GONZALES CAGUIAT RODANTE (US)
NMI FIORESI CAROL (US)
SCHMIDT BRIAN F (US)
VAN KIMMENADE ANITA (US)
ZHU CHAO (US)
| WO2003083125A1 | 2003-10-09 |
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RASHID MUHAMMAD HARUNUR ET AL: "FlaD (sinR) mutations affect SigD-dependent functions at multiple points in Bacillus subtilis", JOURNAL OF BACTERIOLOGY, vol. 178, no. 22, 1996, pages 6640 - 6643, XP055249372, ISSN: 0021-9193
OLMOS J ET AL: "EFFECTS OF THE SINR AND DEGU32 (HY) MUTATIONS ON THE REGULATION OF THE APRE GENE IN BACILLUS SUBTILIS", MOLECULAR AND GENERAL GENETICS, SPRINGER VERLAG, BERLIN, DE, vol. 253, no. 5, 1 January 1997 (1997-01-01), pages 562 - 567, XP000990077, ISSN: 0026-8925, DOI: 10.1007/S004380050358
C. A. VOIGT: "The Bacillus subtilis sin Operon: An Evolvable Network Motif", GENETICS, vol. 169, no. 3, 15 November 2004 (2004-11-15), US, pages 1187 - 1202, XP055249343, ISSN: 0016-6731, DOI: 10.1534/genetics.104.031955
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[0001] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62/092,461, filed December 16, 2014, the entirety of which is hereby incorporated by reference. FIELD OF THE INVENTION
[0002] The present invention is generally related to the fileds of molecular biology, cell biology, microbiology, genetics and protein production. More particularly, in various embodiments disclosed herein, the invention is directed to Gram positive bacterial cells having genetic alterations that result in increased expression of a protein of interest and methods of making and using such cells. REFERENCE TO SEQUENCE LISTING
[0003] The contents of the electronic submission of the text file Sequence Listing, which is named NB40772-WO-PCT_SequenceListing.txt, was created on October 29, 2015 and is 11KB in size, is incorporated herein by reference. BACKGROUND OF THE INVENTION
[0004] Genetic engineering has allowed the improvement of microorganisms used as industrial bioreactors, cell factories and in food fermentations. Gram-positive organisms, including a number of Bacillus species, are used to produce a large number of useful proteins and metabolites (see, e.g., Zukowski, “Production of commercially valuable products,” In: Doi and McGlouglin (eds.) Biology of Bacilli: Applications to Industry, Butterworth-Heinemann, Stoneham. Mass pages 311-337, 1992). Common Bacillus species used in industry include B. licheniformis, B. amyloliquefaciens and B. subtilis. Because of their GRAS (generally recognized as safe) status, strains of these Bacillus species are natural candidates for the production of proteins utilized in the food and pharmaceutical industries. Examples of proteins produced in Gram-positive organisms include enzymes such as α-amylases, neutral proteases, alkaline (or serine) proteases and the like.
