# US 10,370,654 B2 · Processes and host cells for genome, pathway, and biomolecular engineering

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- Number: US 10,370,654 B2
- Document ID: US10370654B2
- Status: Granted
- Kind: B2
- Application number: 15/909,191
- Filed: 2018-03-01
- Published: 2018-11-08
- Granted: 2019-08-06
- Earliest claimed: 2013-08-02
- Assignees: enEvolv, Inc.
- Applicants: enEvolv, Inc.
- Inventors: Jay H. Konieczka; James E. Spoonamore; Ilan N. Wapinski; Farren J. Isaacs; Gregory B. Foley
- Web page: https://patentgrep.com/patent/US10370654B2

## CPC

- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/81: for yeasts
- C12N 15/85: for animal cells
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2330/31: Libraries, arrays
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure provides compositions and methods for genomic engineering.

## Claims

### Claim 1

A method for multiplexed genome engineering of a yeast cell for a desired phenotype, wherein the desired phenotype is one or more of a change in carbon substrate utilization, increased cell growth rate, increased production of a desired chemical, redox cofactor balance, reduced production of one or more undesired byproducts, increased resistance to industrial fermentation, and increased recombinant protein production,
the method comprising:
introducing a first oligonucleotide library into a population of yeast cells, the yeast cells expressing a single-stranded annealing protein (SSAP) and recombinase system wherein said system comprises a member selected from the group consisting of: RecT protein of RecET system, Rad52-like protein, λ Red Beta protein, Sak protein, Erf protein, Rad51-like protein, Sak4 protein, and Gp2.5-like protein, wherein said system further comprises a deletion, inactivation, reduction, or inhibition of a mismatch repair protein,
and the first oligonucleotide library targeting regulatory sequences that control gene expression in the cell wherein said regulatory sequences are selected from the group consisting of promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns;
targeting at least one gene altered by the system with a second oligonucleotide library, or targeting at least one gene with a gene editing system; and
selecting for yeast cells having an improvement in the desired phenotype as a result of oligonucleotide incorporation, wherein the method comprises a co-selection.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first oligonucleotide library or the second oligonucleotide library further targets at least one heterologously expressed gene, wherein said gene encodes an enzyme or a recombinant protein.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first oligonucleotide library or the second oligonucleotide library further targets genes encoding a protein structural motif.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mismatch repair protein is mutS or homolog thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first oligonucleotide library is introduced into the cells by one or more of electroporation, chemical transformation, ballistic transformation, pressure induced transformation, mechanical shear forces, microfluids, carbon nanotubes, nanotube puncture, and induced natural competence mechanisms of an organism.

### Claim 6 (depends on 1)

The method of claim 1, wherein the second oligonucleotide library targets protein coding sequences and/or transcribed sequences.

### Claim 7 (depends on 1)

The method of claim 1, wherein the targeting and selection are conducted in continuous fashion.

### Claim 8 (depends on 1)

The method of claim 1, wherein the yeast cells have a deletion or inactivation of one or more single stranded DNA nucleases.

### Claim 9 (depends on 1)

The method of claim 1, wherein genes altered by the first oligonucleotide library in selected cells are identified by transcriptional or translational analysis or by DNA sequence.

### Claim 10 (depends on 9)

The method of claim 9, wherein genes having altered transcription or translation are targeted for diversification by a second oligonucleotide library, or by a targeted gene editing system.

### Claim 11 (depends on 10)

The method of claim 10, wherein the genes having altered transcription or translation are targeted by a gene editing system selected from the group consisting of CRISPR/Cas9, TALENS, ZFN, or BuD, wherein such gene editing system is expressed and the expression of the gene editing system is inducible or controllable.

### Claim 12 (depends on 1)

The method of claim 1, wherein the system is inducible or regulatable by chemical or thermal means.

### Claim 13 (depends on 1)

The method of claim 1, wherein cells with the desired phenotype are selected using at least one of a microfluidics system, chromatographic analysis, spectroscopic analysis, extraction followed by an analytical chromatographic step, chemical chromogenic assay, flow cytometric analysis and sorting, enzymatic activity assay, zone of inhibition assay, and auxotrophic reporter strain growth assay.

### Claim 14 (depends on 1)

The method of claim 1, wherein the yeast cells are selected from Saccharomyces species (spp.), Schizasaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces sop., Yarrowia spp., and industrial polyploid yeast strains.