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(12) Patent Application Publication (10) Pub. No.: US 2016/0102315 A1 Shasky Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2016/0102315 A1 Shasky Et Al US 201601 02315A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0102315 A1 Shasky et al. (43) Pub. Date: Apr. 14, 2016 (54) METHODS FOR PRODUCING Publication Classification POLYPEPTIDES IN ENZYME-DEFICIENT MUTANTS OF FUSARUMVENENTATUM (51) Int. Cl. CI2N 15/80 (2006.01) (71) Applicant: NOVOZYMES, INC., Davis, CA (US) CI2P 2L/00 (2006.01) (52) U.S. Cl. (72) Inventors: Jeffrey Shasky, Davis, CA (US); Wendy CPC ................. CI2N 15/80 (2013.01); C12P21/00 Yoder, Billingshurst (GB) (2013.01) (57) ABSTRACT (73) Assignee: NOVOZYMES, INC., Davis, CA (US) The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent (21) Appl. No.: 14/974,879 Fusarium venematum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a poly nucleotide encoding the polypeptide and one or more (sev (22) Filed: Dec. 18, 2015 eral) genes selected from the group consisting of pyrC, amy A, and alpA, wherein the one or more (several) genes are Related U.S. Application Data modified rendering the mutant strain deficient in the produc tion of one or more (several) enzymes selected from the group (63) Continuation of application No. 14/168,766, filed on consisting of orotidine-5'-monophosphate decarboxylase, Jan. 30, 2014, now Pat. No. 9.255,275, which is a alpha-amylase, and alkaline protease, respectively, compared continuation of application No. 13/121.254, filed on to the parent Fusarium venematum strain when cultivated Jun. 1, 2011, now Pat. No. 8,647.856, filed as applica under identical conditions; and (b) recovering the polypep tion No. PCT/US2009/059039 on Sep. 30, 2009. tide from the cultivation medium. The present invention also (60) Provisional application No. 61/101,250, filed on Sep. relates to enzyme-deficient mutants of Fusarium venenatum 30, 2008. strains and methods for producing Such mutants. Patent Application Publication Apr. 14, 2016 Sheet 1 of 35 US 2016/01 02315 A1 EcoRI (927) Stul (1440) pyrg promoter pDM156.2 7084 bp EcoRI (4849) \, Stul (2321) EcoRV (41N 18, 97 fs. -- EcoRV (3899) Fig. 1 Patent Application Publication Apr. 14, 2016 Sheet 2 of 35 US 2016/01 02315 A1 Fig. 2 Patent Application Publication Apr. 14, 2016 Sheet 3 of 35 US 2016/01 02315 A1 PEmY23 6722 bp EcoRI (4495) F. Wenenatum A. oryzae pyrg repeat a---------a hpt Fig. 3 Patent Application Publication Apr. 14, 2016 Sheet 4 of 35 US 2016/01 02315 A1 assesassissix. EcoRI (536) A beta lactamase NN Tris 3' UTR W \ BglII (1779) pWTY1470-19-07 pUCOrigin 8117 bp N --- - Ti55 UR EcoRI (4082 Fig. 4 Patent Application Publication Apr. 14, 2016 Sheet 5 of 35 US 2016/01 02315 A1 beta lactamase pWTY1515-2-01 tri5 repeat pUCOrigin 8117 bp & / By II (2386) \ // tri5 repeat Il w ^ Y. s ri55 flank BstZ17 (3987) Fig. 5 Patent Application Publication Apr. 14, 2016 Sheet 6 of 35 US 2016/01 02315 A1 Xhof (22) Promoter gpd Bgll (1054) Beta lactamase HSV1 tik