	US 12,077,762 B1
	Engineered ribosomal promoters for protein production in microorganisms
Cristina Bongiorni, Palo Alto, CA (US); Marc Anton Bernhard Kolkman, Palo Alto, CA (US); Chris Leeflang, Palo Alto, CA (US); Virgil Arthur Rhodius, Palo Alto, CA (US); and Anita Van Kimmenade, Palo Alto, CA (US)
Assigned to DANISCO US INC., Palo Alto, CA (US)
Filed by DANISCO US INC., Palo Alto, CA (US)
Filed on Aug. 8, 2022, as Appl. No. 17/818,258.
Application 17/818,258 is a division of application No. 16/081,082, granted, now 11,447,782, previously published as PCT/US2017/020913, filed on Mar. 6, 2017.
Claims priority of provisional application 62/304,061, filed on Mar. 4, 2016.
Int. Cl. C12N 15/70 (2006.01); C07K 14/32 (2006.01); C12N 9/28 (2006.01); C12N 9/54 (2006.01); C12N 15/75 (2006.01)

CPC C12N 15/75 (2013.01) [C07K 14/32 (2013.01); C12N 9/2417 (2013.01); C12N 9/54 (2013.01); C12Y 302/01001 (2013.01); C12Y 304/21062 (2013.01)]

7 Claims

1. An isolated nucleic acid comprising an engineered complete promoter operably linked to a nucleic acid encoding a protein of interest, the isolated nucleic acid comprising the formula selected from:

5′-UP-1stPro-ORF-3′; (I)

5′-UP-1stPro-UTR-ORF-3′; (II)

5′-UP-1stPro-2ndPro-ORF-3′; (III)

5′-UP-1stPro-2ndPro-UTR-ORF-3′; (IV)

5′-UP-1stPro-UTR-2ndPro-UTR-ORF-3′; (V)

5′-UP-1stPro-2ndPro-3rdPro-ORF-3′; (VI)

5′-UP-1stPro-2ndPro-3rdPro-UTR-ORF-3′; and (VII)

5′-UP-1stPro-2ndPro-UTR-3rdPro-UTR-ORF-3′, (VIII)

wherein UP is a nucleic acid comprising a promoter upstream element, 1^stPro, 2^ndPro and 3^rdPro are the same or different nucleic acids comprising at least a −35/−10 core promoter sequence, UTR is a nucleic acid comprising an untranslated region and ORF is a nucleic acid open reading frame encoding a protein of interest, wherein the UP A element comprises any one of SEQ ID NO:48, or a nucleic acid having at least 95% identity to SEQ ID NO: 48 that retains promoter activity, and wherein the 1^stPro, 2^ndPro and 3^rdPro comprises SEQ ID NO:3, or a nucleic acid having at least 95% identity to SEQ ID NO:3 that retains promoter activity.