IP Library Granted Patent US 11,685,934
Granted Patent B2
US 11,685,934 · App. 16/646,028 · Granted Jun 27, 2023

Methods for genomic integration for

Inventor: Jessica Walter (Albany, CA)
Assignee: Amyris, Inc.
C12N15/905C12N9/22C12N15/11C12N15/815C12N2310/20C12N2800/80
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Quick Facts
Patent No.
US 11,685,934
App. No.
16/646,028
Granted
Jun 27, 2023
Kind
B2
Abstract

The present invention provides high efficiency targeted and marker-less single or simultaneous multiple integrations using nucleases and a stable plasmid in Kluyveromyces host cells.

Claims (12)

1. A method of modifying a target site in a Kluyveromyces host cell genome, the method comprising:

(a) contacting the host cell, which has reduced non-homologous end joining (NHEJ) activity, wherein the NHEJ activity is reduced by integrating a nucleic acid at YKU70 or YKU80 loci. with:

(i) a first linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the host cell, whereby homologous recombination in the host cell results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker and a stability element comprising a centromere sequence (CEN) sequence at least 95% identical to SEQ ID NO: 2 and an autonomously replicating sequence (ARS) consensus sequence at least 90% identical to SEQ ID NO: 3;

(ii) a nuclease capable of cleaving the target site, wherein the nuclease is an RNA guided endonuclease or a meganuclease; and

(iii) a donor DNA molecule capable of homologous recombination at the cleaved target site, whereby homologous recombination in the host cell results in integration of the donor linear nucleic acid at the target site; and

(b) selecting a transformed host cell expressing the selectable marker.

2. The method of claim 1 , wherein the stability element is at least 95% identical to SEQ ID NO: 1.

3. The method of claim 1 , wherein the host cell is K marxianus.

4. The method of claim 1 , wherein the nuclease is an RNA-guided DNA endonuclease.

5. The method of claim 1 , wherein the nucleic acid integrated at YKU70 or YKU80 loci encodes the RNA guided endonuclease.

6. The method of claim 1 , wherein the stability element comprises SEQ ID NO: 1.

7. The method of claim 1 , wherein CEN sequence comprises SEQ ID NO: 2 and the ARS consensus sequence comprises SEQ ID NO: 3.

Assignments (7)
SECURITY INTEREST Recorded May 24, 2024
From: AMYRIS, INC.
To: EUAGORE, LLC
Reel/Frame 067528/0467 →
SECURITY INTEREST Recorded Aug 17, 2023
From: AMYRIS, INC.; AMYRIS CLEAN BEAUTY, INC.; AMYRIS FUELS, LLC; AB TECHNOLOGIES LLC; APRINNOVA, LLC; AMYRIS-OLINKA, LLC; ONDA BEAUTY INC.; UPLAND 1 LLC; AMYRIS ECO-FAB LLC; CLEAN BEAUTY 4U HOLDINGS, LLC; AMYRIS CLEAN BEAUTY LATAM LTDA; INTERFACES INDUSTRIA E COMERCIA DE COSMETICOS LTDA; AMYRIS BIOTECHNOLOGIA DO BRASIL LTDA; AMYRIS EUROPE TRADING B.V. (NETHERLANDS); AMYRIS BIO PRODCUTS PORTUGAL, UNIPESSOAL, LDA; BEAUTY LABS INTERNATIONAL LIMITED; AMYRIS UK TRADING LIMITED
To: EUAGORE, LLC
Reel/Frame 064619/0778 →
SECURITY INTEREST Recorded Aug 3, 2023
From: AMYRIS CLEAN BEAUTY, INC.; AMYRIS FUELS, LLC; AB TECHNOLOGIES LLC; AMYRIS, INC.
To: MUIRISC, LLC
Reel/Frame 064492/0518 →
RELEASE OF SECURITY INTEREST Recorded Feb 14, 2023
From: NAXYRIS S.A.
To: AMYRIS, INC.
Reel/Frame 062760/0753 →
SECURITY INTEREST Recorded Oct 18, 2022
From: AMYRIS, INC.
To: FORIS VENTURES, LLC
Reel/Frame 061703/0499 →
SECURITY INTEREST Recorded Mar 10, 2021
From: AMYRIS, INC., AS GRANTOR
To: NAXYRIS S.A., AS LENDER
Reel/Frame 055547/0529 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 9, 2020
From: WALTER, JESSICA
To: AMYRIS, INC.
Reel/Frame 053180/0450 →