1 Sporosarcina koreensis WP 060206543.1 Pseudomonas thivervalensis WP 053121146.1 lysinibacillus sphaericus WP 010860294.1 Pseudomonas Synxantha WP 057025332.1 lysinibacilus sp. F5 WP 05884.5031.1 bacterium JKG WP 029214447.1 Paenisporosarcina sp. G-14 WP 017380005.1 Gracilibacilius halophilus WP 003467031.1 Bacius Eichenifornis WP 043925819.1 Pseudomonas Syringae WP 024668534.1 lysinibacilus odysseyi WP 036151007.1 bacterium mt3 WP 054949256.1 Anoxybacilius sp. BCO1 WP 042894993.1 Pseudomonas sp. ADP WP 058489589.1 Viridibacilius arvi WP O53416717.1 Pseudomonas Syringae Geobacilius group genomosp, 7 WP 055005936.1 thermodernitrificans WPO29760552.1 Pseudomonas Syringae group genomosp. 3 WP 057415224.1 Geobacilius sp. PA-3 WP 060476126.1 Pseudomonas amygdali WP 005762842.1 Geobacilius sp. G11MC16 WP 008380976, 1 Azospiritum lipoferum WP 014188759.1 Bacius Velezensis WP 029974105.1 Bacius acquimaris WP 052011500.1 ilaiapricum salinum WP 049992575..1 Pseudomonas Syringae group WP 007245942.1 Bacius stratosphericus WP 03996466.1 Pseudomonas Caricapapayae WP 0550.09862.1 Baciliates WP 014114896.1 Bradyrhizobium WP 024580699.1 Solibacius silvestris WP 014823056.1 Oscillochloris trichoides WP 006562625.1 Bhargavaea Cecembernsis WP 0083001.06.1 Bacius enciensis WP 058298109.1 Sporosarcina sp. EUR32.2.2 WP 024534129.1 Pseudomonas savastanoi WP 004665562.1 Bacilius sp. WP 052586469.1 Fig. 14 continued Patent Application Publication Dec. 1, 2016 Sheet 21 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Bacius sp. SG-1 WP 006836445.1 Bacius xiamenensis WP 008360695.1 Pseudomonas awe anae WP 005617735.1 Bacilibacterium VT-13-104 WP 047184596.1 Microbubifer variabilis WP 020415351.1 Bacius sp. DW5-4 WP 034.325145.1 Pseudomonas fuSCOvaginae WP O54064572.1 Bacilius attitudinis WP 047945217.1 Bacius wietnamensis WP 051758539.1 Panomicrobium sp. ES2 WP 052652109.1 Bacius sp., LL01. WP 047970983.1 Geobacilius subterraneus WP 063167279.1 Caldakalibacilius thermarum WP 007503034.1 Geobacius WP 042381875.1 Bacius marisflavi WP 0480.13478.1 Oceanobacilius caeni WP 060668740.1 Pseudomonas Cichorii WP O25259793.1 Lysinibacilius sp. ZYM-1 WP 054612847.1 Aeribacilius pallidus WP 063386559.1 lysinibacilius varians WP 025220363.1 Brevibacterium Bacilius azotoformans WP 035196603.1 frigoritolerans WP 063589832.1 Desmospora sp., 8437 WP 040387746.1 Bacius sp. BSC154 WP 041906541.1 Bacius horikoshii WP 063559773.1 Terribacilius aidingensis WP 038565035.1 Halobacilius halophilus WP 014644096.1 Alicycobacilius pomorum WP 051375075.1 Gracilibacilus Bacius humi WP 0579995.05.1 boraciitolerans WP 035724.544.1 Salimicrobium jeotgali WP 008587369.1 Virgibacilius Soli WPO57982217.1 jeotgalibacilius malaysiensis WP 039810607.1 Bacius sp. 37 MA WP 018394.222.1 Geobacilius sp. Y4.1MC1 WP 0134.00205.1 Panomicrobium glacie WP053167718.1 Pseudomonas agarici WP 060783693.1 Bacilius cihuensis WP 028392653.1 Bacilius sp. Leafa.06 WP O56534732.1 Bacilius cereuS WP 016116829.1 Desulfovibrio desulfuricans WP 041724102.1 Geobacilius sp. JS12 WP 06319.3197.1 Anaerobacius macyae WP O53216218.1 Kurthia massiensis WP 0102884.09.1 Pontibacilius halophilus WP 0268O1400.1 Bacilius sp. Soi1768D1 WP 057215430.1 Geobacilius toebii WP 062755081.1 Bacius Sonorensis WP O06636053.1 Bacilius sp. m3-13 WP 010195203.1 Bacius sp. FAT-20673 WP 063574832.1 Geobacilius thermoglucosidasius WP 042384399.1 lysinibacilius boronitolerans WP 036078490.1 Pontibacilius marinus WP 027447035.1 Bacius sp. Leaf13 WP 056521250.1 Geobacilius sp. WCH70 WP 015864892.1 Bacius sp. 72 WP 051927823.1 Thalassobacilius sp. TM-1 WP 062440761.1 Geobacius sp. JF8 WP 020961.008.1 Bacius sp. CHD6a WP 060666910.1 Bacius butanoivorans WP O53347927.1 Baciliaceae WP 003248477.1 Geobacius sp. C56-T3 WP 013144393.1 Bacius sp., SA1-12 WP 046590138.1 Bacius pseudomycoides WP 006096312.1 Bacius massiliogorillae WPO42345107.1 Roseiflexus sp. RS-1 WP 011954829.1 Fig. 4 continued Patent Application Publication Dec. 1, 2016 Sheet 22 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Pontibacius chungwhensis WP 036782710.1 Bacilius sp. 95MFCvi2.1 WP 018782.033.1 thermogemmatispora carboxidivorans WP 052888923.1 Bacilius cereus group WP 040119032.1 Salinibacilius aidingensis WP 044163325.1 Lysinibacilius fusiformis WP 004225913.1 Bacius sp. X1(2014) WP 038536892.1 Bacilius mycoides WP 041488594.1 Lentibacilius jeotgali WP 01.0532310.1 Lysinibacilius sp. K3 WP 048391047.1 Bacius ginsengihumi WP 035353906.1 Bacilius sp. Soi 745 WP 0572791911 Jeotgalibacilius Soi Cunha et al. 2012 WP 052474929.1 Bacilius sp. FAS-27916 WP 04966.9789.1 Bacius shackletonii WP O55738952.1 hermonicrobium roseum WP 012642614.1 Pontibacilus yanchengensis WP 036821077.1 Geobacilius sp. CAMR5420 WP 033026053.1 Bacius niacini WP 034673537.1 Bacilius sp. 105MF WP 018764645.1 Bacitus sp. 337 WP 026561814.1 Bacius mantiponensis WP 034642812.1 Bacius wireti WP 024027849.1 Bacilius sp. FAR-27245 WP 053367481.1 Bacius cibi WP 029566209.1 Lysinibacilius sp. BF-4 WP 036142602.1 Bacius alveayuensis WP 052659551.1 Lysinibacilius WP 036119793.1 Bacius indicus WP 029278756.1 Opitutus terrae WP 012376455.1 Bacius bataviensis WP 007086491.1 Domibacius robiginosus WP050181303.1 halassobacilius devorans WP 028783548.1 Bacius aminovo rans WP 063975020.1 Chloroflexus aggregans WP 012615533.1 Bacilius sp. 1 NLA3E WP 041580669.1 Bacius fordii WP 018707485.1 Sporosarcina newyorkensis WP 009497990.1 Virgibacilius sp. SK-1 WP 0532.18805.1 Bacius sp. GeB10 WP:006915660.1 Bacius Smithii WP 048623884.1 Paenisporosarcina sp. TG20 WP 019414907.1 Halobacilius kuroshimensis WP 027956472.1 Planococcus antarcticus WP 006831222.1 Geobacilius Bacilius sp. Caldoxylosiyticus WP 017434868.1 UNC437CLA2CVS29 WP 026593876.1 Bacius massilioanorexius WP 019243994.1 Bacilius sp. 123MFChir2 WP 020061371.1 Geobacilius Stearothermophius WP 043905856.1 Haiobacterium sp. CBA1132 WP 058982752.1 Bacius circulans WP 061798785.1 Streptomyces sp. MBT76 WP 058042239.1 Ktedonobacter racemifer WP 007913623.1 Bacilius sp. FA-1383 WP 017153674.1 Jeotgalibacilius aimentarius WP 052474147.1 Bacilius gaemokensis WP 033676253.1 Bacius sp. FJAT-27445 WP 059171493.1 Bacil WP 000616738.1 Sporolactobacilius Bacius WP 009795315.1 aevolacticus WP 023509936.1 Bacius neaisonii WP 016202883.1 Alicycobacilius contaminans WP 051321775.1 Sporosarcina globispora WP O53434007.1 Bacilius cytotoxicus WP 012095948.1 Fig. 14 continued Patent Application Publication Dec. 1, 2016 Sheet 23 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Oceanobacilius picturae WP 036574619.1 Conexibacter woesei WP 035127957.1 Gracilibacillus acisalsi WP 018934096.1 Halobacterium hubeiense WP O59056695.1 Oceanobacilius sp. S5 WP 040979050.1 Bacius sp. H1a WP 025148828.1 Alicyclobacilius macroSporangidus WP 051662824.1 Bacius thuringiensis WP 023523256.1 Bacius sp. NCA33C73SS30 WP 026572089.1 Patulibacter americanus WP 022928.512.1 Bacilius sp. ZYK WP 0177562O2.1 Bacius sp. B14905 WP 04399.0721.1 Bacius psychrosaccharolyticus WP 051387396.1 Planococcus kocurii WP 058385831.1 OrnithinibaciliS contaminans WP O47979667.1 Planococcus sp. CAU13 WP 033543886.1 Anoxybacilius tepidamans WP O274.10481.1 Sporosarcina ureae WP 029055120.1 Oceanobacilius manasiensis Geobacillusicigianus WP 042222742.1 WP 033018318.1 Bacius methanoicus WP 004439139.1 Sporolactobacilius terrae WP 051577709.1 Halobacilus sp. BBL2006 WP 035548017.1 Tuberibacilus calidus WP 027724.185.1 Oceanobacius massiiensis WP 01.0647294.1 Geobacillus vulcani WP 031407519.1 Bacius flexus WP 061784908.1 Bacius Coagulans WP 035188932.1 Chloroflexus sp. Y-396-1 WP 028459931.1 Bacius sp., K2 WP 048374368.1 Ornithinibacius californiensis WP 047983652.1 Kurthia huakui WP O2949.9533.1 Hatobacius WP 035511377.1 Halakalibacilius halophilus WP 027964378.1 Bacilius encimensis WP 063383670.1 Domibacius tundrae WP O52728327.1 Bacilius sp. JS WP 041521409.1 bacterium S5 WP 062354774.1 Planococcus sp. PAMC Anoxybacilius flavithermus WP 006.320635.1 21,323 WP 038703416.1 Bacilius badius WP O63441135.1 Geobacillus kaustophilus WP 044736356.1 Bacius rubiinfantis WP 042354.695.1 Exiguobacterium WP 035412678.1 Streptomyces sp. NRRS Desu fitibacter alkalitolerans WP 051534294.1 83 WP 051844821.1 Bacilius sp. RP1137 WP O29319903.1 Kurthia sp. C3E WP 010304177.1 Balneola vulgaris WP 018127710.1 Roseiflexus castenholzii WP 012122664.1 Bacilius aryabhattai WP 045295385.1 Bacitus anthracis WP 000616727.1 Bacilius niameyensis WP 0621.09560.1 Bacius sp. OxB WP 041070670.1 Bacius sp. FA-25547 WP 057761139.1 Halolamina rubra WP 049981866.1 Bacilius acidiproducens WP 051086254.1 Bacius sp. FAT-27997 WP 049682973.1 Salinarchaeum sp. Harcht Bacius thermoamylovorans WP 034768563.1 Bsk WP_020447809.1 Fig. 14 continued Patent Application Publication Dec. 1, 2016 Sheet 24 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Bacius sp. URhB0009 WP 027322137.1 Bacius sp. GZT WP 062923621.1 Halobacilus sp. BAB-2008 WP O08637585.1 Sporosarcina sp. ZBG7A WP 039044539.1 Bacius sp. A053 WP 040082038.1 Bacilius sp. UMTA18 WP 046199487.1 Halobacteriaceae archaeon Bacius sp. UNC41MFS5 WP 026563219.1 S39 WP 058581597.1 Bacius sp. Soi531 WP 057274095.1 Halopiger djelfamassiliensis WP 049923288.1 Bacius Siamensis WP 016938.462.1 Alicyclobacilius herbarius WP 051343768.1 Bacius farraginis WP 058005647.1 £xiguobacterium indicum WP 058704972.1 Bacius subtilis WP 014477756.1 Matronococcus amylolyticus WP 005555286.1 Bacius Subterraneus WP 044395766.1 lysinibacilius manganicus WP 036190256.1 Bacius gobiensis WP 0536.03894.1 Exiguobacterium sp. BMC-KP WP 053452202.1 Paucisalibacillus sp., E802 WPO42143024.1 slalorubrum sp. BV1 WP 04998.2315.1 Bacius amyloliquefaciens WP 047476771.1 Haloarcua waismortis WP 004.517947.1 Bacius sp. ST10 WP050616161.1 Haiostagnicola S.p.A56 WP 050051196.1 Bacius sp. Root 147 WP 057233,096.1 Haloarcula japonica WP 004592792.1 Streptomyces sp., A exAB Bacius koreensis WP 053400748.1 D23 WP 018554385.1 Bacius sp. 278922107 WP 028411869.1 Alicyclobacilius ferrooxydans WP 054969223.1 Solirubrobacter sp. Pontibacilius litoralis WP 052127216.1 URHOOO82 WP 051323957.1 Lysinibacilius contaminans WP 053582362.1 Haiorubrum hochstenium WP 008580740.1 Saloferax mucosum Bacius sp. JF15 WP 049627228.1 WP 008317500.1 Anoxybacilius kamchatkensis WP 019417289.1 haloarcuta amyolytica WP 008309243.1 *xiguobacterium Bacius sp. FJAT-25496 WP 057772306.1 oxidotolerans WP 029332750.1 Bacius glycinifermentans WP 0483.55295.1 Haloarcuta WP 050033036.1 Bacius sp. FF4 WP 042460803.1 Exiguobacterium acetylicum WP O50677396.1 Bacius sp. SDE1 WP 060964.475.1 haloarcula sp. CBA1127 WP 058995943.1 Bacius atrophaeus WP 01.0789607.1 Halopenitus sp. DYS4 WP 0583.66711.1 Oceanobacilius heyensis WP 011066719.1 Haiorubrum tebenquichense WP 006630090.1 Haadaptatus Bacius megaterium WPO13085283.1 paucihalophilus WP 007978613.1 Bacius sp. Root239 WP 057244921.1 Sotirubrobacter Soli WP 028064223.1 Oscillatoriales Chloroflexus sp. MS-G WP 031458749.1 cyanobacterium MTP1 WP 058833121.1 Anoxybacilus geothermalis WP 044745973.1 Haloarcuta marismortui WP 011223264.1 Lysinibacilius sindu riensis WP 036197348.1 Sporosarcina sp. D27 WP 025.786354.1 Fig. 14 continued Patent Application Publication Dec. 1, 2016 Sheet 25 of 32 US 2016/0348087 A1

Genbank Genbank Microorganism Accession Microorganism Accession Anoxybacius WP 0.09361645.1 Exiguobacterium sp. Leaf187 WP 055966688.1 Bacilius endophyticus WP 019391067.1 Haloarcula hispanica WP 014040312.1 Bacilius cecembensis WP 057988977.1 Haiosimplex carlsbadense WP 006834453.1 Exiguobacterium sp. Bacilius valismortis WP 061571926.1 ZWUOOO9 WP 047395159.1 Bacilius sp. G12015b) WP 058838176.1 Halorhabdus utahensis WP 015788577.1 Bacillus sp. NSP9.1 WP 026588395.1 Planococcus halocryophilus WP 008497280.1 Verrucomicrobia bacterium Bacilius sp. FJAT-27231 WP 049663918.1 SCGC AAA68-F10 WP 038126170.1 Paucisalibacilius globulus WP 026906974.1 Exiguobacterium sp. OS-77 WP 035398779.1 Ureibacus thermosphaericus WP 016837030.1 Halakalicoccus jeotgali WP 008417532.1 Virgibacilius sp. Vm-5 WP 038243188.1 Planococcus donghaensis WP 0084.28950.1 Bacilius Subtilis group WP 013390633.1 Haiorubrum halophium WP O50032715.1 Chloroflexus WP 012259490.1 Exiguobacterium Sibiricum WP 012369533.1 Psychrobacilius sp. FJAT Bacilius sp. EGD-AK10 WP 021480367.1 21,963 WP 056832867.1 Bacius kribbensis WP 0353224.54.1 Bacilius sp. FAT-25509 WP 056473274.1 Bacilius sp., REN51N WP 04.0056994.1 Haiorubrun arcis WP 007992.958.1 Virgibacilius sp. CM-4 WP 021288888.1 Haloarcula sp. SL3 WP053968146.1 Paenisporosarcinasp, HGHOO3O WPO16426536.1 Haloferax mediterranei WP:004.056921.1 Bacilius sp. EB01 WP 043934015.1 Haloarcuta argentinensis WP 005534453.1 Bacilius panaciterrae WP_028399.695.1 Bacius sp. FAR-22090 WP 053588542.1 Bacilius sp., 171095. 106 WP 028410453.1 Halolamina pelagica WP 054583766.1 Ornithinibacilius Scapharcae WP 010097123.1 Geobacilius sp. 12AMOR1 WP 047818914.1 Bacilius nakamurai WP 06152.0043.1 Microcystis aeruginosa WP 004163819.1 Jeotgalibacilius campisalis WP 041060570.1 Halorubrum sp. T3 WP 017343453.1 Bacilius mojavensis WP O29441396.1 Exiguobacterium sp. NG55 WP 035387788.1 Anoxybacilius Suryakundensis WPO554,40175.1 Haiorubrum WP 0.04598556.1 Bacilius sp. FF3 WP 042474379.1 Exiguobacterium marinum WP 026826747.1 Bacillus sp. THO08 WP 046130688.1 Halorubrum aidingense WP 007999743.1 Bacilius sp., AM 13(2015) WP 059375200.1 Bacilius pumius WP044142126.1 Bacilius sp. CMAA 1185 WP 046160765.1 Bacilius sp. FAT-18017 WP 0535936.18.1 Bacilius firmus WP 048011096.1 Fig. 4 continued Patent Application Publication Dec. 1, 2016 Sheet 26 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Pseudomonas WP 003106950.1 Brew undimonas diminuta WP 003165630.1 Pseudomonas aeruginosa WP 023131684.1 Erythrobactersp. NAP1 WP 007164103.1 Pseudomonas aeruginosa group WP 0.09877106.1 Erwinia persicina WP 062742761.1 Microvirguia aerodenitrificans WPO28498980.1 teisingera sp. ANG-M1 WPO39168607.1 Burkholderia cepacia complex WP OO6484772.1 Shinea WP 050742571.1 Burkholderia anthina WP O59584467.1 Sphingomonas sp. KC8 WP 01025831.1 Burkholderia cenocepacia WP 060211908.1 Pseudomonas taeanensis WP 025166139.1 Burkholderia WP 01154.7168.1 Cautobacter sp. OV484 WP 0474.04747.1 Pseudomonas sp. NBRC Burkholderia pseudomailei WP 004.549567.1 135 WPO5491.0491.1 Burkholderia ata WPO11354276.1 Pseudomonas sp. Leaf15 WP 056858237.1 Burkholderia Contaminans Ruegeria pomeroy

