US 201701 74731A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0174731 A1 Kennedy et al. (43) Pub. Date: Jun. 22, 2017 (54) TOXIC PEPTIDE PRODUCTION, PEPTIDE (60) Provisional application No. 61/608,921, filed on Mar. EXPRESSION IN PLANTS AND 9, 2012, provisional application No. 61/698,261, filed COMBINATIONS OF CYSTEINE RCH on Sep. 7, 2012, provisional application No. 61/729, PEPTIDES 905, filed on Nov. 26, 2012. (71) Applicant: Vestaron Corporation, Kalamazoo, MI Publication Classification (US) (51) Int. C. C07K I4/435 (2006.01) (72) Inventors: Robert M. Kennedy, Dexter, MI (US); AOIN 63/02 William Tedford, Calgary (CA): (2006.01) Christopher Hendrickson, Royal Oak, (52) U.S. Cl. MI (US); Robert Venable, Lawrence, CPC ........ C07K 14/43518 (2013.01); A0IN 63/02 MI (US); Catherine Foune, Gobles, MI (2013.01); C07K 2319/55 (2013.01) (US); John McIntyre, Alto, MI (US); (57) ABSTRACT Alvar Carlson, Kalamazoo, MI (US); New insecticidal proteins, nucleotides, peptides, their Lin Bao, Portage, MI (US) expression in plants, methods of producing the peptides, new processes, production techniques, new peptides, new (73) Assignee: Vestaron Corporation, Kalamazoo, MI formulations, and new organisms, a process which increases (US) the insecticidal peptide production yield from yeast expres sion systems. The present invention is also related and (21) Appl. No.: 15/390,153 discloses selected endotoxins we call cysteine rich insecti cidal peptides (CRIPS) which are peptides derived from (22) Filed: Dec. 23, 2016 Bacillus thuringiensis (Bt) and their genes and endotoxins in combination with toxic peptides known as Inhibitor Cystine Knot (ICK) genes and peptides as well as with other types Related U.S. Application Data of insecticidal peptides such as trypsin modulating Oostatic (62) Division of application No. 14/383.841, filed on Sep. factor (TMOF) peptide sequences used in various formula 8, 2014, now Pat. No. 9,567,381, filed as application tions and combinations: of both genes and peptides, useful No. PCT/US2013/030042 on Mar. 8, 2013. for the control of insects. Patent Application Publication Jun. 22, 2017 Sheet 1 of 22 US 2017/0174731 A1 ig. i. Patent Application Publication Jun. 22, 2017. Sheet 2 of 22 US 2017/0174731 A1 *ig. 2. Patent Application Publication Jun. 22, 2017. Sheet 3 of 22 US 2017/0174731 A1 8x8 Patent Application Publication Jun. 22, 2017. Sheet 4 of 22 US 2017/0174731 A1 sersg Sig iriker &rsg Crip irker 3. Fig. 4 Patent Application Publication Jun. 22, 2017 Sheet 5 of 22 US 2017/0174731 A1 Fig. 8 Patent Application Publication Jun. 22, 2017 Sheet 6 of 22 US 2017/0174731 A1 fig. 8 Patent Application Publication Jun. 22, 2017 Sheet 7 of 22 US 2017/0174731 A1 Patent Application Publication Jun. 22, 2017 Sheet 8 of 22 US 2017/0174731 A1 Apo : Yic ki. 8. Fig. 8 Patent Application Publication Jun. 22, 2017 Sheet 9 of 22 US 2017/0174731 A1 3,8 38. 8.2 Rig. 9 Patent Application Publication Jun. 22, 2017. Sheet 10 of 22 US 2017/0174731 A1 Group A Group B Trypsin Cleavage Non-trypsin Cleavage Jun a 3:omega jun a 3::Omega Start with 26mi, split into 3m each treatment of 12g/t or 2g/m. hybrid Spun for 2hr at 3200g in 10kD cutoff fitter, each 3.8m. .4 m reterstate 1. retentate 12.5 in filtrate 2.5 mi filtrate Took 12.5 ml filtrate to spun for 12 hr at 3200g in 3kD Cutoff filter i.3 in reterstate 1. mi retentate 1.0 filtrate 12 mi filtrate Fig. 10 Patent Application Publication Jun. 22, 2017. Sheet 11 of 22 US 2017/0174731 A1 A 20 -3 B SO3 C 500 25 - Retention time (min) Fig. i. Patent Application Publication Jun. 22, 2017. Sheet 12 of 22 US 2017/0174731 A1 8orinatized Yield costigatisor o 8:38.88 Yek (sing:...A ig. 3 Patent Application Publication Jun. 22, 2017. Sheet 13 of 22 US 2017/0174731 A1 2. U-2 K, actis strains & Native K. tactis straits r-rearrera-e-M { 88 SR 88 33 88:888& 8:king of 8.8xxis tassisrepart: ($ ig. 3 Patent Application Publication Jun. 22, 2017. Sheet 14 of 22 US 2017/0174731 A1 :80::sefiy injection; 808ssayg & native U. 27.68 pmi/g 2: 8,823 poig 8 2. tor - 8 & is S. 3 & 8 & 8 & {{ ---& S iose origif Patent Application Publication Jun. 22, 2017. Sheet 15 of 22 US 2017/0174731 A1 8, is igwiec (3.88 . .x. Native i yiek 0.08 s {3,34 2 {{{3- X rrrrrrrrrrrrr. s 2. 3. 88: 8. 3: 2. 3×{{ 38 Yieid (nigi. $ig. 3 Patent Application Publication Jun. 22, 2017. Sheet 16 of 22 US 2017/0174731 A1 383 s 4: { :irrirrigorooocoorj+2 f. pastoris strains o r & M. 2 ... Native ifresssssw.ras.88.888&88&&&&Waa-44SWP pastoris strains: 88: s s: too *: & 3. 80 s & a- & s 88: as so set ''' & e . x X x * * * **** & & 888 & 8.8 & 8:88. 