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1 5 t- SediEon mQ s ea IDqinu) Ne On ct 3A:h6e0 p p ( FrwrNooi E bGpANoG cGlCAdoAdGE uGoAnSGndAA)eAGrAltiiE 1ntTAah5eQCAg; sCUAt w SoCA
DG SA p7A oGHAGSrQEfS -iGSTmGC tSQMACQhCIeVVeCTGA IDrPPDACY sG fSYSGCoiTn NCSGrTETGRGOHC aAVTScVrTPTGVmA: gAwSANADAa4ANACDAeCAAQaPtApPNAVTVAi FnLTTGorlG TIF -AANCTiKdIGeofTAnKGTA 3K eoi Sld,pEt sy cQeponed IsDo ceno N s)dOtrinai:nu1igntRehd - sn se w (c siALQAAtwq
gKA5SGCGaIaVG’HTICcGcLGC-GETgVtGGATCWtLaAAAAcHAcGtDAAAa VItVtGGATgAAcAgSATAgGNaPaLT 2C-GGWGGGaTCNcLITaS5tYT3QGAIACTaVMcNTNaAgAT’YAGITAAA AtADtNMAtStVGSCTNAGAAtAS rA STAoTATtECTeGGQTiAnAAAA IA sTTDAeATCCq NAGAuAOAGGeAC 1GAi:CnACnA2T0GGCcRCAGGAT -eCTACTCCA pTC sGgV
AT ECASPAKTGALSAACTCGVTVCSGGTIAGGATPACSSCASVTGTSCGGGVCSCSATVCAG AGCTPTTAVANAVAAACAAVCGADAQTVGATGCCGTK TLSVRTACNAGTAGYYCKCDS ASTMAEGLGSGITKCSCIAGCVGETEAQTSALCRARAAAAANAICVGEALAKALTKTQGQ ADCATKATCICYAVGKATTGTSGNGQTRCSGGCLTVTYAPAFGKGVTYCLAAALASALGE ASAH NGAT
TQ SAI CDGE p oVTAGA
CEGFrS
CEEL TEfS
GKiCT
TRmGTF
TK tQSI
TALhAASAS
TECHeK
CeQTC
AV IMAA
CFrSH
CEADS AR sGAA
TL fVP
ATToiCTA
AD NnSCGN
GDTEArTL
AARTOAPP GYTY aWTPGY
CGMCVcvACTHAT
AQCKm:TE
CD gTMV
TTDCaA5G
TTeGGGGCCVAG
5CSCKTSeLNA
AGTTStpR
TrTNTAA
CSGKiC RGAnA
GWsAC
CQo TlDTPTSC
AQTCECiKLGA
TGCKeeGTfeG
TRnTLCVGQ
GKTLTWGiA
GTATGTTACCACGTGCGGGGGAGGAT2GAAATGT0AGTATGCTAAA ATAGGCAGGC
c5SaKa’LaGa-EaLaNtIa cTtNgtTgVccTaQ-gKaAa 33gLcFaA5a’aGgYgAgtDaYaQacSgG
AAGGAACAAAGTTCTATTATGTAATGGAACTTAAAGGCCGATACATAAAGCTAATCTGAA GAAAAAATGTTGGAAGGAGACAGATCATATAAATTAA AGACGTCTAGATGGGTAAAT C3 SCT,uGA3GEAt sGa0AeAQGnGATnTAtGsT IG sTD cTeTioCTlAeCd N sGATntiGOAnrTtCAatTTugiTGCiA:A mniGnoA3etGTGRtAThA sdnAaAunAT -A GeAgCTtAcA G ( i mAaTqCA snCAew Qo SLALtVAEeVPNiNQLnDDAYG s IGGPDeRKSDqY NLSuDOTeSNA 1GnA:yVT7K5GcETRSAeAS AISIT pGFmNVNrK
AL8GWGTN -SYAQFTPGNASDTIIKLQKSKTSMHDSQGSHWNIPVTNALMHIKIW -WYKDNE 3LNY LV SQcIEyIePESaFVTQsNKDeSEE IAY CVDMVQKC p NIISArONoGIAYce 1HDt:NPGHe8tAq0GTaYRNiTGuEnIt PTAYeWEeNEKe sSAnLQT
SVTMGGLFYHQKFQLSLEIKRRGQKTKIFFLFRINVDVYQFIWSAADPLVSIAGWSDPDL KKNTTARKDYRFHETLPGTVQDANGDWMILQVDSQFVKLTAGDFEGNMDQNAQVNTDKVG SGLD AYGSSDEKAGSEIAFNYNYNRLQVAVGQSTYVNNSDAVGLYIQPLKEGMQFVKKIADNWF NELPAGVVNDPRRGNGVIQA5SVTKYVNVNKTASDDARHAWGGIELVSTNGSDDTSEHET DALGT WFIGRDVSMISSSKETSRHAGCSFDQYQSTHTDLAKTIDVDGSAPTTKIGDSLNYNRSVD FMAFENLTVDSKFKLIEFNTEGGFIVAISEVNGRDASGWHAIDFRATKGTDDTKAVSIKQ WAPI HNGDNSVNTNSNAGVQGKQLQMFVTIFKARSFEWGFTMDIQPVNQGPGRQAVVYENKMRG RGIQNEEPSTGDYVTHRNLNQNSRSVAIFNSGITGNH