WP 039366337.1 WP 011047295.1 Candidatus Fomicrobium Burkholderia cepacia WP 05952.5391.1 marinum WP 046475.955.1 Burkholderia thailandensis WP 0.09897990.1 Ensifer sp. Br316 wP 018234899.1 Burkholderia okahomensis WP 010108308.1 Cautobacter wP 056050097.1 Pseudomonas fluorescens WP 016979925.1 Rhizobium sp. Leaf341 WP 062692519.1 Pseudomonas sp. 2 92 (2010) WP 028616070.1 Rhizobium WP 062470505.1 Pseudomonas azotoformans WP 061436824.1 Ensifer sojae WP 034859346.1 Pseudomonas sp. FH1 WP 033901146.1 Ruegeria mobilis WP OO5628022.1 Pseudomonas thiverwaensis WP O53121148.1 Sedimentitaea nanhaiensis WP 027263471.1 Pseudomonas Synxantha WP 057025331.1 Porphyrobacter cryptus WP 027441928.1 Pseudomonas sp. CHMO2 WP O25854584.1 Porphyrobacter sp. AAP60 WP 054117689.1 Pseudomonas mandeii WP 033056094.1 Ewingeila americana WP 0347896.90.1 Pseudomonas fluorescens group WP 043050251.1 Chromobacterium vaccinii WP 046155509.1 Alcanivorax dieseloei WP 014994845.1 Ruminococcus abus WP 043538032.1 Nitrococcus mobilis WP 005.004372.1 Variovorax paradoxus WP 042576834.1 Pseudomonas sp., 2(2015) WPO45198124.1 Parvularcua bermudensis WPO13300785.1 Rubelimicrobium Pseudomonas sp. PH1b WP O25129887.1 thermophilum WP 021097656.1 Pseudomonas sp. PAMC 25886 WP 010169498.1 Dechloromonas aromatica WP 011289796.1 Fig. 15 Patent Application Publication Dec. 1, 2016 Sheet 27 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Pseudomonas putida WP 023535597.1 Varioworax boronicumulans WP 062477800.1 Pseudomonas helieri WP 048388689.1 Sphingomonas sp. MM-1 WP 015457563.1 Pseudomonas sp., Os17 WP 060839303.1 Phaeobacter inhibens WP 061049393.1 Pseudomonas sp. ABAC61 WP 058436409.1 Variovorax sp. Root473 WP O56580651.1 Pseudomonas protegens WP 041752761.1 Silicibacter sp. TrichCH4B WP O09177420.1 Pseudomonas sp. St29 WP 060843902.1 Phaeospirium notischianum WP 040566020.1 Pseudomonas sp. PAMC Wibrio WP 029223919.1 26793 WP 017477817. Zymobacter pamae WP 027706238.1 Pseudomonas simiae WP 047543395.1 Vibrio spendidus WP 017095663.1 Sinorhizobium arboris WP O27999396.1 Vibrio nigripulchritudo WP 022598767.1 Caulobacter sp. APO7 WP 007669693.1 Vibrio win ificus WP 011031756.1 Porphyrobacter mercurialis WPO39096260.1 Pseudomonas sp. ADP WP 058489588.1 Oceanicola sp. S124 WP 01.0137633.1 Pseudogubenkiania Pseudomonas fuscovaginae WPO54061580.1 ferrooxidans WP 021478802.1 Desulfovibrio desulfuricans Erythrobacter gangjinensis WP 012624972.1 WP 047005672.1 Pseudoalteromonas luteo violacea WP 063364351.1 Klebsiela Oxytoca WP 004131235.1 Pseudomonas entomophia WP 011533710.1 Labrenzia aggregata WP 006935802.1 Pseudomonas sp. KG01 WP 048731723.1 Caulobacter sp. K31 WP 012287297.1 Pseudomonas cichorii WPO2S2597.94.1 Erythrobacter tongus WP 051698842.1 Pseudomonas rhizosphaerae WP 043188773.1 Pseudomonas endophytica WP O55101787.1 Providencia Azospirillum lipoferum WP 014188758.1 burhodogranariea WP 008911142.1 Pseudomonas fulva WP 042556657.1 Caulobacter sp. CCH5-E12 WP 062099535.1 Brenneria sp. EniD312 WP 009114600.1 Ruegeria atlantica WP 058276921.1 tonsdalea quercina WP 026739756.1 Brew undimonas aveniform is WP 029086159.1 Azospirillum sp. B506 WP 042693433.1 Phaeobacter gallaeciensis WP 014879244.1 Pseudomonas monteii WP 060393461.1 Erythrobacter atlanticus WPO48885888.1 Pseudomonas Syringae WP 017708113.1 Ensifer sp. USDA 6670 WP 029959387.1 Pseudomonas Syringae group WP 0.0576284.0.1 He ea balneolensis WP 026940740.1 Pseudomonas amygda WP 005738477.1 Sinorhizobium meioti WP 010968657.1 Sinorhizobium sp., CCBAU Pseudomonas Savastanoi WP 019741432.1 0.563 WP 037425973.1 Fig. 15 continued Patent Application Publication Dec. 1, 2016 Sheet 28 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Pseudomonas Syringae group genOmoSp. 3 WP 054091058.1 Ruegeria sp., CECT 5091 WP 058284.168.1 Microbubifer variabilis WP 02041535.0.1 Ruegeria sp. ANG-R WP 039538859.1 Tolypothrix campylonemoides WP 041041287.1 Cautobacter vibrioides WPO35017242.1 Rhodobacteraceae bacterium Nodositinea nodulosa WP 017298636.1 KH WP 008755607.1 Leptolyngbya sp. NES-2104 WP O59001045.1 Caulobacter Segnis WP 0130784.09.1 Agrobacterium turnefaciens WPO352254.56.1 Klebsiela WP 004.871378.1 Calothrix sp. PCC 71.03 WP 019493169.1 Yersinia WP 050084882.1 Cellvibrio sp. OA-2007 WP 062064.078.1 Pseudomonas libanensis WP 057012799.1 Scytonema tolypothrichoides WP 048868701.1 Rhodobacter sp. SW2 WP 008031693.1 Pantoea sp. RT-P-b WPO49853162.1 Labrenzia sp. DG1229 WPO35899651.1 Stanieria cyanosphaera WP 015195188.1 Ruegeria halocynthiae WP 037310730.1 Pseudomonas chlororaphis WP 009049359.1 Pantoea WP 045815650.1 Ruinea imosa WP 02852.5382.1 Mannheimia varigena WP O25216860.1 Pedobacter sp. W48 WP 043904841.1 Enterobacteriaceae WP 049084995.1 Pedobacter sp. PACM 27299 WP 062550966.1 Pseudomonas fragi WP 0167794O7.1 Pantoea rodasii WP 039334894.1 Cautobacter sp. Root055 WP O56724929.1 Crinalium epipsammum WP 015201559.1 Acidovorax deafieldii WP 060977469.1 Brenneria goodwinii WP 048636391.1 Cautobacter henricii WP 062145109.1 Pseudomonas frederiksbergensis WP 039591.223.1 Sinorhizobium fred WP 037432167.1 Snodgrassella avi WP 0374.73754.1 Rhizobium giardinii WP 018324O75.1 Pedobacter sp. R20-19 WP 029287121.1 Porphyrobacter sp. HL-46 WP 036800189.1 Pseudomonasagarici WP 017133639.1 Erythrobacter marinus WP 047093931.1 Bradyrhizobium WP 024580698.1 Altererythrobacter marensis WP 047806000.1 Hassalia byssoidea WP 039743314.1 Kordimonas gwangyangensis WP 020399.032.1 Type-E Symbiont of Plautia stati WP 058962445.1 Pseudomonas sp. 313 WP 017639561.1 Pseudomonas kilonensis WP 046062292.1 Yersinia intermedia WP 005183468.1 Alcanivorax hongdengensis WP 008930023.1 Ensifer WP O25425846.1 Oscillatoria nigro-viridis WP 01517.9369.1 Ruegeria Conchae WP 010442824.1 Microcoleus vaginatus WP O06635331.1 Paenibacilius zanthoxyli WP O25690613.1 Fig. 5 continued Patent Application Publication Dec. 1, 2016 Sheet 29 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Pseudomonas sp. GM60 WP 008035544.1 Labrenzia sp. CP4 WP 06249.1890.1 Pedobacter sp. Hv1. WP 055131283.1 Labrenzia WP 031269713.1 Serratia ply muthica WP OO6316853.1 Novosphingobium barchaimi WP 058735897.1 Serratia WP 037395580.1 Xenophilus azovorans WP 038209862.1 Dysgonomonas Pseudomonas sp. GM80 WP 008085501.1 Capnocytophagoides WP 026625288.1 Serratia sp. C-1 WP 062789569.1 Cautobacter sp. Root056 WP 057183767.1 Pseudomonas sp. 45MFCO3.1 WP 019648555.1 Thermopetrobacter sp. C1 WP 038O34810.1 Pseudomonas sp. GM48 WP 007992678.1 Thalassospira lucentensis WP 022734.083.1 Pseudomonas sp. GM18 WP 007937000.1 Arthrobacter sp. H14 WP 026535687.1 Synechocystis sp. PCC 7509 WP 0289541911 Brew undimonas abyssa is WP 021697031.1 Pseudomonas sp. OF5 WP 030131028.1 Parwularcula Oceani WP 031555700.1 bacterium UASB.4 WP045505933.1 Blastomonas sp. AAP53 WP 017670274.1 Pseudomonas sp. GM.79 WP 008074041.1 Labrenzia alexandrii WP 055672130.1 Pseudomonas sp. CF161 WP 043230378.1 Pseudomonas deceptionensis WP O48359292.1 Type-D symbiont of Plautia Alcanivorax WP 063521418.1 sta WP 058970.253.1 Pseudomonas sp. Root329 WP 056741929.1 Erythrobacter WP 0506.00626.1 Paudibacterium Gilliamea apicola WP 034883414.1 yongneupense WP 051229,471.1 Pseudomonas sp. GM49 WP 007993749.1 Cautobacter sp., Root1.472 WP 056761772.1 Aquabacterium parvum WP 058086343.1 Ensifer adhaerens WP 053248748.1 Acidithiobacilius thiooxidans WP 024893728.1 Sphingomonas sp., 35-24ZXX WP 033923881.1 Pseudomonas sp. GM41 (2012) WP 008154661.1 Achromobactersp. Ria WP 043546625.1 Pseudomonas Pseudorhodobacter brassicacearum WP O25213967.1 wandonensis WP O50522986.1 Leptolyngbya boryana WP 017288016.1 Aphanizomenon fos-aquae WP 039203516.1 Pseudomonas sp. 11/12A WP 047527806.1 Sphingomonas endophytica WP O58756259.1 Pseudomonas sp. GM 102 WP 007905729.1 Devosia sp. A16 WP 055048936.1 Candidatus Solibacter usitatus WP 011686428.1 Rhizobium sp. Root483D2 WP 060636130.1 Myxosarcina sp. G1 WP 036484294.1 Ensifer sp, WSM1721 WP O26622024.1 Alcanivorax sp. 19-m-6 WPO35233016.1 Enterobacter cancerogenus WP 034824240.1 Pseudomonas sp. GM50 WP 0080084.59.1 Cystobacter fuscus WP 002621816.1 Fig. 15 continued Patent Application Publication Dec. 1, 2016 Sheet 30 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Acaryochloris sp. CCMEE 54 WP 010467794.1 Sphingomonas sp. Y57 WP 047167465,1. Pseudomonas sp. G5 (2012) WP 020800719.1 Sinorhizobium sp., PC2 WP O46119906.1 Acaryochloris marina WP 012162817.1 Sphingomonas jasps WPO37503921.1 Thalassospira sp. MCCC Pseudoalteroinonas WP 0421491.86.1 1AO148 WP 062953912.1 Pantoea sp. A4 WP 026042421.1 Ensifer sp. TW10 WP 026613300,1 Alcanivorax jadensis WP O35250216.1 Fulvimarina pelagi WP O40488894.1 Bacteria WP O09562838.1 Rhizobium sp. CFO97 WP 037119901.1 Pseudarthrobacter chlorophenolicus Pseudomonas sp. RT-P-q WP 0594041.96.1 WPO15937239.1 Serratia grimesii WP 037426347.1 Devosia WP 055878182.1 Scytonema millei WP 039714778.1 Coccidioides in mitis RS XP 001248209.2 Calothrix sp. 336/3 WP 035158367.1 Biastomonas sp. AAP25 WP 054134089.1 Chroococcidiopsis thermais WP 015152152.1 Sphingomonas sp. Ag1. WP 046409195.1 beta proteobacterium L13 WP 017510054.1 Oceanicaulis sp. Hi-87 WP 036514352, 1 Alcanivorax sp. H0083 WP 063518558.1 Penicilium digitatum Pd1 XP 014534104.1 Pseudopedobacter Saitans WPO13633880.1 Maritaiea myrionectae WPO2783.5180.1 Pseudomonas gingeri WP 0171241.51.1. Sinorhizobium sp. GL28 WP O58323580.1 Vogesella sp. EB WP 047967847.1 Erythrobacter sp., SD-21 WP OO6832002.1 Costridiales bacterium W22-28 WPO25484850.1 Sinorhizobium/Ensifer group WPO57243140.1 Cellvibrio sp., BR WP 007642494.1 Afifelia pfennigii WP O51631353.1 Hungateia hatheway WP 039892.287.1 Croceicoccus naphthovorans WP 04782.0461.1 Tatumella sp. UCD D Suzuki WP O25903659.1 Arthrobacter nitrophenolicus WP 035752355.1 tatumea ptyseos WP 029990876.1 Sphingomonas WP 056359867.1 Niabella aurantiaca WP 018629826.1 Thalassospira WPO37991166.1 achnociostridium WP 024296203.1 Anabaena cylindrica WP 015215725.1 Oraconibacteriurn Corynebacterium Sediminis WP 045033031.1 haotolerans WPO4874.2456.1 Desulfowibrio frigidus WP 031479260.1 Ensifer Sp.ZNCOO28 WP 043613157.1 Acinetobacter brisou WP 004902992.1 Brevundimonas sp. Leaf363 WP O561.03760.1 Pseudorhodobacter aquimaris WP 050528496.1 Serratia fonticola WPO24485202.1 Fig. 15 continued Patent Application Publication Dec. 1, 2016 Sheet 31 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession Aftererythrobacter Rubrobacter aplysinae WP 0478666.23.1 epoxidivorans WP 061923692.1 Acinetobacter sp. ANC alpha proteobacterium 3789 WP 004749213.1 2015 WP 038280972.1 atumella morbirosei WP 038O17563.1 Arthrobacter sp. Leaf137 WP O56O79511.1 Altererythrobacter troitSensis WP 057882776.1 Sphingomonas sp. Root710 WP 056378476.1 Dinoroseobacter shibae WP 012177956.1 Sphingomonas wittichii WP 037526704.1 Loktaneta vestfoldensis WP 0263.52351.1 Corynebacterium freneyi WP O52O54332.1 Sphingopyxis WP 003044487.1 Sphingomonas sp., SRS2 WP 046195859.1 Acinetobacter WP 005173592.1 Porphyrobacter sp., AAP82 WPO17664013.1 Sediminimonas AgaricuS bisporus war. qiaohouensis WP 026756407.1 bisporus H97 XP OO6455.303.1 Agaricus bisporus war, Clostridium clariflavum WP 014254971.1 burnettii JB137-S8 XP 00733O894.1 uncultured Sulfuricurvum sp. RFRC-1 WP 015653945.1 Erythrobacter vulgaris WP 040963667.1 Lysinibacillus macroides WP 0539934.07.1 Sphingomonas sp. Leaf231 WPO56631646.1 Dysgonomonas sp. HGC4 WP 050708384.1 Cucumibacter marinus WP 029039849.1 alpha proteobacterium Mf Leisingera sp. ANG-M7 WP 039183947.1 1,05b.01 WP 029638849.1 Sphingopyxis sp. A083 WP053811316.1 Brew undimonas sp., Leaf280 WP 0557.55221.1 Costridium sp. DL-VH} WP 0.09170495.1 Sinorhizobium WP 011974380.1 Lactococcus raffinoactis WP 061.774007.1 Arthrobacter enciensis WP 058267449.1 Acinetobacter woffii WP OO4280839.1 Nocardia otitidiscaviarum WP 039817058.1 Leisingera WP 019294976.1 Citromicrobium WP 010237878.1 Pantoea anthophila WP 046102129.1 Rhizobium sp. OK665 WP 037105052.1 Pseudorhodobacter antarcticus WP 050519835.1 Rhizobium sp. Root482 WP 056330571.1 Kaistia granuli WP 018185327.1 titoreibacter arenae WP 021100.138.1 Bradyrhizobium elkani WP 051003151.1 Bradyrhizobium tropiciagri WP 050420370.1 Pseudomonas Aquabacterium sp. NY1 WP 052162578.1 psychrotolerans WP O58768510.1 Halothiobacius neapolitanus WP 012824911.1 Erythrobacter litoralis WP 011414053.1 Dysgonomonasgadei WP OO680.1167.1 Oceanicau is alexandrii WP_022700009.1 Type-FSymbiont of Plautia sta WP 058957646.1 Phaeobacter WPO40172007.1 Roseibium sp. TrichSKD4 WP 0.09758811.1 Fig. 5 continued Patent Application Publication Dec. 1, 2016 Sheet 32 of 32 US 2016/0348087 A1