3: 8& 88: O percert: 8king of 8, pastoris transferrants (8% ig. 6 Patent Application Publication Jun. 22, 2017. Sheet 17 of 22 US 2017/0174731 A1 Normalized Yield Distribution Av3+2 strains www.swaxss'''''''.....:Native Av3 stairs s S Norralized Yield (At.sec?i Fig. 3; Patent Application Publication Jun. 22, 2017. Sheet 18 of 22 US 2017/0174731 A1 &Six 8. AyS-28:a:::::: 20 :aixi sistasis. 8 58. 80 { 33 4R 30: 80 3. eft:8:tie &nking of 8.3actis tra:story.arts & Fig. 8 Patent Application Publication Jun. 22, 2017. Sheet 19 of 22 US 2017/0174731 A1 24 hour Percent wortality Vs, Age of larvae 333 x. 834-33: 3;wa& 888 its : 38 or 3 was rest its 72 hour larvae Results 7 : a 36----''''''' Hour larvae Resuits" Patent Application Publication Jun. 22, 2017. Sheet 20 of 22 US 2017/0174731 A1 18, 24 and 48 Hours %. Mortality vs. Treatment using 72 hour larvae 0.00 18 hours % Mortality Results 900 --------- 3O.OO 8:324 hours %.% Mortality Results 70.00 - 3 48 hours 9%. Motality Resuits 60,00 --------M.-- S0.00 40,30 30.00 20,00 0.00 0.00 - Untreated 2) 0.125% MSO 4} 10ppt SDP 5} 0.3mg/ml. 7)0.3mg/m. Control Water 1234.65- Dipel Dipe DF + 10ppt i. 0.25% MSO SOp 2.34605 --0.25% MSO Fig. 20 Patent Application Publication Jun. 22, 2017 Sheet 21 of 22 US 2017/0174731 A1 Feeding Damage with 120hr Crya resistart diamotackX 8 oth 3.30 po 8 C tige &y: 3.36: 3: Aw3 Fig. 3 Patent Application Publication Jun. 22, 2017. Sheet 22 of 22 US 2017/0174731 A1 111212 Pxylostella HD-1 Res. %. Mortality vs. Treatment on Romaine lettuce 8 24 Hour 90 8 48 hour 80 70 60 50 40 30 20 O 1) Jntreated 2) 0.15mg/rn Dipei 3} 1 ppt SDP 609 iii. 4) ppt. SDP 609 Control Water EF fil Fi; +0.15mg/ml Dipe 3F Fig. 22 US 2017/0174731 A1 Jun. 22, 2017 TOXIC PEPTIDE PRODUCTION, PEPTIDE exception, peptides derived from Bacillis thuringiensis or EXPRESSION IN PLANTS AND Bt. And now there is concern over rising insect resistance to COMBINATIONS OF CYSTEINE RICH Bt proteins. PEPTIDES 0007 Bt proteins, or Bt peptides, are effective insecti cides used for crop protection in the form of both plant REFERENCE TO RELATED APPLICATIONS incorporated protectants and foliar sprays. Commercial for mulations of Bt proteins are widely used to control insects 0001. This application is a divisional application of U.S. at the larval stage. ICK peptides include many molecules patent application Ser. No. 14/383.841, filed on Sep. 8, 2014, that have insecticidal activity. Such ICK peptides are often which is a 371 of PCT Application No. PCT/US2013/ toxic to naturally occurring biological target species, usually 030042, filed Mar. 8, 2013, which claims the benefit of insects or arachnids of some type. Often ICK peptides can earlier filed U.S. Provisional Application Ser. No. 61/608, have arthropod origins such as the venoms of Scorpions or 921, filed on Mar. 9, 2012, U.S. Provisional Application Ser. spiders. Bt is the one and only source organism of commer No. 61/644.212, filed on May 8, 2012, U.S. Provisional cially useful insecticidal peptides. Other classes and types of Application Ser. No. 61/698.261, filed on Sep. 7, 2012, and potential peptides have been identified, such as Trypsin U.S. Provisional Application Ser. No. 61/729,905, filed Nov. modulating Oostatic factor (TMOF) peptides. TMOF pep 26, 2012, the entire contents of which are incorporated tides have to be delivered to their physiological site of action herein by reference. in various ways, and TMOF peptides have been identified as a potential larvicides, with great potential, see D. Borovsky, SEQUENCE LISTING Journal of Experimental Biology 206, 3869-3875, but like 0002 This application incorporates in its entirety the nearly all other insecticidal peptides, TMOF has not been Sequence Listing entitled “FAM X US 225312 361200 commercialized or widely used by farmers and there are SEQ LIST 2014 0908 ST25.txt” (5,585,417 bytes), reasons for this. which was created on Mar. 8, 2013, and filed electronically 0008. The ability to successfully produce insecticidal herewith. peptides on a commercial scale, with reproducible peptide formation and folding, at a reasonable and economical price, can be challenging. The wide variety, unique properties and FIELD OF THE INVENTION special nature of insecticidal peptides, combined with the 0003 New insecticidal proteins, nucleotides, peptides, huge variety of possible production techniques, can present their expression in plants, methods of producing the pep an overwhelming number of approaches to peptide applica tides, new processes, production techniques, new peptides, tion and production, but few, if any, are commercially new formulations, and combinations of new and known Successful.