HindIII (3076 Fig. 6 Patent Application Publication Apr. 14, 2016 Sheet 7 of 35 US 2016/01 02315 A1 Xhol (22) Promoter gpd Beta lactamase HSV1 k HindII (3080) Fig. 7 Patent Application Publication Apr. 14, 2016 Sheet 8 of 35 US 2016/01 02315 A1 Pst (1271) HSV-1 tik Gene Fig. 8 Patent Application Publication Apr. 14, 2016 Sheet 9 of 35 US 2016/01 02315 A1 trpCTerminator HSV-1 tik gpd Promoter Fig. 9 Patent Application Publication Apr. 14, 2016 Sheet 10 of 35 US 2016/01 02315 A1 pUCOrigin siccias. BamH (253) picillin pJfyS1540-75-5 5805 bp Kanamycin - Bgll (21.77) Bgll (3158 i-roin Fig. 10 Patent Application Publication Apr. 14, 2016 Sheet 11 of 35 US 2016/01 02315 A1 BamH (200) a'-? sf EcoRITi53' (536) UTR tri55 Repeat EcoRI (2417) pJfyS1579-1-13 10041 bp EcoRI (60067 N / -\O Bstz17 (5911 “I Bgll (4310)( Ti55 Fig.11 Patent Application Publication Apr. 14, 2016 Sheet 12 of 35 US 2016/01 02315 A1 pUCOrigin - BamH (253) Ampicillin pJfyS1579-8-6 6379 bp Kanamycin Bgll (3732 Y- st Bgll (2751) f1 Origin T7Promoter Fig. 12 Patent Application Publication Apr. 14, 2016 Sheet 13 of 35 US 2016/01 02315 A1 BamH (200) tris 3' UTR pUcOR pJfyS1579-21-16 12539 bp Fig. 13 Patent Application Publication Apr. 14, 2016 Sheet 14 of 35 US 2016/01 02315 A1 pAllo 1492-24 3637 bp Nicol (1521) Fig. 14 Patent Application Publication Apr. 14, 2016 Sheet 15 of 35 US 2016/01 02315 A1 hpt OFR(hygr beta lactamase pJfyS1579-35-2 5515 bp / Big II (2124) is...:* 328 Repeat 2 kanimycin resistance an Sbf (2375) Fig. 15 Patent Application Publication Apr. 14, 2016 Sheet 16 of 35 US 2016/01 02315 A1 Asci (11) Repeat 1 pJfyS1579-41-11 8014 bp g g A. Fig. 16 Patent Application Publication Apr. 14, 2016 Sheet 17 of 35 US 2016/01 02315 A1 pUC ori Not (11) ? pyrg 5' flank 3. old-Repeat 1 beta lactamas pJfyS1604-55-13 kanimycin resistan 9289 bp NaI(6137) : pyrg 3'Flank N Repeat 2 N. Fig. 17 Patent Application Publication Apr. 14, 2016 Sheet 18 of 35 US 2016/01 02315 A1 FWen R P'u-6 s hpt pJfyS1579-93-1 --EcoR (2155) 8746 bp i f; Fig. 18 Patent Application Publication Apr. 14, 2016 Sheet 19 of 35 US 2016/01 02315 A1 Not I (11) & amyA5 flank beta lactamase 1 s-sanel (1237) kanimycin resistance pJfyS1604-17-2 9961 bp Fig. 19 Patent Application Publication Apr. 14, 2016 Sheet 20 of 35 US 2016/01 02315 A1 Beta lactanase Not (416) t EcoRI (423) M BspU11 (2816) FOSP387 Terminator Fig. 20 Patent Application Publication Apr. 14, 2016 Sheet 21 of 35 US 2016/01 02315 A1 Scal (9134 gaAPromoter Lactose Oxidase Fig. 21 Patent Application Publication Apr. 14, 2016 Sheet 22 of 35 US 2016/01 02315 A1 A A. glaA promoter f; SnaB (1874) pEJG65 9541 bp BARgene Candidaantarcticalipase A Afi (5152 Fig. 22 Patent Application Publication Apr. 14, 2016 Sheet 23 of 35 US 2016/01 02315 A1 betalactamase EcoR Site (423) W. Paciste (278) Pad Site (2802) Fig. 23 Patent Application Publication Apr. 14, 2016 Sheet 24 of 35 US 2016/01 02315 A1 beta Notl (416) - ffs. EcoRI (423) ^ f FV. AMGPromoter f E. pEJG49 Not (6930 9189 bp BAR EcoRI (4918 -- as:F.O. SP387Paci Terminator (3859) Fig. 24 Patent Application Publication Apr. 14, 2016 Sheet 25 of 35 US 2016/01 02315 A1 pEmY15 9166 bp Fig. 25 Patent Application Publication Apr. 14, 2016 Sheet 26 of 35 US 2016/01 02315 A1 glaApromoter SnaBI (1874) AfII (4800 SP387e Fig. 26 Patent Application Publication Apr. 14, 2016 Sheet 27 of 35 US 2016/01 02315 A1 Scal (9274). beta lactamase F. venenatum py > Afi (5152) SP387 terminator Fig. 27 Patent Application Publication Apr. 14, 2016 Sheet 28 of 35 US 2016/01 02315 A1 beta lactamase Not (416) frt Egli (650) gaA promoter Not I (7635 pDM258 9879 bp Lactose oxidase Fig. 28 Patent Application Publication Apr. 14, 2016 Sheet 29 of 35 US 2016/01 02315 A1 Fig. 29 Patent Application Publication Apr. 14, 2016 Sheet 30 of 35 US 2016/01 02315 A1 Fig. 30 Patent Application Publication Apr. 14, 2016 Sheet 31 of 35 US 2016/01 02315 A1 Asci (11) pJfyS1698-65-15 hpt OFR(hygr) kanimycin resistance 9060 bp Fig. 31 Patent Application Publication Apr. 14, 2016 Sheet 32 of 35 US 2016/01 02315 A1 pUCori Natl (19) efs 5alpAflank beta - / 1 - Repeat 1 s kanimycin resistance pJfyS1698–72-10 9996 bp 8 Fig. 32 Patent Application Publication Apr. 14, 2016 Sheet 33 of 35 US 2016/01 02315 A1 WY1OS-1-36 fyS1763-30-29 gfyS1763-30-46 JfyS1763-30 (alpA-) (alpA-} 46 (alpA-) Fig. 33 Patent Application Publication Apr. 14, 2016 Sheet 34 of 35 US 2016/01 02315 A1 pJfyS1879-32-2 kanimycin 9026 bp Not I (5875) 3 dips1 flank Fig. 34 Patent Application Publication Apr. 14, 2016 Sheet 35 of 35 US 2016/01 02315 A1 Elim Not (19) 5 dips1 flank hpt ORF (hygr) pJfyS111 97.52 bp Noti (6601 \ 3 dist flank N.N Fig. 35 US 2016/01 02315 A1 Apr. 14, 2016 METHODS FOR PRODUCING to the parent Fusarium venematum strain when cultivated POLYPEPTIDES IN ENZYME-DEFICIENT under identical conditions; and MUTANTS OF FUSARUMVENENTATUM 0011 (b) recovering the polypeptide from the cultivation medium. CROSS-REFERENCE TO RELATED 0012. In one aspect of the methods of producing a APPLICATIONS polypeptide, the mutant strain further comprises one or both 0001. This application is a continuation of U.S. applica of the genes tri5 and dips 1, wherein the one or both genes are tion Ser. No. 14/168,766, filed on Jan. 30, 2014, which is a modified rendering the mutant strain deficient in the produc continuation of U.S. application Ser. No. 13/121,254, filed on tion of one or both enzymes trichodiene synthase and cyclo Jun. 1, 2011, now U.S. Pat. No. 8,647.856, which is a 35 hexadepsipeptide synthetase, respectively, compared to the U.S.C. S371 national application of PCT/US2009/059039, parent Fusarium venenatum Strain when cultivated under filed on Sep. 30, 2009, which claims the benefit of U.S. identical conditions. Provisional Application No. 61/101,250, filed on Sep. 30, 0013 The present invention also relates to mutants of a 2008, which applications are fully incorporated herein by parent Fusarium venematum strain, comprising a polynucle otide encoding a polypeptide and one or more (several) genes reference.
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