Microorganism Genbank Accession Microorganism Genbank Accession eisingera sp. ANG-M6 WP 0391943.16.1 abrenzia aba WP 055675296.1 Bradyrhizobium viridifuturi WP 050629432.1 Pseudomonas Stutzeri WP 045164245.1 Brevundimonas Acinetobacter gerneri WP 004370294.1 naejangsanensis WPO24353237.1 Acinetobacter sp. HR7 WP 034585714.1 Rhizobium sp. Leaf371 WP 062595152.1 Kaistia adipata WP 0290753.59.1 Pseudomonas nitroreducens WP O24762244.1 Pantoea sp. Sc1 WP O09089455.1 Sphingopyxis alaskensis WP 041383.077.1 Sphingopyxis terrae WP 062902254.1 Phaeospirium fulvum WP 051185933.1 Bradyrhizobium embra pense WP 050400635.1 Raouiteia ornithinolytica WP 041145659.1 Raouiteia terrigena WP 045858268.1 Ruegeria sp. TM1040 WP 0115396.77.1 Sinorhizobium americanum WP 037378402.1 Shinella sp. DiD12 WP 023515461.1 leisingera sp. ANG-Vp WPO391846O7.1 Erythrobacter sp. 475 WP 034954745.1 Fig. 15 continued US 2016/0348087 A1 Dec. 1, 2016

GENETICALLY ENGINEERED SUMMARY OF THE INVENTION MCROORGANISMS FOR THE PRODUCTION OF CHORISMATE-DERVED 0009. The invention provides a genetically engineered PRODUCTS microorganism capable of producing chorismate-derived products. In particular, the invention provides a genetically CROSS REFERENCE TO RELATED engineered microorganism capable of producing at least one APPLICATIONS chorismate-derived product, wherein the bacterium com prises at least one of (a) an exogenous chorismate pyruvate 0001. This application claims the benefit of U.S. Provi lyase (EC 4.1.3.40), (b) an exogenous isochorismate Syn sional Patent Application No. 62/167,101 filed May 27, thase (EC5.4.4.2), (c) an exogenous isochorismate pyruvate 2015, the entirety of which is incorporated herein by refer lyase (EC 4.2.99.21), and (d) a prephenate synthase (EC CCC. 5.4.99.5) comprising a disruptive mutation. In particular embodiments, the genetically engineered microorganism is a FIELD OF THE INVENTION C1-fixing bacterium, Such as a Clostridium bacterium, capable of producing at least one chorismate-derived prod 0002 The present invention relates to genetically engi uct by fermentation of a C1-containing gaseous Substrate. neered microorganisms and methods for the production of 0010 For example, the chorismate pyruvate lyase may be chorismate-derived products by microbial fermentation, par ubiC, the isochorismate synthase may be pchA, the isocho ticularly by microbial fermentation of a gaseous Substrate. rismate pyruvate lyase may be pchB, and the prephenate synthase may be phea. The disruptive mutation in prephen BACKGROUND OF THE INVENTION ate synthase may reduce or eliminate the expression or 0003. The current generation of biologically-produced activity of the prephenate synthase. Such a disruptive muta commodity chemicals that use either food or non-food crops tion may yield a bacterium that produces a reduced amount to produce Sugar or cellulose-based feedstocks have draw of prephenate or prephenate-derived products compared to a backs relating to land use, food security, Supply volatility, parental bacterium and/or a bacterium that produces Sub and environmental issues. stantially no tyrosine or phenylalanine. 0004. It has long been recognized that catalytic processes 0011. The microorganism of the invention may comprise may be used to convert gases containing carbon monoxide at least one nucleic acid encoding at least one of (a) the (CO) and/or carbon dioxide (CO) and hydrogen (H) into a exogenous chorismate pyruvate lyase, (b) the exogenous variety of fuels and chemicals. However, microorganisms isochorismate synthase, (c) the exogenous isochorismate may also be used to biologically convert Such gases into pyruvate lyase, and (d) the prephenate synthase comprising fuels and chemicals. Biological processes have several a disruptive mutation. In certain embodiments, the nucleic advantages over catalytic processes, including higher speci acid is codon optimized for expression in Clostridium. ficity, higher yields, lower energy costs, and greater catalyst 0012. The chorismate-derived product may be any prod resistance to poisoning. uct produced directly or indirectly from chorismate. In 0005 CO is a major free energy-rich byproduct of the particular, the chorismate-derived product may comprise a incomplete combustion of organic materials such as coal or 6-membered carbon ring, for example, a benzene or cyclo oil and oil-derived products. For example, the steel industry hexane ring, Substituted with a carboxyl group or carboxy in Australia is reported to produce and release into the late anion and further substituted with one or more OH atmosphere over 500,000 tonnes of CO annually. groups and/or one or more NH groups. Chorismate-derived products include, but are not limited to, para-hydroxyben 0006. The ability of microorganisms to grow on CO as a Zoic acid, salicylate, 2-aminobenzoate, dihydroxybenzoate, sole carbon source was first discovered in 1903. This was and 4-hydroxycyclohexane carboxylic acid. later determined to be a property of microorganisms that use the acetyl coenzyme A (acetyl-CoA) biochemical pathway 0013. In one embodiment, the microorganism of the of autotrophic growth, also known as the Wood-Ljungdahl invention expresses a chorismate pyruvate lyase of ubiC and pathway. A large number of anaerobic microorganisms produces a chorismate-derived product of para-hydroxyben including carboxydotrophic, photosynthetic, methanogenic, Zoic acid. In one embodiment the microorganism of the and acetogenic microorganisms have been shown to metabo invention further expresses feedback-insensitive DAHP syn lize CO to various end products, namely CO. H. methane, thase. n-butanol, acetate, and ethanol. 0014. In one embodiment, the microorganism of the 0007. The aromatic compound para-hydroxybenzoic acid invention expresses an isochorismate synthase of pchA and (pHBA) is a major monomer used in liquid crystal polymers an isochorismate pyruvate lyase of pchB and produces a and also used as a precursor for the production of parahy chorismate-derived product of salicylate. In one embodi droxybenzoates or parahydroxybenzoic esters, commonly ment the microorganism of the invention further expresses referred to as parabens. Liquid crystal polymers include feedback-insensitive DAHP synthase. Kevlar and Vectran, which have multiple uses. Parabens and 0015. In one embodiment, the microorganism of the their salts are used in a range of industries including the invention comprises a prephenate synthase comprising a cosmetic, pharmaceutical and food industries. They are disruptive mutation and produces a one or more of choris effective preservatives and can be used for their bactericidal mate-derived product of 2-aminobenzoate, 2,3-dihydroxy and fungicidal properties in cosmetic and food formulations. benzoate, 3,4-dihydroxybenzoate and 4-hydroxycyclo 0008 Accordingly, there remains a need for additional hexane carboxylic acid. microorganisms and methods for producing pHBA and other 0016. In one embodiment, the microorganism of the high-value chorismate-derived products. invention produces at least one chorismate-derived product US 2016/0348087 A1 Dec. 1, 2016

not produced by a parental microorganism or a greater and 192 hour time points. Three replicate cultures were amount of at least one chorismate-derived product than a sampled for the negative control strain (C. autoethanogenium parental microorganism. LZ1561) and the two biological replicates of C. autoetha 0017. In one embodiment, the bacterium of the invention nogenium LZ1561 carrying pARO 01. FIG.9b shows mean is derived from a C1-fixing parental bacterium. In a pre of n=3 technical replicates +1 SD. ferred embodiment, the bacterium of the invention is derived 0029 FIG. 10 is a graph showing production of new from a parental bacterium selected from the group consisting aromatic compounds in a genetically engineered of Clostridium autoethanogenium, Clostridium ljungdahli, Clostridium bacterium comprising a disruptive mutation in and Clostridium ragsdalei. In a particularly preferred a nucleic acid encoding phea. The ApheA strain produces embodiment, the bacterium of the invention is derived from 4-hydroxy cyclohexane carboxylic acid, 2-aminobenzoic a parental bacterium of Clostridium autoethanogenium acid, and 3,4-dihydroxybenzoic acid, while the control strain deposited under DSMZ accession number DSM23693. (LZ1561) does not. 0018. The invention further provides a method of pro 0030 FIG.11a is a graph showing biomass growth of the ducing a fermentation product, comprising fermenting the salicylate production strain with and without induction of microorganism of the invention in the presence of a C1-con the salicylate biosynthetic pathway. taining gaseous Substrate. Generally, the fermentation prod 0031 FIG. 11b is a graph showing the difference in uct is a chorismate-derived product. In a preferred embodi accumulation of Salicylate in liquid cultures of the test strain ment, the gaseous Substrate comprises at least one C1 carbon with and without induction of the salicylate biosynthetic SOUC. pathway. 0032 FIG. 12 is a graph showing concentration of 4-hy BRIEF DESCRIPTION OF THE DRAWINGS droxy cyclohexane carboxylic acid, 2-aminobenzoic acid, 0019 FIG. 1 is a diagram showing production of choris and 3,4-dihydroxybenzoic acid produced by fermentation of mate via a native shikimate pathway in Clostridia. an engineered Clostridium bacterium comprising a disrup 0020 FIG. 2 is a diagram showing the pathway for tive mutation in a nucleic acid encoding phe A. production of pHBA in a genetically engineered Clostridium 0033 FIG. 13 is a table identifying exemplary sources of bacterium. chorismate pyruvate lyase (EC 4.1.3.40). 0021 FIG. 3 is a diagram showing the pathway for 0034 FIG. 14 is a table identifying exemplary sources of production of Salicylate in a genetically engineered isochorismate synthase (EC 5.4.4.2). Clostridium bacterium. 0035 FIG. 15 is table of identifying exemplary sources 0022 FIG. 4 is a diagram showing the pathway for of isochorismate pyruvate lyase (EC 4.2.99.21). production of aromatic products in a genetically engineered Clostridium bacterium comprising a disruptive mutation in DETAILED DESCRIPTION OF THE a nucleic acid encoding phe A. INVENTION 0023 FIG. 5 is a graph of a standard curve showing 0036 Clostridia natively produce chorismate, which quantitation of authentic pHBA standards. serves as a precursor to the aromatic amino acids tryptophan, 0024 FIG. 6a is a graph showing the total ion count of tyrosine, and phenylalanine, from phosphoenolpyruvate and authentic standards (i) authentic standard of pHBA (trim erythrose-4-phosphate via the shikimate pathway (FIG. 1). ethylsilyated) prepared in Supernatant from C. autoethano This pathway is described in detail in Bentley, Crit Rev genum LZ1561 culture medium, (ii) authentic standard of Biochem Mol Biol, 25.5: 307-384, 1990. The invention pHBA (trimethylsilyated) prepared in water, and (iii) mass provides a genetically engineered bacterium capable of spectrum of trimethylsilyated pHBA. producing at least one chorismate-derived product by fer 0025 FIG. 6b is a graph showing selected ion monitoring mentation of a gaseous Substrate. of fermentation samples and Standards: (i) C. autoethano 0037. The inventors have demonstrated that chorismate genum LZ1561 without pARO 01 plasmid, (ii) and (iii) derived products can be sustainably produced and recovered samples from C. autoethanogenium LZ1561 bearing pARO from a C1-carbon source. The invention provides a method 01 plasmid, (iv) authentic standard of pHBA, and (v) total of producing at least one chorismate-derived product using ion count comparison between NIST database entry for a C1-containing gaseous Substrate as the main carbon and pHBA and pHBA peak from LZ1561/pARO 01. energy source. In this way, the present invention has a 0026 FIG. 7 is a diagram of a pARO 01 plasmid. The number of advantages over processes that rely on Sugar- or chorismate pyruvate lyase (ubiC) and feedback-insensitive cellulose-based substrates. For example, Sugar- or cellulose DAHP synthase (aroG*) are under control of the Wood based Substrates are typically also useful for food (e.g. Sugar Ljungdahl promoter (Pwl). Other shuttle vector features are cane) and their intensive land use has negative environmen also shown. tal consequences. Further, the invention provides an alter 0027 FIG. 8 is a graph showing biomass accumulation in native method for the production of chorismate-derived test strains. Biomass was estimated by measuring the absor products, optionally via the use of waste gases (e.g. CO from bance of culture samples at 600 nm at different time points. industrial processes). Thus, the invention provides a source Data points represent the mean of n=3 replicate cultures +1 of revenue from waste gases and, furthermore, captures the standard deviation. LZ1561 refers to untransformed C. carbon in those waste gases to reduce the carbon emissions autoethanogenium LZ1561. p ARO 01(1) and pARO 01(2) that would occur if the gases were flared to the atmosphere. are biological replicates of C. autoethanogenium LZ1561 0038 Heterotrophic microorganisms such as E. coli and transformed with the p ARO 01 plasmid. S. cerevisiae produce relatively high levels of ATP through 0028 FIGS. 9a and 9b are graphs showing p-hydroxy glycolysis. In contrast, microorganisms which use C1-car benzoate accumulation in test strains. FIG. 9a shows quan bon sources (e.g., CO or CO) have poor ATP availability. tification of pHBA detected in each sample at 24, 96, 144, For example, analysis of the reaction kinetics in a typical US 2016/0348087 A1 Dec. 1, 2016 carboxydotrophic microorganism C. autoethanogenium 0043. The UbiC enzyme or ubiC gene may also be gives a predicted ATP yield when producing pHBA, a modified (e.g., mutated) to enhance solubility, stability, or chorismate-derived product) of -0.4 ATP per mol of CO other gene/enzyme properties. Such modifications may fixed. As such, it would not be expected that any pHBA result in increased product titers. Example 4 describes an would be produced due to the energy constraints. Similarly experimental protocol to engineer a UbiC enzyme to it would not be expected that other chorismate-derived decrease product inhibition through retention of para-hy products would be produced by a carboxydotrophic micro droxybenzoic acid. One particular modification involves organism due to the metabolic burden of producing Such engineering the ubiC gene to express a UbiC enzyme with compounds under autotrophic conditions. The inventors two Surface-active serines instead of cysteines. The serine have Surprisingly shown however that a number of choris residues result in less protein aggregation and, in turn, mate-derived products can be produced from a gaseous improved solubility. Accordingly, in a particular embodi substrate. Further, said products can be produced from ment, the UbiC enzyme comprises a mutation to replace at industrial waste gases which provide practical, economic, least one surface-active cysteine with a serine. and environmental benefits over other substrates. 0044. In alternative embodiments, the chorismate pyru 0039. In particular, the invention provides genetically vate lyase (EC 4.1.3.40) may be or may be derived, for engineered microorganisms capable of producing at least example, from any of the sources identified in FIG. 13. one chorismate-derived product by introducing at least one 0045 Introduction of an exogenous chorismate pyruvate of (a) a nucleic acid encoding an exogenous chorismate lyase (e.g., ubiC) or a nucleic acid encoding an exogenous pyruvate lyase, (b) a nucleic acid encoding an exogenous chorismate pyruvate lyase (e.g., ubiC) results in production isochorismate synthase (a.k.a., isochorismate mutase), (c) a of para-hydroxybenzoic acid, a chorismate-derived product, nucleic acid encoding an exogenous isochorismate pyruvate by the microorganism of the invention. The production of lyase, and (d) a nucleic acid encoding a prephenate synthase para-hydroxybenzoic acid is illustrated in FIG. 2. C1 fixing comprising a disruptive mutation. In a preferred embodi bacteria including the species Acetobacterium woodii, Alka ment, the genetically engineered microorganism is a C1-fix libaculum bacchi, Blautia producta, Butyribacterium meth ing bacterium capable of producing at least one chorismate ylotrophicum, Clostridium aceticum, Clostridium autoetha derived product by fermentation of a gaseous Substrate. In nogenium, Clostridium carboxidivorans, Clostridium preferred embodiments the C1-fixing bacterium is a coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium bacterium. Clostridium ljungdahli, Clostridium magnum, Clostridium 0040. A “chorismate-derived product” or “product ragsdalei, Clostridium scatologenes, Eubacterium limosum, derived from chorismate' or similar terms encompass prod Moorella thermautotrophica, Moorella thermoacetica, Oxo ucts produced directly or indirectly from chorismate (or bacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, chorismic acid). Chorismate-derived products typically Sporomusa sphaeroides, and Thermoanaerobacter kivui, do comprise a 6-membered carbon ring, for example, a benzene not natively produce para-hydroxybenzoic acid. In fact, or cyclohexane ring, Substituted with a carboxyl group or since ubiquinone is generally only produced in aerobically carboxylate anion and further substituted with one or more respiring microorganisms, chorismate pyruvate lyase is not OH groups and/or one or more NH2 groups. Specifically, typically found in carboxydotrophic microorganisms. chorismate-derived products include, but are not limited to, Although it may be expected that the diversion of choris para-hydroxybenzoic acid, Salicylate, 2-aminobenzoate, 2.3- mate to produce pHBA instead of amino acids would have dihydroxybenzoate, 3,4-dihydroxybenzoate, and 4-hydroxy detrimental effects on the growth or survival of the micro cyclohexane carboxylic acid. organism, the inventors have shown that the microorganism is not affected to a degree that significantly compromises 0041. The microorganism of the invention may comprise Survival and growth under standard conditions. an exogenous chorismate pyruvate lyase enzyme (EC 4.1. 0046 Para-hydroxybenzoic acid may also be referred to, 3.40) that catalyzes the conversion of chorismate to para for example, as pHBA, 4-hydroxybenzoic acid, p-hydroxy hydroxybenzoic acid and pyruvate in the first committed step of ubiquinone biosynthesis. The enzyme may be benzoic acid, or para-hydroxybenzoate. References to any of derived from any microorganism having Such an enzyme. these terms, as used herein, encompass both the acid and The enzyme may be a UbiC enzyme. The UbiC enzyme may anion forms of the molecule. be derived from , , Cit robacter freundii, or any other microorganism having a HO O O O UbiC enzyme. In one embodiment, the UbiC enzyme is derived from Escherichia coli and comprises SEQ ID NO: 1 or a functionally equivalent variant thereof. 0042. Similarly, the microorganism of the invention may comprise a nucleic acid encoding an exogenous chorismate pyruvate lyase. The nucleic acid may be a chorismate pyruvate lyase gene derived from any microorganism having OH OH Such a gene. The chorismate pyruvate lyase gene may be a para-hydroxybenzoic acid para-hydroxybenzoate ubiC gene. The ubiC gene may be derived from Escherichia coli, Klebsiella Oxytoca, Citrobacter freundii, or any other microorganism having a ubiC gene. In one embodiment, the 0047. The microorganism of the invention may comprise ubiC gene is derived from Escherichia coli and comprises an exogenous isochorismate synthase enzyme, also referred SEQID NO: 2 or a codon-optimized or functionally equiva to as isochorismate mutase, (EC 5.4.4.2) that catalyzes the lent variant thereof. conversion of chorismate to isochorismate. The enzyme may US 2016/0348087 A1 Dec. 1, 2016 be derived from any microorganism having Such an enzyme. Sporomusa sphaeroides, and Thermoanaerobacter kivui, do The enzyme may be a PChA enzyme. The PChA enzyme may not natively produce salicylate. be derived from Pseudomonas aeruginosa or any other 0054 Salicylate may also be referred to, for example, as microorganism having a PChA enzyme. In one embodiment, 2-hydroxybenzoate, salicylic acid, or 2-hydroxybenzoic the PehA enzyme is derived from Pseudomonas aeruginosa acid. References to any of these terms, as used herein, and comprises SEQ ID NO: 3 or a functionally equivalent encompass both the acid and anion forms of the molecule. variant thereof. 0048 Similarly, the microorganism of the invention may comprise a nucleic acid encoding an exogenous isochoris mate synthase. The nucleic acid may be an isochorismate synthase gene derived from any microorganism having Such OH OH a gene. The isochorismate synthase gene may be a pchA gene. The pchA gene may be derived from Pseudomonas aeruginosa or any other microorganism having apchagene. In one embodiment, the pchA gene is derived from Salicylate Salicylic acid Pseudomonas aeruginosa and comprises SEQID NO. 4 or a codon-optimized or functionally equivalent variant thereof. (d) Prephenate Synthase Comprising a Disruptive Mutation 0049. In alternative embodiments, the isochorismate syn thase (EC 5.4.4.2) may be or may be derived, for example, 0055. The microorganism of the invention may comprise from any of the sources identified in FIG. 14. a prephenate synthase enzyme (EC 5.4.99.5) comprising a 0050. The microorganism of the invention may comprise disruptive mutation. Prephenate synthase typically catalyzes an exogenous isochorismate pyruvate lyase enzyme (EC the conversion of chorismate to prephenate (i.e., a choris 4.2.99.21) that catalyzes the conversion of isochorismate to mates sprephenate mutase reaction). Accordingly, a pre salicylate and pyruvate. The enzyme may be derived from phenate synthase enzyme comprising a disruptive mutation any microorganism having Such an enzyme. The enzyme is unable or less able to catalyze the conversion of choris may be a PChB enzyme. The PChB enzyme may be derived mate to prephenate. The prephenate synthase comprising a from Pseudomonas aeruginosa or any other microorganism disruptive mutation may be phe A comprising a disruptive having a PChB enzyme. In one embodiment, the PChB mutation. The prephenate synthase may also be referred to enzyme is derived from Pseudomonas aeruginosa and com as chorismate mutase. prises SEQ ID NO: 5 or a functionally equivalent variant 0056. In some embodiments, the phea may be a bifunc thereof. tional enzyme that carries out both prephenate synthase (i.e., chorismate mutase) (EC 5.4.99.5) and prephenate dehy 0051 Similarly, the microorganism of the invention may dratase (EC 4.2.1.51) reactions. In microorganisms where comprise a nucleic acid encoding an exogenous isochoris these two reactions are carried out by separate enzymes, mate pyruvate lyase. The nucleic acid may be an isochoris knocking out EC 5.4.99.5 activity will result in significantly mate pyruvate lyase gene derived from any microorganism decreased or eliminated production of prephenate or com having Such a gene. The isochorismate pyruvate lyase gene pounds downstream of prephenate, while knocking out EC may be a pchB gene. The pchB gene may be derived from 4.2.1.51 activity alone would not achieve the same pheno Pseudomonas aeruginosa or any other microorganism hav type, since prephenate may still be produced. In one embodi ing a pchB gene. In one embodiment, the pchB gene is ment, the phe A is derived from Clostridium autoethanoge derived from Pseudomonas aeruginosa and comprises SEQ num and comprises SEQ ID NO: 11 or a functionally ID NO: 6 or a codon-optimized or functionally equivalent equivalent variant thereof variant thereof. 0057 Similarly, the microorganism of the invention may 0.052. In alternative embodiments, the isochorismate comprise a nucleic acid encoding a prephenate synthase pyruvate lyase (EC 4.2.99.21) may be or may be derived, for comprising a disruptive mutation. The nucleic acid may be example, from any of the sources identified in FIG. 15. a phe A gene comprising a disruptive mutation. In one 0053 Introduction of (1) an exogenous isochorismate embodiment, the disruptive mutation is a knockout mutation synthase (e.g., pchA) and (2) an exogenous isochorismate of a pheA gene. In one embodiment, the phe A gene is pyruvate lyase (e.g., pchB) results in production of Salicy derived from Clostridium autoethanogenium and comprises late, a chorismate-derived product, by the microorganism of SEQ ID NO: 10 or a codon-optimized or functionally the invention. The production of salicylate is illustrated in equivalent variant thereof. FIG.3, whereby chorismate is converted to isochorismate by 0.058 Disrupting prephenate synthase results in reduced isochorismate synthase and then further converted to Salicy or eliminated production of phenylalanine and tyrosine. late and pyruvate by isochorismate pyruvate lyase. C1 fixing Surprisingly, disrupting prephenate synthase also results in bacteria including the species Acetobacterium woodii, Alka the production of additional products that are not typically libaculum bacchii, Blautia producta, Butyribacterium meth produced or that are produced only at very low levels. ylotrophicum, Clostridium aceticum, Clostridium autoetha 0059. In particular, the introduction of a disruptive muta nogenium, Clostridium carboxidivorans, Clostridium tion to prephenate synthase (e.g., phe A) or a nucleic acid coskatii, Clostridium drakei, Clostridium formicoaceticum, encoding prephenate synthase (e.g., phe A) results in pro Clostridium ljungdahli, Clostridium magnum, Clostridium duction of one or more of 2-aminobenzoate, dihydroxyben ragsdalei, Clostridium scatologenes, Eubacterium limosum, Zoate, and 4-hydroxycyclohexane carboxylic acid, all cho Moorella thermautotrophica, Moorella thermoacetica, Oxo rismate-derived products, by the microorganism of the bacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, invention. The production pathways of these products is US 2016/0348087 A1 Dec. 1, 2016 illustrated in FIG. 4. Many microorganisms, including spe cies of Clostridia Such as Clostridium autoethanogenium, O O O OH Clostridium liungdahli, and Clostridium ragsdalei, do not natively produce these products or only produce very low OH OH levels of these products. 0060 Exemplary sources for phe A are provided. How ever, it should be appreciated that other suitable sources for OH OH phe A may be available The prephenate dehydratase be or 2,3-dihydroxybenzoate 2,3-dihydroxybenzoic acid may be derived, for example, from any of the following O O HO O Sources, the sequences of which are publically available: OH OH

Genbank Description Microorganism accession OH OH bifunctional chorismate Acetobacterium woodi AFA49374.1 3,4-dihydroxybenzoate 3,4-dihydroxybenzoic acid, mutasef Protocatechuic acid prephenate dehydratase prephenate dehydratase Bialitia producia WP 033143345. prephenate dehydratase Clostridium aceticum WP 0448231 68. 0063 4-hydroxycyclohexane carboxylic acid may also be prephenate dehydratase Clostridium AGY75132.1 autoethanogenim referred to, for example, as cis-4-hydroxycyclohexane car bifunctional chorismate Ciostridium WP 007060905. boxylic acid or 4-hydroxycyclohexane-1-carboxylate. Ref mutasef carboxidivorans erences to any of these terms, as used herein, encompass prepnenate denydratase both the acid and anion forms of the molecule. bifunctional chorismate Ciostridium coskati WP 063600678. mutasef prepnenate denydratase HO O O O bifunctional chorismate Ciostridium drakei WP 032076381. prepnenate denydratase bifunctional chorismate Clostridium iungdahli WP 063554005.

prepnenate denydratase prepnenate denydratase Clostridium magnum KZL8937.0.1 bifunctional chorismate Clostridium scatologenes WP 029159263. OH OH enydratase 4-hydroxycyclohexane 4-hydroxycyclohexane Etibacterium inosum WP 058695931. carboxylic acid carboxylate Oxobacter pfennigii WP 054874911. enydratase Sporomisa ovata EQB25731.1 enydratase Thermoanaerobacter WP 049685.038. 0064. In another embodiment, the microorganism of the kiviti invention further comprises a nucleic acid encoding a feed back-insensitive DAHP synthase DAHP synthase catalyses the first committed step in the shikimate pathway (FIG. 1) in 0061 2-aminobenzoate may also be referred to, for which erythrose-4-phosphate and phosphoenolpyruvate are example, as 2-aminobenzoic acid, o-aminobenzoic acid, converted to 3-deoxy-D-arabinoheptosonate-7-phosphate. anthranilic acid, anthranilate, or vitamin L1. References to The inventors believe that this step in the pathway is subject any of these terms, as used herein, encompass both the acid to feedback inhibition by aromatic amino acids (tryptophan, and anion forms of the molecule. phenylalanine, tyrosine) as described for E. coli (Hu et al. J. Basic Microbiol. 2003, 43:399-406). Accordingly, the inventors have, based on this prior art, developed a feed back-insensitive DAHP synthase, which is believed to reduce the risk of flux to chorismate-derived products being reduced by this feedback inhibition. Nucleic acids encoding appropriate DAHP synthases are known to those of skill in the art. However, by way of example, the nucleic acid encoding a DAHP synthase may be derived from Escheri chia coli, Clostridium beijerinckii, or Saccharomyces cer 2-aminobenzoate 2-aminobenzoic acid evisiae. In one embodiment, the DAHP synthase may be feedback-insensitive DAHP synthase from Escherichia coli, having the nucleic acid sequence of SEQID NO: 7 and the 0062 Dihydroxybenzoate may be referred to, for amino acid sequence of SEQ ID NO: 8. The feedback example, as 2.3-dihydroxybenzoate, 2,3-dihydroxybenzoic insensitive DAHP synthase may be introduced on the same acid, 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid or vector as a gene encoding one of the aforementioned Protocatechuic acid. References to any of these terms, as enzymes or on a different vector. The feedback-insensitive used herein, encompass both the acid and anion forms of the DAHP synthase may have its own promoter or may follow molecule. the promoter for one of the aforementioned enzymes in a US 2016/0348087 A1 Dec. 1, 2016

bicistronic arrangement, wherein a single promoter drives accounts for at least 10% of all fermentation products the transcription of a single mRNA that encodes both the produced by the microorganism of the invention, such that enzyme and the feedback-insensitive DAHP synthase. the microorganism of the invention has a selectivity for the 0065. In one embodiment, the microorganism of the target chorismate-derived product of at least 10%. In another invention comprises an exogenous chorismate pyruvate embodiment, the target chorismate-derived product accounts lyase enzyme (EC 4.1.3.40), and an exogenous feedback for at least 30% of all fermentation products produced by the insensitive DAHP synthase. In particular embodiments the microorganism of the invention, Such that the microorgan microorganism comprises an exogenous UbiC enzyme, and ism of the invention has a selectivity for the target choris an exogenous feedback-insensitive DAHP synthase. In a mate-derived product of at least 30%. specific embodiment, the invention comprises exogenous 0072 The invention further provides a method of pro ubiC gene having the nucleic acid sequence of SEQID NO: ducing a fermentation product, specifically a chorismate 1, and an exogenous feedback-insensitive DAHP synthase derived product, comprising fermenting the microorganism having the nucleic acid sequence of SEQ ID NO: 7. In one of the invention in the presence of a gaseous Substrate. embodiment, the microorganism comprising both an exog 0073. The invention also provides chorismate-derived enous chorismate pyruvate lyase enzyme and an exogenous products produced by fermenting a microorganism of the feedback-insensitive DAHP synthase demonstrates greater invention in the presence of a gaseous Substrate. production of para-hydroxybenzoic acid compared to a microorganism without a feedback-insensitive DAHP syn DEFINITIONS AND BACKGROUND thase. 0074 The term “genetic modification' or “genetic engi 0066 Similarly, the microorganism of the invention may neering broadly refers to manipulation of the genome or comprise a nucleic acid encoding both an exogenous cho nucleic acids of a microorganism. Methods of genetic modi rismate pyruvate lyase and feedback-insensitive DAHP syn fication of include, for example, heterologous gene expres thase. Sion, gene or promoter insertion or deletion, nucleic acid 0067. In one embodiment, the microorganism of the mutation, altered gene expression or inactivation, enzyme invention comprises (i) an exogenous isochorismate mutase, engineering, directed evolution, knowledge-based design, (EC 5.4.4.2), (ii) an isochorismate pyruvate lyase enzyme random mutagenesis methods, gene shuffling, and codon (EC 4.2.99.21), and (iii) an exogenous feedback-insensitive optimization. DAHP synthase. In particular embodiments the microorgan 0075 "Recombinant indicates that a nucleic acid, pro ism comprises an exogenous PehA enzyme, an exogenous tein, or microorganism is the product of genetic modifica PchB enzyme, and an exogenous feedback-insensitive tion, engineering, or recombination. Generally, the term DAHP synthase. In one embodiment, the microorganism “recombinant” refers to a nucleic acid, protein, or microor comprising an exogenous feedback-insensitive DAHP syn ganism that contains or is encoded by genetic material thase demonstrates greater production of salicylic acid com derived from multiple sources, such as two or more different pared to a microorganism without a feedback-insensitive strains or species of microorganisms. As used herein, the DAHP synthase. term “recombinant may also be used to describe a micro 0068. Similarly, the microorganism of the invention may organism that comprises a mutated nucleic acid or protein, comprise a nucleic acid encoding both an exogenous cho including a mutated form of an endogenous nucleic acid or rismate pyruvate lyase and feedback-insensitive DAHP syn protein. thase. 0076 “Endogenous” refers to a nucleic acid or protein 0069. In another embodiment, the microorganism of the that is present or expressed in the wild-type or parental invention does not comprise a feedback-insensitive DAHP microorganism from which the microorganism of the inven synthase and instead merely comprises an endogenous tion is derived. For example, an endogenous gene is a gene DAHP synthase. Where production or natural concentration that is natively present in the wild-type or parental micro of aromatic amino acids is expected to be low enough so as organism from which the microorganism of the invention is to not induce feedback inhibition, it is not necessary to derived. In one embodiment, the expression of an endog introduce a feedback-insensitive DAHP synthase. enous gene may be controlled by an exogenous regulatory 0070 The microorganism of the invention may produce element, such as an exogenous promoter. chorismate-derived products at any concentration or in any 0077 “Exogenous” refers to a nucleic acid or protein that amount. In one embodiment, the microorganism of the is not present in the wild-type or parental microorganism invention produces chorismate-derived products at a con from which the microorganism of the invention is derived. centration of at least about 5 mg/L. 10 mg/L, 15 mg/L. 20 In one embodiment, an exogenous gene or enzyme may be mg/L, 30 mg/L, 50 mg/L, 75 mg/L. 100 mg/L, 200 mg/L, derived from a heterologous (i.e., different) strain or species 500 mg/L, 750 mg/L, 1 g/L, 1.5 g/L or 2 g/L. In one and introduced to or expressed in the microorganism of the embodiment, the microorganism of the invention produces invention. In another embodiment, an exogenous gene or at least one chorismate-derived product at a concentration of enzyme may be artificially or recombinantly created and at least 10 mg/L, 50gm/L. 100 mg/L, 500 mg/L, 800 mg/L, introduced to or expressed in the microorganism of the or 1 g/L invention. Exogenous nucleic acids may be adapted to 0071. Furthermore, the microorganism of the invention integrate into the genome of the microorganism of the may be engineered to produce products at a certain selec invention or to remain in an extra-chromosomal state in the tivity or at a minimum selectivity. In one embodiment, a microorganism of the invention, for example, in a plasmid. target chorismate-derived product accounts for at least about (0078 “Enzyme activity” refers broadly to enzymatic 5%, 10%, 15%, 20%, 30%, 50%, or 75% of all fermentation activity, including, but not limited, to the activity of an products produced by the microorganism of the invention. In enzyme, the amount of an enzyme, or the availability of an one embodiment, the target chorismate-derived product enzyme to catalyze a reaction. Accordingly, “increasing US 2016/0348087 A1 Dec. 1, 2016 enzyme activity includes increasing the activity of an may include allelic variants, fragments of a gene, mutated enzyme, increasing the amount of an enzyme, or increasing genes, polymorphisms, and the like. Homologous genes the availability of an enzyme to catalyze a reaction. from other microorganisms are also examples of function 0079. “Mutated” refers to a nucleic acid or protein that ally equivalent variants. These include homologous genes in has been modified in the microorganism of the invention species such as Clostridium acetobutylicum, Clostridium compared to the wild-type or parental microorganism from beijerinckii, or Clostridium ljungdahli, the details of which which the microorganism of the invention is derived. In one are publicly available on websites such as Genbank or embodiment, the mutation may be a deletion, insertion, or NCBI. Functionally equivalent variants also include nucleic Substitution in a gene encoding an enzyme. In another acids whose sequence varies as a result of codon optimiza embodiment, the mutation may be a deletion, insertion, or tion for a particular microorganism. A functionally equiva Substitution of one or more amino acids in an enzyme. lent variant of a nucleic acid will preferably have at least 0080. In particular, a “disruptive mutation' is a mutation approximately 70%, approximately 80%, approximately that reduces or eliminates (i.e., “disrupts) the expression or 85%, approximately 90%, approximately 95%, approxi activity of a gene or enzyme. The disruptive mutation may mately 98%, or greater nucleic acid sequence identity (per partially inactivate, fully inactivate, or delete the gene or cent homology) with the referenced nucleic acid. A func enzyme. The disruptive mutation may be a knockout (KO) tionally equivalent variant of a protein will preferably have mutation. The disruptive mutation may be any mutation that at least approximately 70%, approximately 80%, approxi reduces, prevents, or blocks the biosynthesis of a product mately 85%, approximately 90%, approximately 95%, produced by an enzyme. The disruptive mutation may approximately 98%, or greater amino acid identity (percent include, for example, a mutation in a gene encoding an homology) with the referenced protein. The functional enzyme, a mutation in a genetic regulatory element involved equivalence of a variant nucleic acid or protein may be in the expression of a gene encoding an enzyme, the evaluated using any method known in the art. introduction of a nucleic acid which produces a protein that I0085 Nucleic acids may be delivered to a microorganism reduces or inhibits the activity of an enzyme, or the intro of the invention using any method known in the art. For duction of a nucleic acid (e.g., antisense RNA, siRNA, example, nucleic acids may be delivered as naked nucleic CRISPR) or protein which inhibits the expression of an acids or may be formulated with one or more agents, such enzyme. The disruptive mutation may be introduced using as liposomes. The nucleic acids may be DNA, RNA, cDNA, any method known in the art. or combinations thereof, as is appropriate. Restriction I0081) “Codon optimization” refers to the mutation of a inhibitors may be used in certain embodiments. Additional nucleic acid, such as a gene, for optimized or improved vectors may include plasmids, viruses, bacteriophages, cos translation of the nucleic acid in a particular strain or mids, and artificial chromosomes. In a preferred embodi species. Codon optimization may result in faster translation ment, nucleic acids are delivered to the microorganism of rates or higher translation accuracy. In a preferred embodi the invention using a plasmid. By way of example, trans ment, the genes of the invention are codon optimized for formation (including transduction or transfection) may be expression in Clostridium, particularly Clostridium achieved by electroporation, ultrasonication, polyethylene autoethanogenium, Clostridium liungdahli, or Clostridium glycol-mediated transformation, chemical or natural com ragsdalei. In a further preferred embodiment, the genes of petence, protoplast transformation, prophage induction, or the invention are codon optimized for expression in conjugation. In certain embodiments having active restric Clostridium autoethanogenium LZ1561, which is deposited tion enzyme systems, it may be necessary to methylate a under DSMZ accession number DSM23693. nucleic acid before introduction of the nucleic acid into a 0082 “Overexpressed’ refers to an increase in expres microorganism. sion of a nucleic acid or protein in the microorganism of the I0086. Furthermore, nucleic acids may be designed to invention compared to the wild-type or parental microor comprise a regulatory element, such as a promoter, to ganism from which the microorganism of the invention is increase or otherwise control expression of a particular derived. Overexpression may be achieved by any means nucleic acid. The promoter may be a constitutive promoter known in the art, including modifying gene copy number, or an inducible promoter. Ideally, the promoter is a Wood gene transcription rate, gene translation rate, or enzyme Ljungdahl pathway promoter, a ferredoxin promoter, a pyru degradation rate. Vate:ferredoxin oxidoreductase promoter, an Rnf complex 0083. The term “variants’ includes nucleic acids and operon promoter, an ATP synthase operon promoter, or a proteins whose sequence varies from the sequence of a phosphotransacetylase/acetate kinase operon promoter. reference nucleic acid and protein, such as a sequence of a I0087. A “microorganism' is a microscopic organism, reference nucleic acid and protein disclosed in the prior art especially a bacterium, archea, virus, or fungus. The micro or exemplified herein. The invention may be practiced using organism of the invention is typically a bacterium. As used variant nucleic acids or proteins that perform Substantially herein, recitation of “microorganism’ should be taken to the same function as the reference nucleic acid or protein. encompass “bacterium.” For example, a variant protein may perform Substantially the I0088 A “parental microorganism' is a microorganism same function or catalyze substantially the same reaction as used to generate a microorganism of the invention. The a reference protein. A variant gene may encode the same or parental microorganism may be a naturally-occurring micro Substantially the same protein as a reference gene. A variant organism (i.e., a wild-type microorganism) or a microorgan promoter may have Substantially the same ability to promote ism that has been previously modified (i.e., a mutant or the expression of one or more genes as a reference promoter. recombinant microorganism). The microorganism of the 0084. Such nucleic acids or proteins may be referred to invention may be modified to express or overexpress one or herein as “functionally equivalent variants.” By way of more enzymes that were not expressed or overexpressed in example, functionally equivalent variants of a nucleic acid the parental microorganism. Similarly, the microorganism of US 2016/0348087 A1 Dec. 1, 2016

the invention may be modified to contain one or more genes more of CO, CO, CH, CHOH, or CHO. Preferably, the that were not contained by the parental microorganism. In C1-carbon source comprises one or both of CO and CO. A one embodiment, the parental microorganism is Clostridium “C1-fixing microorganism' is a microorganism that has the autoethanogenium, Clostridium liungdahli, or Clostridium ability to produce one or more products from a C1-carbon ragsdalei. In a preferred embodiment, the parental micro Source. Typically, the microorganism of the invention is a organism is Clostridium autoethanogenium LZ1561, which C1-fixing bacterium. In a preferred embodiment, the micro is deposited under DSMZ accession DSM23693. organism of the invention is derived from a C1-fixing 0089. The term “derived from indicates that a nucleic microorganism identified in Table 1. acid, protein, or microorganism is modified or adapted from 0092 An “anaerobe' is a microorganism that does not a different (e.g., a parental or wild-type) nucleic acid, require oxygen for growth. An anaerobe may react nega protein, or microorganism, so as to produce a new nucleic tively or even die if oxygen is present. Typically, the acid, protein, or microorganism. Such modifications or microorganism of the invention is an anaerobe. In a pre adaptations typically include insertion, deletion, mutation, ferred embodiment, the microorganism of the invention is or substitution of nucleic acids or genes. Generally, the derived from an anaerobe identified in Table 1. microorganism of the invention is derived from a parental 0093. An "acetogen' is a microorganism that produces or microorganism. In one embodiment, the microorganism of is capable of producing acetate (or acetic acid) as a product the invention is derived from Clostridium autoethanogenium, of anaerobic respiration. Typically, acetogens are obligately Clostridium liungdahli, or Clostridium ragsdalei. In a pre anaerobic bacteria that use the Wood-Ljungdahl pathway as ferred embodiment, the microorganism of the invention is their main mechanism for energy conservation and for derived from Clostridium autoethanogenium LZ1561, which synthesis of acetyl-CoA and acetyl-CoA-derived products, is deposited under DSMZ accession DSM23693. such as acetate (Ragsdale, Biochim Biophy's Acta, 1784: 0090 The microorganism of the invention may be further 1873-1898, 2008). Acetogens use the acetyl-CoA pathway classified based on functional characteristics. For example, as a (1) mechanism for the reductive synthesis of acetyl the microorganism of the invention may be or may be CoA from CO, (2) terminal electron-accepting, energy derived from a C1-fixing microorganism, an anaerobe, an conserving process, (3) mechanism for the fixation (assimi acetogen, an ethanologen, a carboxydotroph, and/or a lation) of CO in the synthesis of cell carbon (Drake, methanogen. Table 1 provides a representative list of micro Acetogenic Prokaryotes, In: The Prokaryotes, 3rd edition, p. organisms and identifies their functional characteristics. 354, New York, N.Y., 2006). All naturally occurring aceto TABLE 1. C1-fixing Anaerobe Acetogen Ethanologen Autotroph Carboxydotroph Methanotroph Acetobacterium woodii Aikaibaculum bacchi Bialitia producia Butyribacterium methylotrophicum Cliostridium aceticum Clost ridium attoethanogentin Cliostridium carboxidivorans Cliostridium coskati Cliostridium drakei Clostridium formicoaceticum Clostridium iungdahli -- Clostridium magnum --f Clostridium ragsdaiei Clost ridium Scatologenes Eubacterium inosum Moorella thermoautotrophica -- Moorella thermoacetica (formerly Clostridium thermoaceticum) Oxobacter pfennigii Sporomisa ovata Sporomisa Silvacetica --f Sporomisa sphaeroides Thermoanaerobacter kiviti Acetobacterium woodi can produce ethanol from fructose, but not from gas, * It has not been investigated whether Closiridium magnum can grow on CO. One strain of Moorella thermoacetica, Moorella sp, HUC22-1, has been reported to produce ethanol from gas, * It has not been investigated whether Sporomusa ovata can grow on CO. It has not been investigated whether Sporomusa Silvacetica can grow on CO. It has not been investigated whether Sporomusa Sphaeroides can grow on CO.

0091 “C1' refers to a one-carbon molecule, for example, gens are C1-fixing, anaerobic, autotrophic, and non-metha CO, CO., CH, or CH-OH. “C1-oxygenate” refers to a notrophic. Typically, the microorganism of the invention is one-carbon molecule that also comprises at least one oxygen an acetogen. In a preferred embodiment, the microorganism atom, for example, CO, CO, or CH-OH. “C1-carbon of the invention is derived from an acetogen identified in Source” refers a one carbon-molecule that serves as a partial Table 1. or Sole carbon source for the microorganism of the inven 0094. An "ethanologen' is a microorganism that pro tion. For example, a C1-carbon Source may comprise one or duces or is capable of producing ethanol. Typically, the US 2016/0348087 A1 Dec. 1, 2016 microorganism of the invention is an ethanologen. In a organization and number of these genes and proteins has preferred embodiment, the microorganism of the invention been found to be the same in all species (Kopke, Curr Opin is derived from an ethanologen identified in Table 1. Biotechnol, 22: 320-325, 2011). 0095. An “autotroph” is a microorganism capable of 0102 Thus, in summary, many of the characteristics of growing in the absence of organic carbon. Instead, auto Clostridium autoethanogenium, Clostridium liungdahli, or trophs use inorganic carbon sources, such as CO and/or CO. Clostridium ragsdalei are not specific to that species, but are Typically, the microorganism of the invention is an auto rather general characteristics for this cluster of C1-fixing, troph. In a preferred embodiment, the microorganism of the anaerobic, acetogenic, ethanologenic, and carboxydotrophic invention is derived from an autotroph identified in Table 1. members of the genus Clostridium. However, since these 0096. A “carboxydotroph” is a microorganism capable of species are, in fact, distinct, the genetic modification or utilizing CO as a sole source of carbon. Typically, the manipulation of one of these species may not have an microorganism of the invention is a carboxydotroph. In a identical effect in another of these species. For instance, preferred embodiment, the microorganism of the invention differences in growth, performance, or product production is derived from a carboxydotroph identified in Table 1. may be observed. 0097. A “methanotroph” is a microorganism capable of 0103) The microorganism of the invention may also be utilizing methane as a sole source of carbon and energy. In derived from an isolate or mutant of Clostridium autoetha certain embodiments, the microorganism of the invention is nogenium, Clostridium liungdahli, or Clostridium ragsdalei. derived from a methanotroph. Isolates and mutants of Clostridium autoethanogenium 0098. More broadly, the microorganism of the invention include JA1-1 (DSM100.61) (Abrini, Arch Microbiol, 161: may be derived from any genus or species identified in Table 345-351, 1994), LBS1560 (DSM19630) (WO 2009/ 1. In a preferred embodiment, the microorganism of the 064200), and LZ1561 (DSM23693). Isolates and mutants of invention is a Clostridium bacterium. Clostridium liungdahlii include ATCC 49587 (Tanner, Int J 0099. In a preferred embodiment, the microorganism of Syst Bacteriol, 43: 232-236, 1993), PETCT (DSM13528, the invention is derived from the cluster of Clostridia ATCC 55383), ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593, comprising the species Clostridium autoethanogenium, 886), C-01 (ATCC 55.988) (U.S. Pat. No. 6,368.819), O-52 Clostridium ljungdahli, and Clostridium ragsdalei. These (ATCC 55989) (U.S. Pat. No. 6,368.819), and OTA-1 (Ti species were first reported and characterized by Abrini, Arch rado-Acevedo, Production of bioethanol from synthesis gas Microbiol, 161: 345-351, 1994 (Clostridium autoethanoge using Clostridium ljungdahlii, PhD thesis, North Carolina num), Tanner, Int J System Bacteriol, 43: 232-236, 1993 State University, 2010). Isolates and mutants of Clostridium (Clostridium ljungdahli), and Huhnke, WO 2008/028055 ragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (Clostridium ragsdalei). (WO 2008/028055). 0100. These three species have many similarities. In 0104 “Substrate” refers to a carbon and/or energy source particular, these species are all C1-fixing, anaerobic, aceto for the microorganism of the invention. Typically, the Sub genic, ethanologenic, and carboxydotrophic members of the strate is gaseous and comprises a C1-carbon Source, for genus Clostridium. These species have similar genotypes example, CO, CO., and/or CH. Preferably, the substrate and phenotypes and modes of energy conservation and comprises a C1-carbon source of CO or CO+CO. The fermentative metabolism. Moreover, these species are clus Substrate may further comprise other non-carbon compo tered in clostridial rRNA homology group I with 16S rRNA nents, such as H, N, or electrons. DNA that is more than 99% identical, have a DNA G+C 0105. The substrate generally comprises at least some content of about 22-30 mol %, are gram-positive, have amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, similar morphology and size (logarithmic growing cells 70, 80,90, or 100 mol % CO. The substrate may comprise between 0.5-0.7x3-5 um), are mesophilic (grow optimally at a range of CO, such as about 20-80, 30-70, or 40-60 mol % 30-37° C.), have similar pH ranges of about 4-7.5 (with an CO. Preferably, the substrate comprises about 40-70 mol % optimal pH of about 5.5-6), lack cytochromes, and conserve CO (e.g., steel mill or blast furnace gas), about 20-30 mol % energy via an Rnf complex. Also, reduction of carboxylic CO (e.g., basic oxygen furnace gas), or about 15-45 mol % acids into their corresponding alcohols has been shown in CO (e.g., syngas). In some embodiments, the Substrate may these species (Perez, Biotechnol Bioeng, 110:1066-1077, comprise a relatively low amount of CO, such as about 1-10 2012). Importantly, these species also all show strong auto or 1-20 mol % CO. The microorganism of the invention trophic growth on CO-containing gases, produce ethanol typically converts at least a portion of the CO in the substrate and acetate (or acetic acid) as main fermentation products, to a product. and produce Small amounts of 2,3-butanediol and lactic acid 0106 The substrate may comprise some amount of H. under certain conditions. For example, the substrate may comprise about 1, 2, 5, 10. 0101 However, these three species also have a number of 15, 20, or 30 mol % H. In some embodiments, the substrate differences. These species were isolated from different may comprise a relatively high amount of H2. Such as about Sources: Clostridium autoethanogenium from rabbit gut, 60, 70, 80, or 90 mol % H. In further embodiments, the Clostridium liungdahli from chicken yard waste, and Substrate comprises Substantially no H. Clostridium ragsdalei from freshwater sediment. These spe 0107 The substrate may comprise some amount of CO. cies differ in utilization of various Sugars (e.g., rhamnose, For example, the substrate may comprise about 1-80 or 1-30 arabinose), acids (e.g., gluconate, citrate), amino acids (e.g., mol % CO. In some embodiments, the substrate may arginine, histidine), and other Substrates (e.g., betaine, buta comprise less than about 20, 15, 10, or 5 mol % CO. In nol). Moreover, these species differ in auxotrophy to certain another embodiment, the Substrate comprises Substantially Vitamins (e.g., thiamine, biotin). These species have differ no CO. ences in nucleic and amino acid sequences of Wood-Ljung 0108. Although the substrate is typically gaseous, the dahl pathway genes and proteins, although the general substrate may also be provided in alternative forms. For US 2016/0348087 A1 Dec. 1, 2016 example, the Substrate may be dissolved in a liquid Saturated 0113 Typically, the culture is performed in a bioreactor. with a CO-containing gas using a microbubble dispersion The term “bioreactor” includes a culture/fermentation generator. By way of further example, the substrate may be device consisting of one or more vessels, towers, or piping adsorbed onto a solid Support. arrangements, such as a continuous stirred tank reactor 0109 The substrate and/or C1-carbon source may be a (CSTR), immobilized cell reactor (ICR), trickle bed reactor waste gas obtained as a byproduct of an industrial process or (TBR), bubble column, gas lift fermenter, static mixer, or from Some other source. Such as from automobile exhaust other vessel or other device Suitable for gas-liquid contact. fumes or biomass gasification. In certain embodiments, the In some embodiments, the bioreactor may comprise a first industrial process is selected from the group consisting of growth reactor and a second culture/fermentation reactor. ferrous metal products manufacturing, such as a steel mill The substrate may be provided to one or both of these manufacturing, non-ferrous products manufacturing, petro reactors. As used herein, the terms “culture' and "fermen leum refining processes, coal gasification, electric power tation” are used interchangeably. These terms encompass production, carbon black production, ammonia production, both the growth phase and product biosynthesis phase of the methanol production, and coke manufacturing. In these culture/fermentation process. embodiments, the substrate and/or C1-carbon source may be 0114. The culture is generally maintained in an aqueous captured from the industrial process before it is emitted into culture medium that contains nutrients, vitamins, and/or the atmosphere, using any convenient method. minerals sufficient to permit growth of the microorganism. 0110. The substrate and/or C1-carbon source may be Preferably the aqueous culture medium is an anaerobic syngas, Such as Syngas obtained by gasification of coal or microbial growth medium, Such as a minimal anaerobic refinery residues, gasification of biomass, or reforming of microbial growth medium. Suitable media are well known in natural gas. The composition of the Substrate may have a the art. significant impact on the efficiency and/or cost of the reac 0115 The culture/fermentation should desirably be car tion. For example, the presence of oxygen (O2) may reduce ried out under appropriate conditions for production of the the efficiency of an anaerobic fermentation process. Depend target product. Reaction conditions to consider include pres ing on the composition of the Substrate, it may be desirable Sure (or partial pressure), temperature, gas flow rate, liquid to treat, scrub, or filter the substrate to remove any undesired flow rate, media pH, media redox potential, agitation rate (if impurities. Such as toxins, undesired components, or dust using a continuous stirred tank reactor), inoculum level. particles, and/or increase the concentration of desirable maximum gas Substrate concentrations to ensure that gas in components. the liquid phase does not become limiting, and maximum 0111. The microorganism of the invention may be cul product concentrations to avoid product inhibition. In par tured to produce one or more products. For instance, ticular, the rate of introduction of the substrate may be Clostridium autoethanogenium produces or can be engi controlled to ensure that the concentration of gas in the neered to produce ethanol (WO 2007/117157), acetate (WO liquid phase does not become limiting, since products may 2007/117157), butanol (WO 2008/115080 and WO 2012/ be consumed by the culture under gas-limited conditions. 053905), butyrate (WO 2008/115080), 2,3-butanediol (WO 0116 Operating a bioreactor at elevated pressures allows 2009/151342), lactate (WO 2011/112103), butene (WO for an increased rate of gas mass transfer from the gas phase 2012/024522), butadiene (WO 2012/024522), methyl ethyl to the liquid phase. Accordingly, it is generally preferable to ketone (2-butanone) (WO 2012/024522 and WO 2013/ perform the culture/fermentation at pressures higher than 185123), ethylene (WO 2012/026833), acetone (WO 2012/ atmospheric pressure. Also, since a given gas conversion 115527), isopropanol (WO 2012/115527), lipids (WO 2013/ rate is, in part, a function of the Substrate retention time and 036147), 3-hydroxypropionate (3-HP) (WO 2013/180581), retention time dictates the required volume of a bioreactor, isoprene (WO 2013/180584), fatty acids (WO 2013/ the use of pressurized systems can greatly reduce the Volume 191567), 2-butanol (WO 2013/185123), 1,2-propanediol of the bioreactor required and, consequently, the capital cost (WO 2014/0369152), and 1-propanol (WO 2014/0369152). of the culture/fermentation equipment. This, in turn, means In addition to one or more target products, the microorgan that the retention time, defined as the liquid volume in the ism of the invention may also produce ethanol, acetate, bioreactor divided by the input gas flow rate, can be reduced and/or 2,3-butanediol. when bioreactors are maintained at elevated pressure rather 0112 “Selectivity” refers to the ratio of the production of than atmospheric pressure. The optimum reaction conditions a target product to the production of all fermentation prod will depend partly on the particular microorganism used. ucts produced by a microorganism. The microorganism of However, in general, it is preferable to operate the fermen the invention may be engineered to produce products at a tation at a pressure higher than atmospheric pressure. Also, certain selectivity or at a minimum selectivity. In one since a given gas conversion rate is in part a function of embodiment, a target product account for at least about 5%, Substrate retention time and achieving a desired retention 10%, 15%, 20%, 30%, 50%, or 75% of all fermentation time in turn dictates the required volume of a bioreactor, the products produced by the microorganism of the invention. In use of pressurized systems can greatly reduce the Volume of one embodiment, the target product accounts for at least the bioreactor required, and consequently the capital cost of 10% of all fermentation products produced by the microor the fermentation equipment. ganism of the invention, such that the microorganism of the 0117 Target products may be separated or purified from invention has a selectivity for the target product of at least a fermentation broth using any method or combination of 10%. In another embodiment, the target product accounts for methods known in the art, including, for example, fractional at least 30% of all fermentation products produced by the distillation, evaporation, pervaporation, gas stripping, phase microorganism of the invention, such that the microorgan separation, and extractive fermentation, including for ism of the invention has a selectivity for the target product example, liquid-liquid extraction. In certain embodiments, of at least 30%. target products are recovered from the fermentation broth by US 2016/0348087 A1 Dec. 1, 2016

continuously removing a portion of the broth from the -continued bioreactor, separating microbial cells from the broth (con veniently by filtration), and recovering one or more target Amount per 1.0 L of Wolfe's products from the broth. Alcohols and/or acetone may be Wolfe's vitamin solution component vitamin Solution recovered, for example, by distillation. Acids may be recov Biotin 2 mg ered, for example, by adsorption on activated charcoal. Folic acid 2 mg Separated microbial cells are preferably returned to the Pyridoxine hydrochloride 10 mg Thiamine HCI 5 mg bioreactor. The cell-free permeate remaining after target Riboflavin 5 mg products have been removed is also preferably returned to Nicotinic acid 5 mg the bioreactor. Additional nutrients (such as B vitamins) may Calcium D-(+)-pantothenate 5 mg be added to the cell-free permeate to replenish the medium Vitamin B12 0.1 mg before it is returned to the bioreactor. P-aminobenzoic acid 5 mg Thioctic acid 5 mg EXAMPLES Amount per 100 mL of reducing Reducing agent solution component agent solution 0118. The following examples further illustrate the NaOH 0.9 g invention but, of course, should not be construed to limit its Cysteine-HCl 4 g Scope in any way. NaS 4 g Example 1 0119 This example describes general methods for cul Example 2 turing C. autoethanogenium and C. liungdahli. 0120 C. autoethanogenium DSM10061 and DSM23693 0.122 This example demonstrates the construction of a (a derivate of DSM10061) and C. ljungdahlii DSM 13528 strain comprising a p-hydroxybenzoate expression plasmid. were sourced from DSMZ (The German Collection of I0123. The nucleotide sequence for chorismate pyruvate Microorganisms and Cell Cultures, Inhoffenstra?e 7 B. lyase (ubiC) (SEQ ID NO: 1) was optimized (SEQ ID NO: 3.8124 Braunschweig, Germany). 2) according to the C. autoethanogenium codon-usage table by GeneArt and cloned into the pMTL8315 expression 0121 Strains were grown at 37° C. in PETC medium at vector (FIG. 7) under control of the Wood-Ljungdahl path pH 5.6 using standard anaerobic techniques (Hungate, Meth way promoter (US 2011025.6600). The coding sequence for ods Microbiol, 3B: 117-132, 1969: Wolfe, Adv. Microbiol a feedback-insensitive mutant 3-deoxy-D-arabino-heptu Physiol. 6: 107-146, 1971). Fructose (heterotrophic growth) losonate-7-phosphate (DAHP) synthase (aroG) (SEQ ID or 30 psi CO-containing steel mill gas (collected from New NO: 8) was also included, following ubiC in a bicistronic Zealand Steel site in Glenbrook, NZ; composition: 44% CO, format (FIG. 7). The plasmid pARO 01 (SEQ ID NO: 9) 32% N, 22% CO, 2% H) in the headspace (autotrophic was transformed into C. autoethanogenium LZ1561 growth) was used as substrate. For solid media, 1.2% bacto (DSM23693) via conjugation with E. coli strain CA434 as agar (BD, Franklin Lakes, N.J. 07417, USA) was added. donor. Donor Strains were grown overnight in LB media supplemented with 25 ug/mL chloramphenicol and 100 Amount per 1.0 L of ug/mL spectinomycin. Cells from 1.5 mL of culture were PETC medium component PETC medium harvested by centrifugation and washed in phosphate buff ered saline (PBS). Inside an anaerobic workstation, the NHCI 1 g KC 0.1 g donor cell pellet was resuspended in 200 uL of exponentially MgSO4·7H2O 0.2 g growing recipient LZ1561. The conjugation mixture was NaCl 0.8 g. spotted on PETC-MES agar medium and incubated at 37°C. KH2PO 0.1 g After 24 hours the cells were scraped from the conjugation CaCl2 0.02 g plate and spread on PETC-MES agar medium supplemented Trace metal solution (see below) 10 ml Wolfe's vitamin solution (see below) 10 ml with 7.5ug thiamphenicol/mL (Sigma) and 10 ug trimethop Yeast extract (optional) 1 g rim/mL (Sigma). Three plasmid-bearing colonies (i.e. bio ResaZurin (2 g/L stock) 0.5 ml logical triplicates) isolates were grown in PETC-MES liquid NaHCO 2 g Reducing agent solution (see below) 0.006-0.008% (v/v) medium containing 7.5 ug thiamphenicol/mL and with a gas Fructose (for heterotrophic growth) 5 g blend that simulates steel mill off gas as the carbon source (50% CO, 10% H, 30% CO, 10% N, subsequently Amount per 1.0 L of trace referred to as “mill gas” in this application). Trace metal solution component metal solution (0.124 Liquid cultures were grown in 10 mL PETC-MES Nitrillotriacetic acid 2 g medium in serum bottles containing thiamphenicol and mill MnSOHO 1 g gas at 22 psi. Samples were taken daily to measure biomass Fe(SO4)2(NH)6H2O 0.8 g. (FIG. 8) and pHBA (FIG. 9a and FIG.9b). CoCl6HO 0.2 g ZnSO7H2O 0.2 mg 0.125 To measure pHBA, samples (100 uL) were spiked CuCl2.H2O 0.02 g with 10 uL 0.1N NaOH, frozen, and then freeze dried. The NaMoCl2.H2O 0.02 g samples were then derivatised with 100 uL BSTFA+TCMS Na-SeO. 0.02 g NiCl26H2O 0.02 g (99:1) and pyridine 100 uL. The samples were then incu Na-WO2H2O 0.02 g bated at 60° C. for 30 minto form trimethylsilyl derivatives of the carboxylic acid functional group. Details of GC-MS method are: Inj. Vol. 1 uL: Inj. T 250° C.; split ratio 10:1. US 2016/0348087 A1 Dec. 1, 2016

Initial T 50° C. (hold 5 min); final T 220° C. (20° C./min): Example 5 const. flow 1 mL/min (He carrier gas); column Zebron ZB-5MS 30 mx0.25 mmx0.25 um. Varian Ion Trap 4000 I0134. This example demonstrates the construction of a operated in full scan mode 40-400 m/z. Tune PFTBA strain comprising a salicylate expression plasmid. 0126. In FIG. 9a, LZ1561 (the control strain) has three I0135. The nucleotide sequences for pchA (SEQ ID NO: technical replicates (i.e., grown and sampled three times). 4) and pchB (SEQ ID NO: 6) were codon optimized and Two biological replicates of LZ1561 with p ARO 01, were cloned into the expression vector under control of a tetra also prepared, each with three technical replicates. “Tech cycline-inducible promoter. The plasmid is transformed into nical replicate” refers to growing and sampling each Strain C. autoethanogenium LZ1561 (DSM23693) via conjugation in separate experiments, while “biological replicate” refers with E. coli strain CA434 as donor. Donor strains were to reproducing the strain from Scratch. In this way, the grown overnight in LB media Supplemented with 25 ug/mL biological replicates account for background biological chloramphenicol and 100 ug/mL spectinomycin. Cells from variation in the microorganism, while technical replicates 1.5 mL culture were harvested by centrifugation and washed account for variation due to technical aspects including in phosphate buffered saline (PBS). Inside an anaerobic culture, sampling, and analysis methods. FIG. 9a shows that workstation, the donor cell pellet was resuspended in 200 uL pHBA was produced repeatedly in separate instances. FIG. of exponentially growing recipient C. autoethanogenium. 8 and FIG.9b give an overall representation of growth and The conjugation mixture was spotted on PETC-MES agar pHBA productivity. medium and incubated at 37° C. After 24 hours the cells were scraped from the conjugation plate and spread on Example 3 PETC-MES agar medium supplemented with 7.5 ug thiam phenicol/mL (Sigma) and 10 ug trimethoprim/mL (Sigma). 0127. This example demonstrates the production of p-hy Three plasmid-bearing colonies (i.e. biological triplicates) droxybenzoate via gas fermentation. isolates were grown in PETC-MES liquid medium contain 0128. C. autoethanogenium harbouring plasmid paRO ing 7.5ug thiamphenicol/mL and with mill gas as the carbon 01 (SEQID NO: 9) were grown on mill gas as described in SOUC. Example 1. GC-MS analysis, performed as in Example 1, of the culture determined that pHBA was produced by the I0136. Liquid cultures were grown in 10 mL PETC-MES bacterium expressing chorismate pyruvate lyase. The linear medium in serum bottles containing thiamphenicol and mill range for analysis of pHBA using this method spanned gas at 22 psi. 0-12.5 mg/mL (FIG. 5). 0.137 Biomass was monitored spectrophotometrically. At 0129 pHBA was validated by comparison to retention OD600 nm=0.3 expression of the salicylate biosynthetic time and characteristic fragment ions of an authentic pHBA pathway was induced by addition of 40 ng anhydrotetracy standard and predicted characteristic ions from the NIST cline/mL. Duplicate cultures (technical replicates of the mass spectrometry database (FIG. 6). three biological triplicates) were grown without the addition 0130 pHBA production was observed in all cultures of anhydrotetracylcine such that the salicylate biosynthetic expressing the chorismate-pyruvate lyase encoded on the pathway remained uninduced. Samples were taken daily pMTL8315 expression vector. The peak titre of pHBA 0.138 Salicylate concentrations were measured using gas observed in any one culture was 17 mg pHBA/L after eight chromatography mass spectrometry analysis (GCMS), days (FIG. 9b). No pHBA was observed in the control employing a Thermo Scientific ISQLT GCMS equipped an sample without the expression vector. Agilent CP-SIL 5CB-MS (50 mx0.25 umx0.25um) column 0131 Detectable levels of pHBA were produced by the and autosampler. Samples were prepared by diluting 300 uL genetically engineered bacterium and present in the culture. of sample with 600 uL of acetonitrile and 50 uL 0.1N NaOH. The samples were vortexed then centrifuged for 3 minutes at Example 4 14,000 rpm, 800 uL of the supernatant was transferred to a glass vial and the sample was dried in a Thermo SpeedVacR. 0132) This example demonstrates an experimental pro Once dry, the samples were then Suspended in a solution of tocol for increasing the production of pHBA through 100 ul of pyridine containing 22 mg/ml methoxyamine HCl enzyme engineering. then heated in a sealed glass vial for 60 minutes at 60° C. 0.133 UbiC is subject to product inhibition through reten After which, 300 uL N.O-Bistrifluoroacetamide (BSTFA) tion of pHBA. The nucleic acid sequence encoding ubiC was added then heated in a sealed glass vial for 60 minutes may be modified Such that amino acids involved in product at 60° C. Samples were transferred to an autosampler for retention by the enzyme are mutated and release of product analysis using a 1.5 LL injection, a split ration of 20 to 1, and is enhanced. To do this, the amino acids involved in pHBA an inlet temperature of 250° C. Chromatography was per binding are identified by analysis of existing structures with formed with an oven program of 80°C. (no hold) to a ramp bound product. Product inhibition is then minimised by of 3° C./min to 140° C. to a ramp of 20°C/min to 230° C. mutating the amino acids involved in pHBA binding and with a 4-min final hold. The column flow rate was 38 retention. To identify enzymes with the greatest catalytic cm/min, with helium as the carrier gas. The MS ion source efficiency for pHBAyield, a targeted library of ubiC mutants was kept at 280° C. Quantitation, was performed using 267 can be produced where different combinations of pHBA m/Z for a quantification ion with 135 and 45 m/z used as binding amino acids are altered, and these mutant enzymes qualifier ions. can be analysed with an enzyme assay. Improved mutants 0.139 FIG.11a shows a comparison of biomass growth in are then expressed in C. autoethanogenium LZ1561 to Vali the induced and un-induced samples. FIG. 11b shows that date the strains with most improved pHBA productivity. salicylate was produced repeatedly. US 2016/0348087 A1 Dec. 1, 2016

Example 6 mL/min/L at 4 hours intervals to the maximum flow rate that 0140. This example demonstrates knockout of phe A for the target CO uptake can be achieved. The NaS was added enhanced production of chorismate-derived products. over the course of the fermentation with an initial pump rate 0141 phea (e.g. from C. autoethanogenium, CAETHG of 0.3 mL/h and later increased in 0.2 mL/h increments when 0905 (CP006763. 1:973789 . . . 974925)) is a gene that the HS concentration in the headspace dropped below encodes the enzyme prephenate synthase. Prephenate Syn 200-ppm. The CO and H consumption and CO, production thase catalyses the conversion of chorismate to prephenate, along with the HS concentration were measured hourly which is a precursor to the aromatic amino acids phenyl using gas chromatography (GC). Liquid samples were taken alanine and tyrosine. pheA function was knocked out by from the fermenter at regular intervals over the course of the disrupting the gene using the ClosTron method (Heap et al., fermentation to determine cell mass and metabolite concen J Microbiol Methods. 2010, 80(1):49-55). The ClosTron trations using HPLC. plasmid pMTL007C-E2 was generated by DNA2.0 and 0.147. After starting up in batch mode, the fermenter was transformed into C. autoethanogenium LZ1561 turned to continuous when the OD reached a value of 2. The (DSM23693) via conjugation with E. coli strain CA434 as media and nutrient inflow rates were controlled by one or donor. Donor Strains were grown overnight in LB media more precision peristaltic pumps (Masterflex L/S digital supplemented with 25 g/mL chloramphenicol. Cells from drive pumps) while the fermenter volume was held constant 1.5 mL culture were harvested by centrifugation and washed by using a level probe that triggers a pump to remove in phosphate buffered saline (PBS). Inside an anaerobic fermentation broth from the CSTR. The dilution rate was set workstation, the donor cell pellet was resuspended in 200 uL in one step to 0.5 day' and further increased to 1 day then of exponentially growing recipient C. autoethanogenium to 1.7 day' at 24 hour intervals. LZ1561. The conjugation mixture was spotted on PETC 0.148. An additional equipment was added to the fermen agar media and incubated at 37° C. After 24 hours the cells tation was a hollow fibre membrane (GE Healthcare) with a were scraped and resuspended in 500 uL PBS and spread on pore size of 0.2 um and a surface area of 1,200 cm. The PETC agar media supplemented with 7.5 ug/mL thiam membrane was used to increase the cell concentration in the phenicol (Sigma) and 10 g/mL trimethoprim (Sigma). fermentation. The fermentation broth was pumped at high Plasmid-bearing isolates were grown in PETC-MES liquid speed through the membrane and returned back to the medium containing 7.5 g thiamphenicol/mL and with mill fermenter while a stream of cell-free filtrate was pumped to gas as the carbon Source. the filtrate tank at a slower rate than the media pump rate. 0142 Colonies were streaked on PETC solid media con This allowed the retention time of the bacteria cell in the taining the antibiotic clarithromycin (5 g/mL). This step fermenter to increase. selected for integration of the intron retargeting sequence 0149. As shown in FIG. 10, three new compounds were into the genome. Integration of the intron sequence into the identified using GC-MS. These compounds were cis-4- target site results in an 1800 base pair insertion in the hydroxycyclohexane carboxylic acid, 3,4-dihydroxybenzoic genome, which was screened for with colony PCR The PCR acid, and 2-aminobenzoic acid. These compounds were only product of the positive ClosTron mutants were purified and detected in this phea::CT culture and were not detected in sequenced to confirm the insertion site. the parental strain (LZ1561) culture. 0143 Liquid cultures were grown in 10 mL PETC-MES 0150 3.4 dihydroxy benzoic acid, 2-aminobenzoic acid medium in serum bottles containing clarithromycin and mill and cis-4-hydroxycyclohexanecarboxylic acid concentra gas at 22 psi. Glycerol stock was prepared from this serum tions were measured using gas chromatography (GC) analy bottle sis, employing an Agilent 6890N GC equipped a Agilent 014.4 Bioreactor experiments were carried out in a 2 L CP-SIL 5CB-MS (50 mx0.25 umx0.25 um) column, BioFlo 115 water jacket system (New Brunswick Scientific autosampler and a flame ionization detector (FID). Samples Corp., Edison, N.J.) with a working volume of 1.5 L. The were prepared by diluting 400 uL of sample with 400 uL of CSTR system was equipped with two six-bladed Rushton acetonitrile, followed by a 3 minute centrifugation at 14,000 impellers and baffles enhance the mixing of fermentation rpm; the Supernatant was transferred to a glass vial and the broth and the gas to liquid mass transfer. A pH and an sample was dried in a Thermo SpeedVacR). Once dry, the oxidation-reduction potential (ORP) electrode (Broadley samples were then suspended in a solution of 400 uL of James Corporation) were inserted through the headplate and N.O-Bistrifluoroacetamide (BSTFA) and pyridine (3:1 ratio) their readings were recorded at 5 min intervals. pH was and heated in a sealed glass vial for 60 minutes at 60° C. maintained at 5.0 by automated addition of a 5M solution Samples were transferred to an autosampler for analysis of ammonium hydroxide. using a 1 uL injection, a split ration of 30 to 1, and an inlet 0145 The inoculum was prepared from a glycerol stock. temperature of 250° C. Chromatography was performed One mL of glycerol stock was transferred into 50 mL of with an oven program of 70° C. (no hold) to a ramp of 3 PETC media with 22 psi mill gas as carbon source. The C./min to 110° C. to a ramp of 15° C./min to 230° C., culture was incubated at 37° C. for on a shaker two to three followed by a final ramp of 40° C./min to 310° C. with a days until a visible growth was observed. The culture was 3-min hold. The column flow rate was 1.8 ml/min, with then used to inoculate 200 mL of fresh media in 1 L-Schott helium as the carrier gas. The FID was kept at 320°C., with bottle and mill gas was added to a pressure of 22 psi. The hydrogen at 40 ml/min, air at 400 ml/min, and helium at 20 Schott bottle was incubated for another 24 to 36 hours before ml/min as the makeup gas. being transferring to the fermenters. 0151 FIG. 12 shows the concentration of cis-4-hydroxy 0146 The agitation was set at 200 rpm and the gas flow cyclohexane carboxylic acid, 3,4-dihydroxybenzoic acid, was set at 35 mL/min/L. After one day, the stirring rate was and 2-aminobenzoic acid over the course of the fermentation increased by 25 rpm at 4 hours intervals to the maximum run. As shown in FIG. 12, compound cis-4-hydroxycyclo value of 900 rpm. The gas flow was increased by 25 hexanecarboxylic acid increased to a concentration of about US 2016/0348087 A1 Dec. 1, 2016

0.9 g/L on day 6 of the fermentation. 2-aminobenzoic acid should not be taken as, an acknowledgement that that prior accumulated to a concentration of about 0.45 g/L on day 8-9 art forms part of the common general knowledge in the field of the fermentation. 3,4-dihydroxybenzoic acid was pro of endeavour in any country. duced in Smaller amounts, peaking at a concentration of 0155 The use of the terms “a” and “an and “the and around 0.3 g/L between days 6-8. A total accumulation of similar referents in the context of describing the invention cis-4-hydroxycyclohexanecarboxylic acid, 2-aminobenzoic (especially in the context of the following claims) are to be acid and 3,4-dihydroxybenzoic acid of >1.3 g/L was construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by con observed on day 6. text. The terms “comprising,” “having,” “including,” and 0152 Little is known in literature about the production of “containing are to be construed as open-ended terms (i.e., cis-4-hydroxycyclohexanecarboxylic acid. There is only one meaning “including, but not limited to') unless otherwise report that cis-4 hydroxycyclohexanecarboxylic acid was noted. Recitation of ranges of values herein are merely detected in a child’s urine sample using GC-MS. It was intended to serve as a shorthand method of referring indi hypothesized that the compound was a by-product of enteric vidually to each separate value falling within the range, bacterial metabolism (Kronick, Clinica Chimica Acta, 132: unless otherwise indicated herein, and each separate value is 205-208, 1983). It seems likely that this compound is a incorporated into the specification as if it were individually direct product of chorismate or prephanate as the reaction recited herein. All methods described herein can be per mechanism may be explained by a cleavage of the pyruvate formed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use molecule followed by a reduction requiring a further 2.5 H. of any and all examples, or exemplary language (e.g., “Such molecules that may be provided through NAD(P)H. as') provided herein, is intended merely to better illuminate 0153 2-Aminobenzoic acid is a known intermediate in the invention and does not pose a limitation on the scope of the chorismate to tryptophan pathway. Anthranilate synthase the invention unless otherwise claimed. No language in the catalyses the amination followed by the aromatization of specification should be construed as indicating any non chorismate to obtain the aromatic backbone of the trypto claimed element as essential to the practice of the invention. phan molecule. It is known that the gene expression of 0156 Preferred embodiments of this invention are anthranilate synthase is highly regulated and Subjected to described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art feedback inhibition by the end product tryptophan (Dosse upon reading the foregoing description. The inventors laere, Crit Rev. Microbiol, 27: 75-131, 2001). 2-Aminoben expect skilled artisans to employ Such variations as appro Zoic acid was only secreted into the fermentation broth when priate, and the inventors intend for the invention to be growth ceased indicating that it is an overflow product that practiced otherwise than as specifically described herein. was no longer reacted away when growth had stopped. Accordingly, this invention includes all modifications and 0154 All references, including publications, patent appli equivalents of the Subject matter recited in the claims cations, and patents, cited herein are hereby incorporated by appended hereto as permitted by applicable law. Moreover, reference to the same extent as if each reference were any combination of the above-described elements in all individually and specifically indicated to be incorporated by possible variations thereof is encompassed by the invention reference and were set forth in its entirety herein. The unless otherwise indicated herein or otherwise clearly con reference to any prior art in this specification is not, and tradicted by context.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 11

SEO ID NO 1 LENGTH: 1.65 TYPE PRT ORGANISM: Escherichia coli

<4 OOs SEQUENCE: 1

Met Ser His Pro Ala Luell Thir Glin Lell Arg Ala Luell Arg Phe 1. 1O 15

Glu Il e Pro Ala Glu Pro Glin Lell Lell Asp Trp Luell Tell Lell Glu 2O 25 3 O

Asp Se r Met Thir Arg Phe Glu Glin Glin Gly Thir Wall Ser Wall 35 4 O 45

Thir Me t Ile Arg Glu Gly Phe Wall Glu Glin Asn Glu Ile Pro Glu Glu SO 55 60

Leul Pro Luell Luell Pro Lys Glu Ser Arg Luell Arg Glu Ile Lell 65 70

Luell Ala Asp Gly Glu Pro Trp Lell Ala Gly Arg Thir Wall Wall Pro 85 90 95 US 2016/0348087 A1 Dec. 1, 2016 15

- Continued Val Ser Thir Lieu. Ser Gly Pro Glu Lieu Ala Lieu Gln Llys Lieu. Gly Lys 1OO 105 11 O Thr Pro Leu Gly Arg Tyr Lieu Phe Thr Ser Ser Thr Lieu. Thr Arg Asp 115 12 O 125 Phe Ile Glu Ile Gly Arg Asp Ala Gly Lieu. Trp Gly Arg Arg Ser Arg 13 O 135 14 O Lieu. Arg Lieu. Ser Gly Llys Pro Lieu. Lieu. Lieu. Thr Glu Lieu. Phe Lieu Pro 145 150 155 160 Ala Ser Pro Leu Tyr 1.65

<210s, SEQ ID NO 2 &211s LENGTH: 273 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2 titt.ccc.gc.gc gctgggaaat cct agagatt Ctttgagagc ccttggaaaa tatataggcg 6 O cgttgcgcga aaatticgc.gc gcttgcaaaa ggct Ctcgga gagattic ccc ttagagagat 12 O atagaggata ggggg.ccgga ggatatatag ggtttgggala agagagatct gcgc.gc.gctg 18O aggggaggag agagagat.cg Ctctagat.cg at agcggata gacct ctaga gatcc.cgc.gc 24 O gatagaga aa gggcct Ct c agagat.cgcg caa 273

<210s, SEQ ID NO 3 &211s LENGTH: 476 212. TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa

<4 OOs, SEQUENCE: 3 Met Ser Arg Lieu Ala Pro Lieu. Ser Glin Cys Lieu. His Ala Lieu. Arg Gly 1. 5 1O 15 Thir Phe Glu Arg Ala Ile Gly Glin Ala Glin Ala Lieu. Asp Arg Pro Val 2O 25 3O Lieu Val Ala Ala Ser Phe Glu Ile Asp Pro Lieu. Asp Pro Lieu. Glin Val 35 4 O 45 Phe Gly Ala Trp Asp Asp Arg Glin Thr Pro Cys Lieu. Tyr Trp Glu Glin SO 55 6 O Pro Glu Lieu Ala Phe Phe Ala Trp Gly Cys Ala Lieu. Glu Lieu. Glin Gly 65 70 7s 8O His Gly Glu Glin Arg Phe Ala Arg Ile Glu Glu Asn Trp Glin Lieu. Lieu. 85 90 95 Cys Ala Asp Ala Val Val Glu Gly Pro Lieu Ala Pro Arg Lieu. Cys Gly 1OO 105 11 O Gly Phe Arg Phe Asp Pro Arg Gly Pro Arg Glu Glu. His Trp Glin Ala 115 12 O 125

Phe Ala Asp Ala Ser Lieu Met Lieu Ala Gly Ile Thr Val Lieu. Arg Glu 13 O 135 14 O Gly Glu Arg Tyr Arg Val Lieu. Cys Gln His Lieu Ala Lys Pro Gly Glu 145 150 155 160

Asp Ala Lieu Ala Lieu Ala Ala Tyr His Cys Ser Ala Lieu. Lieu. Arg Lieu 1.65 17O 17s

Arg Glin Pro Ala Arg Arg Arg Pro Ser Gly Pro Thr Ala Gly Ala Glin 18O 185 19 O US 2016/0348087 A1 Dec. 1, 2016 16

- Continued

Gly Asp Ala Ser Ala Glin Glu Arg Arg Glin Trp Glu Ala Lys Val Ser 195 2OO 2O5 Asp Ala Val Ser Ser Val Arg Glin Gly Arg Phe Gly Llys Val Val Lieu 21 O 215 22O Ala Arg Thr Glin Ala Arg Pro Lieu. Gly Asp Ile Glu Pro Trp Glin Val 225 23 O 235 24 O Ile Glu. His Lieu. Arg Lieu Gln His Ala Asp Ala Glin Lieu. Phe Ala Cys 245 250 255 Arg Arg Gly Asn Ala Cys Phe Lieu. Gly Ala Ser Pro Glu Arg Lieu Val 26 O 265 27 O Arg Ile Arg Ala Gly Glu Ala Lieu. Thir His Ala Lieu Ala Gly. Thir Ile 27s 28O 285 Ala Arg Gly Gly Asp Ala Glin Glu Asp Ala Arg Lieu. Gly Glin Ala Lieu 29 O 295 3 OO Lieu. Asp Ser Ala Lys Asp Arg His Glu. His Glin Lieu Val Val Glu Ala 3. OS 310 315 32O Ile Arg Thr Ala Lieu. Glu Pro Phe Ser Glu Val Lieu. Glu Ile Pro Asp 3.25 330 335 Ala Pro Gly Lieu Lys Arg Lieu Ala Arg Val Glin His Lieu. Asn Thr Pro 34 O 345 35. O Ile Arg Ala Arg Lieu Ala Asp Ala Gly Gly Ile Lieu. Arg Lieu. Lieu. Glin 355 360 365 Ala Lieu. His Pro Thr Pro Ala Val Gly Gly Tyr Pro Arg Ser Ala Ala 37 O 375 38O Lieu. Asp Tyr Ile Arg Glin His Glu Gly Met Asp Arg Gly Trp Tyr Ala 385 390 395 4 OO Ala Pro Lieu. Gly Trp Lieu. Asp Gly Glu Gly Asn Gly Asp Phe Lieu Val 4 OS 41O 415 Ala Lieu. Arg Ser Ala Lieu. Lieu. Thr Pro Gly Arg Gly Tyr Lieu. Phe Ala 42O 425 43 O Gly Cys Gly Lieu Val Gly Asp Ser Glu Pro Ala His Glu Tyr Arg Glu 435 44 O 445 Thir Cys Lieu Lys Lieu. Ser Ala Met Arg Glu Ala Lieu. Ser Ala Ile Gly 450 45.5 460 Gly Lieu. Asp Glu Val Pro Lieu. Glin Arg Gly Val Ala 465 470 47s

<210s, SEQ ID NO 4 &211s LENGTH: 1431 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas aeruginosa <4 OOs, SEQUENCE: 4 atgagc.cggc tiggcgc.ccct gagcc agtgc ctgcacgc.ct tcc.gcggcac Cttctgagcgc 6 O gccatcggcc aggcgcaggc gct catcgt CC9gtgctgg togcggcatc gttcgagat C 12 O gacc cattgg accc.gctgca gg tatt cqgt gcctgggacg accggcaaac gcc ctgcctg 18O tactgggaac agc.ccgagct ggcgttct tc gcc tdgggct gcgcc ctgga gctgcaaggc 24 O cacggcgaac agcgct tcgc ccggat.cgag gaaaactggc aattgctctg. c9ccgacgc.c 3OO gtggtcgagg gcc.cgctggc gcc.gc.gc.ctg. tcggcggat tcc.gctt.cga t cc.gc.gcggc 360 cc.gc.gc.gagg aac actggca agc ctitcgcc gatgc.ca.gcc tatgct cqc cqgcatcacc 42O

US 2016/0348087 A1 Dec. 1, 2016 18

- Continued cgcttcaagg C cagdgaggc ggcgatt.ccg gcgc.ccgagc gggtc.gc.cgc gatgctic ccc 18O gagggcgc.cc gctgggc.cga ggaaaacgga Ctcgacgc.gc cct tcgt.cga gggactgttc 24 O gcgcagat catcc actggta catcgc.cgag Cagat Caagt actggcgc.ca gacacggggit 3OO gcc.gcatga 309

<210s, SEQ ID NO 7 &211s LENGTH: 350 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 7 Met Asn Tyr Glin Asn Asp Asp Lieu. Arg Ile Lys Glu Ile Lys Glu Lieu. 1. 5 1O 15 Lieu Pro Pro Val Ala Lieu. Lieu. Glu Lys Phe Pro Ala Thr Glu Asn Ala 2O 25 3O Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Lieu Lys 35 4 O 45 Gly Asn Asp Asp Arg Lieu. Lieu Val Val Ile Gly Pro Cys Ser Ile His SO 55 6 O Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Lieu. Lieu Ala Lieu. Arg 65 70 7s 8O Glu Glu Lieu Lys Asp Glu Lieu. Glu Ile Val Met Arg Val Tyr Phe Glu 85 90 95 Llys Pro Arg Thir Thr Val Gly Trip Lys Gly Lieu. Ile Asn Asp Pro His 1OO 105 11 O Met Asp Asn. Ser Phe Glin Ile Asn Asp Gly Lieu. Arg Ile Ala Arg Llys 115 12 O 125 Lieu. Lieu. Lieu. Asp Ile Asn Asp Ser Gly Lieu Pro Ala Ala Gly Glu Phe 13 O 135 14 O Lieu. Asp Met Ile Thr Pro Glin Tyr Lieu Ala Asp Leu Met Ser Trp Gly 145 150 155 160 Ala Ile Gly Ala Arg Thir Thr Glu Ser Glin Val His Arg Glu Asp Ala 1.65 17O 17s Ser Gly Lieu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr 18O 185 19 O Ile Llys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys 195 2OO 2O5 Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn. Thir Ser 21 O 215 22O Gly Asn Gly Asp Cys His Ile Ile Lieu. Arg Gly Gly Lys Glu Pro Asn 225 23 O 235 24 O Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Lieu. Asn Lys Ala 245 250 255 Gly Leu Pro Ala Glin Val Met Ile Asp Phe Ser His Ala Asn Ser Ser 26 O 265 27 O

Lys Glin Phe Llys Lys Glin Met Asp Val Cys Ala Asp Val Cys Glin Glin 27s 28O 285 Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His 29 O 295 3 OO

Lieu Val Glu Gly Asn Glin Ser Lieu. Glu Ser Gly Glu Pro Lieu Ala Tyr 3. OS 310 315 32O US 2016/0348087 A1 Dec. 1, 2016 19

- Continued Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala 3.25 330 335 Lieu. Lieu. Arg Glin Lieu Ala Asn Ala Val Lys Ala Arg Arg Gly 34 O 345 35. O

<210s, SEQ ID NO 8 &211s LENGTH: 1053 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 8 atgaattatc aaaatgatga tittaagaata aaagaaatta aagaattatt acctic ct gta 6 O gctittattag aaaaatttico togcaactgaaaatgcagcaa atact gtagc acatgcaaga 12 O aaagcaatac ataaaatact taaaggtaat gatgatagat tattagtagt aataggacct 18O tgtagtatac atgatcctgt agcagcaaaa gaatatgcaa citagacttitt agcattalaga 24 O gaagaattaa aagatgaatt agaaatagta atgagagtat attittgaaaa acctagaact 3OO actgtaggat ggaaaggact tataaatgat cot catatgg ataatagittt toaaataaat 360 gatggacitta gaatagcaag aaaattactt ttagatataa atgatagtgg attacctgca 42O gctggtgaat ttittagatat gataactic ct caatatt tag cagatttaat gagttgggga 48O gcaattggag caagaactac taaagttcaa gtacatagag aagatgcaag tacttagt 54 O tgtc.ctgtag gatttaaaaa tigaactgat ggalactataa aagtagcaat agatgcaata 6OO aatgcagctg gtgcacct cattgttittctt agtgtaacaa aatggggaca tagtgcaata 660 gtaaat act a gtggaaatgg tattgtcat ataat actta gaggtggaaa agaacctaat 72 O tatt Ctgcaa alacatgtagc agaagtaaaa gaaggactta ataaagctgg act tcctgca 78O cagg taatga tagatttittc. tcatgcaaat agtag taaac aatttaagaa acaaatggat 84 O gtatgtgcag atgitatgtca gcaaatagct ggaggtgaaa alagcaataat tagtaatg 9 OO gtagaaagtic atttagtaga aggtaatcaa agtttagaaa gtggtgaac C tittagctitat 96.O ggaaaaagta taactgatgc atgtatagga tigggaagata Ctgatgcact tct taga caa 1 O2O

Cttgcaaatg cagtaaaagc aagaa.gagga taa 1053

<210s, SEQ ID NO 9 &211s LENGTH: 6561 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid <4 OOs, SEQUENCE: 9 cctgcaggat aaaaaaattig tagataaatt ttataaaata gttittatcta caattitttitt 6 O at Caggaaac agctatgacc gcggcc.gcag at agt catala tagttccaga at agttcaat 12 O ttagaaatta gactaaactt caaaatgttt gttaaatata taccaaact a gtatagatat 18O tttittaaata citggacittaa acagtagtaa tittgcctaaa aaattittitt c aatttitttitt 24 O aaaaaatcct tttcaagttg tacattgtta tdgtaatatg taattgaaga agittatgtag 3OO taat attgta aacgtttctt gattitttitta catccatgta gtgcttaaaa aaccaaaata 360 tgtcacatgc aattgtatat ttcaaataac aatatttatt ttct cqttaa attcacaaat 42O aatttattaa taatat caat aaccalagatt at acttaaat gigatgttitat tttittaa.cac 48O US 2016/0348087 A1 Dec. 1, 2016 20

- Continued ttittatagta aatatattta ttt tatgtag taaaaaggitt ataattataa ttg tattitat 54 O tacaattaat taaaataaaa at agggittitt agg taaaatt aagttattitt aagaagtaat 6OO tacaataaaa attgaagtta ttgctittaag gagggaatta t t catatgag to atcctgca 660 cittact caac ttagagcatt aagatattitt aaagaaatac ctdcattaga acct caatta 72 O ttagattggit tattacttga agatag tatg actaaaagat ttgaacaa.ca gggaaaaact 78O gtaagtgtaa citatgataag agaaggattt gtagaacaaa atgaaat acc taagaatta 84 O c ctitt attac ctaaagaaag tagatattgg ttalagagaaa tattactittg togcagatggit 9 OO gaac Cttggit tagctggaag aactgtagta Cctgtaagta Ctttalagtgg acctgaactt 96.O gcacttcaaa aattaggaaa aactic ctitta ggaagat atc tittttac tag tag tactitta O2O actagagatt ttatagaaat taa.gagat gcaggattat ggggaagaag aagtagatta O8O agattaagtg gaaaac ctitt attact tact gaattatttic titcctgcaa.g. tcc totttat 14 O taagaatticg agctcggtag gaggt cagaa taattatca aaatgatgat ttalagaataa 2OO aagaaattaa agaatt atta cct cotgtag ctitt attaga aaaattt cot gcaactgaaa 26 O atgcagcaaa tactgtagca catgcaagaa aag caataca taaaatactt aaagg taatg 32O atgatagatt attagtagta at aggacctt gtagtataca tdatcCtgta gcagcaaaag 38O aatatgcaac tag acttitta gcattaagag aagaattaaa agatgaatta gaaatagtaa 44 O tgagagtata ttittgaaaaa cct agaacta Ctgtaggatg gaaaggactt ataaatgat C SOO ct catatgga taatagittitt caaataaatg atggact tag aatagcaaga aaattactitt 560 tagatataaa tdatagtgga ttacctgcag ctggtgaatt tittagatatgata actic ct c 62O aatatttagc agatttaatgagttggggag caattggagc aagaact act gaaagt caag 68O tacatagaga agatgcaagt ggact tagtt gtc.ctgtagg atttaaaaat ggaactgatg 74 O gaactataaa agtagcaata gatgcaataa atgcagotgg togcacct cat tdttitt citta 8OO gtgtaacaaa atggggacat agtgcaatag taaatactag tigaaatggit gattgtcat a 86 O taat act tag aggtggaaaa galacctaatt attctgcaaa acatgtagca gaagtaaaag 92 O aaggacittaa taaagctgga citt cotgcac agg taatgat agatttittct catgcaaata 98 O gtag taalaca atttaagaala caaatggatg tatgtgcaga tigtatgtcag caaatagctg 2O4. O gaggtgaaaa agcaataatt ggagtaatgg tagaaagtica tttagtagaa ggtaatcaaa 21OO gtttagaaag titgaacct ttagct tatg gaaaaagtat alactgatgca totataggat 216 O gggalagatac tatgcactt Cttagacaac ttgcaaatgc agtaaaag.ca agaagaggat 222 O aactictagag ticgacgtcac gcgt.c catgg agat Ctcgag gcctgcagac atgcaa.gctt 228O ggcactggcc gtcgttttac aacgt.cgtga Ctgggaaaac C ctggcgitta cccaact taa 234 O tcgc.cttgca gca catcc cc ctitt.cgc.cag Ctggcgtaat agcgaagagg ccc.gcaccga 24 OO tcgc.cct tcc caa.cagttgc gcagoctgaa tigcgaatgg cqctagdata aaaataagaa 246 O gcctgcattt gcaggcttct tatttittatg gcgc.gc.cgcc attatttittt togaacaattg 252O acaatt catt tottatttitt tattalagtga tagt caaaag gcatalacagt gctgaataga 2580 aagaaattta cagaaaagaa aattatagaa tittag tatga ttaattatac to atttatga 264 O atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata 27 OO gacaagttga aaaatttaat aaaaaaataa gtcct cagot cittatatatt aagctaccala 276 O US 2016/0348087 A1 Dec. 1, 2016 21

- Continued cittagtatat aagccaaaac ttaaatgtgc taccalacaca toaa.gc.cgtt agagaactict 282O atctatagca at atttcaaa totaccaca tacaa.gagaa acattaact a tatatatto a 288O attitatgaga titat cittaac agatataaat gtaaattgca ataagtaaga tittagaagtt 294 O tatagoctitt gtg tattgga agcagtacgc aaaggcttitt ttatttgata aaaattagaa 3 OOO gtatattt at tttitt cataa ttaattitatgaaaatgaaag gigggtgagca aagtgacaga 3 O 6 O ggaaag cagt at cittatcaa ataacaaggt attagcaata t cattattga ctittagcagt 312 O aaac attatg acttittatag tecttgtagc taagtag tac gaaaggggga gctittaaaaa 318O gctic cittgga atacatagaa tt cataaatt aattitatgaa aagaaggg.cg tatatgaaaa 324 O cittgtaaaaa ttgcaaagag titt attaaag at actgaaat atgcaaaata catt.cgttga 33 OO tgatt catga taaaac agta gcaacctatt gcagtaaata caatgagtica agatgtttac 3360 atalaagggaa agt ccaatgt attaattgtt Caaagatgaa ccgatatgga tiggtgtgc.ca 342O taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt 3480 atgcaaggag ctittaaaaaa got catgitaa agaagagtaa aaagaaaaaa taatttattt 354 O attaatttaa tattgagagt gcc.gacacag tatgcactaa aaaatatatic tdtggtgtag 36OO tgagc.cgata caaaaggata gttcact cqca ttitt cataat a catct tatgttatgattat 366 O gtgtcggtgg gaCttcacga caaaaccca caataaaaaa agagttcggg gtagggittaa 372 O gCatagttga ggcaactaala caatcaagct aggatatgca gtagcagacic gta aggt ct 378 O tgtttaggtg togttgtaata catacgct at taagatgtaa aaatacggat accaatgaag 384 O ggaaaagtat aatttittgga tigtagtttgt ttgttcatct atgggcaaac tacgt.ccaaa 3900 gcc.gtttcca aatctgctaa aaagtatat c ctittctaaaa toaaagt caa gitatgaaatc 396 O ataaataaag tittaattittg aagttatt at gat attatgt ttittct atta aaataaatta 4 O2O agtatataga at agtttaat aatagtatat acttaatgtgataagtgtct gacagtgtca 4 O8O

Cagaaaggat gattgttatggattataagc ggc.cggc.cag tiggcaagtt gaaaaattica 414 O caaaaatgtg gtataatat c tttgttcatt agagcigataa acttgaattt gagagggaac 42OO ttagatggta tittgaaaaaa ttgataaaaa tagttggaac agaaaagagt attittgacca 426 O c tactittgca agtgtacctt gtacctacag catgaccgtt aaagtggata t cacacaaat 432O aaaggaaaag ggaatgaaac tatat cotgc aatgctittat tatattgcaa tdattgtaaa 438 O cc.gc.cattca gagtttagga C9gcaatcaa t caagatggit gaattgggga tatatgatga 4 44 O gatgatacca agctatacaa tattt cacaa tdatactgaa acatttitcca gcc tittggac 4500 tgagtgtaag totgactitta aat catttitt agcagattat gaaagtgata cqcaacggta 456 O tggaaacaat catagaatgg aaggaaagcc aaatgct cog gaaaacattt ttaatgitatic 462O tatgat accq toggtcaacct tcgatggctt taatctgaat ttgcagaaag gatatgatta 468O tittgattic ct attitt tacta toggggaaata ttataaagaa gataacaaaa ttatact tcc 474. O tittggcaatt caagttcatc acgcagtatgtgacggattt cacatttgcc gttttgtaaa 48OO cgaattgcag gaattgataa atagittaact tcaggtttgt ctd taactaa aaacaagtat 486 O ttaa.gcaaaa a catcgtaga aatacggtgt tttttgttac cctaagttta aactic cttitt 492 O tgataatcto atgaccaaaa toccittaacg tdagttitt.cg titccact gag cqt cagaccc 498O cgtagaaaag atcaaaggat cittcttgaga t cotttittitt citgcgcgitaa totgctgctt 5040

US 2016/0348087 A1 Dec. 1, 2016 23

- Continued t cct ctacag gtggcatc.cc acaggitttat gat cittatag gagaatatga ctitttacata 54 O gttggagaaa aatgtattga agtaaatcac aattt attag gagtaaaggg agcgt.ctatt 6OO tccgatataa aagaagttta ttct catagt caa.gcattta togcaaagtag taaatttctg 660 gagaaacaca agaattggaa gctaaatc cc tattittaata cagotagaag togccaaatat 72 O ataagtgagc aaaatgttaa gagtaaagct gct at agcaa gtaaaaatgc agcaaaactt 78O tatggactitg atataataga aaaaaatata aattataa.ca gcaataatta cactagattit 84 O ataataatag gaaaaaatat agaaagtgat aaacaacgtg acaagataag tatattgatt 9 OO actctg.ccgc atgaac cagg aactic tittat aatgttittga agtattt coa tdaaaataac 96.O ttgaatatga ctaaaataga gttcaaggcct ataataaata aatcc tiggca gtactitctitt 1 O2O tacattgatt ttaatggaaa tattatggat aaagatacta gg tatgctitt aaatggtata 108 O gaagaagaaa gogcat attt taalacttittggggaattaca aaggagattgtttittag 1137

<210s, SEQ ID NO 11 &211s LENGTH: 378 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum

<4 OOs, SEQUENCE: 11 Met Glu Asp Lieu. Glu Tyr Lieu. Arg Asp Glu Ile Asn Lys Ile Asp Llys 1. 5 1O 15 Glu Met Ile Glu Lieu Phe Glu Lys Arg Ala Llys Val Ser Arg Llys Val 2O 25 3O Ala Glu Tyr Lys Met Glu Asn. Ser Met Asp Ile Lieu. Asp Llys Ser Arg 35 4 O 45 Glu Glu Glu Val Ile Llys Val Asn Lieu Lys Asn Lieu Lys Asp Llys Ser SO 55 6 O Ile Lys Asp Glu Thir Lys Ile Phe Lieu Lys Asn. Wal Met Glu Ile Ser 65 70 7s 8O Arg Asn. Ile Glin Lys Arg Glu Phe Lys Glin Ser Ser Lys Ser Ser Glu 85 90 95 Ile Llys Pro Lys Gly Glin Asn. Ser Asp Lieu. Phe Lys Ile Gly Phe Glin 1OO 105 11 O Gly Val Pro Ala Ser Phe Ser His Glu Ala Leu Lleu. Glu Tyr Phe Gly 115 12 O 125 Asn Glu Ser Glu Ala Lieu. Asn. Phe Glu Ser Phe Lys Asp Val Phe Glu 13 O 135 14 O Ala Lieu Lys Asn Gly Ala Ile Llys Tyr Gly Val Lieu Pro Ile Glu Asn 145 150 155 160 Ser Ser Thr Gly Gly Ile Pro Glin Val Tyr Asp Lieu. Ile Gly Glu Tyr 1.65 17O 17s Asp Phe Tyr Ile Val Gly Glu Lys Cys Ile Glu Val Asn His Asn Lieu 18O 185 19 O

Lieu. Gly Wall Lys Gly Ala Ser Ile Ser Asp Ile Lys Glu Val Tyr Ser 195 2OO 2O5

His Ser Glin Ala Phe Met Glin Ser Ser Llys Phe Lieu. Glu Lys His Lys 21 O 215 22O

Asn Trp Llys Lieu. ASn Pro Tyr Phe Asn. Thir Ala Arg Ser Ala Lys Tyr 225 23 O 235 24 O

Ile Ser Glu Glin Asn. Wall Lys Ser Lys Ala Ala Ile Ala Ser Lys Asn US 2016/0348087 A1 Dec. 1, 2016 24

- Continued

250 255

Ala Ala Luell Gly Lell Asp Ile Ile Glu Asn Ile Asn Tyr 26 O 265 27 O

Asn. Ser Asn. Asn Thir Arg Phe Ile Ile Ile Gly Lys Asn Ile Glu 28O 285

Ser Asp Glin Arg Asp Llys Ile Ser Ile Luell Ile Thir Luell Pro His 29 O 295 3 OO

Glu Pro Gly. Thir Lieu. Tyr Asn Val Lieu. Llys Tyr Phe His Glu Asn Asn 3. OS 310 315 32O

Lieu. Asn Met Thr Lys Ile Glu Ser Arg Pro Ile Ile Asn Lys Ser Trp 3.25 330 335

Glin Tyr Phe Phe Ile Asp Phe Asn Gly Asn. Ile Met Asp Asp 34 O 345 35. O

Thir Tyr Ala Lieu. Asn Gly Ile Glu Glu Glu Ser Ala Tyr Phe Lys 355 360 365

Lell Luell Gly Asn Lys Gly Asp Phe 37 O 375

1. A genetically engineered C1-fixing bacterium capable stituted with a carboxyl group or carboxylate anion and of producing at least one chorismate-derived product, further substituted with one or more OH groups and/or one wherein the bacterium comprises at least one of or more NH groups. a. an exogenous chorismate pyruvate lyase (EC 4.1.3.4.0), 13. The bacterium of claim 1, wherein the chorismate b. an exogenous isochorismate synthase (EC 5.4.4.2). derived product is selected from the group consisting of c. an exogenous isochorismate pyruvate lyase (EC 4.2. para-hydroxybenzoic acid, salicylate, 2-aminobenzoate, 99.21), and dihydroxybenzoate, 4-hydroxycyclohexane carboxylic acid, d. a prephenate synthase (EC 5.4.99.5) comprising a and salts and ions thereof. disruptive mutation. 14. The bacterium of claim 1, wherein the bacterium 2. The bacterium of claim 1, wherein the bacterium is a expresses a chorismate pyruvate lyase of ubiC and produces Clostridium bacterium capable of producing at least one a chorismate-derived product of para-hydroxybenzoic acid. chorismate-derived product by fermentation of a gaseous substrate. 15. The bacterium of claim 1, wherein the bacterium 3. The bacterium of claim 1, wherein the chorismate expresses an isochorismate synthase of pchA and an isocho pyruvate lyase is ubiC. rismate pyruvate lyase of pchB and produces a chorismate 4. The bacterium of claim 1, wherein the isochorismate derived product of salicylate. synthase is pchA. 16. The bacterium of any one of claims 14 and 15, 5. The bacterium of claim 1, wherein the isochorismate wherein the bacterium further expresses a feedback-insen pyruvate lyase is pchB. sitive DAHP synthase. 6. The bacterium of claim 1, wherein the prephenate 17. The bacterium of claim 1, wherein the bacterium synthase is phe A. comprises a prephenate synthase comprising a disruptive 7. The bacterium of claim 1, wherein the disruptive mutation and produces a chorismate-derived product of mutation reduces or eliminates the expression or activity of 2-aminobenzoate, 2,3-dihydroxybenzoate, or 4-hydroxycy the prephenate synthase. clohexane carboxylic acid. 8. The bacterium if claim 7, wherein the bacterium 18. The bacterium of claim 1, wherein the bacterium produces a reduced amount of prephenate or prephenate produces at least one chorismate-derived product not pro derived products compared to a parental bacterium. duced by a parental bacterium. 9. The bacterium of claim 7, wherein the bacterium produces Substantially no tyrosine or phenylalanine. 19. The bacterium of claim 1, wherein the bacterium 10. The bacterium of claim 1, wherein the bacterium produces a greater amount of at least one chorismate-derived comprises at least one nucleic acid encoding at least one of product than a parental bacterium. a. the exogenous chorismate pyruvate lyase, 20. The bacterium of claim 1, wherein the bacterium is b. the exogenous isochorismate synthase, derived from a parental bacterium selected from the group c. the exogenous isochorismate pyruvate lyase, and consisting of Clostridium autoethanogenium, Clostridium d. the prephenate synthase comprising a disruptive muta liungdahli, and Clostridium ragsdalei. tion. 21. The bacterium of claim 20, wherein the Clostridium 11. The bacterium of claim 10, wherein the nucleic acid autoethanogenium is Clostridium autoethanogenium is codon optimized for expression in Clostridium. DSM23693. 12. The bacterium of claim 1, wherein the chorismate 22. The bacterium of claim 1, wherein the gaseous sub derived product comprises a 6-membered carbon ring Sub strate comprises at least one of CO, CO, and H. US 2016/0348087 A1 Dec. 1, 2016 25

23. A method of producing a fermentation product, com prising fermenting the bacterium of claim 1 in the presence of a gaseous Substrate to produce a fermentation product. 24. The method of claim 23, wherein the gaseous sub strate comprises at least one of CO, CO, and H. k k k k k