Crispr-cas system for clostridium genome engineering and recombinant strains produced thereof

Patent metadata

Field	Value
Publication number	US-2020283746-A1
Application number	US-202016811733-A
Country	US
Kind code	A1
Filing date	Mar 6, 2020
Priority date	Mar 7, 2019
Publication date	Sep 10, 2020
Grant date	—

Abstract

A system for modifying the genome of Clostridium strains is provided based on a modified endogenous CRISPR array. The application also describes Clostridium strains modified for enhanced butanol production wherein the modified strains are produced using the novel CRISPR-Cas system.

First claim

What is claimed is:

1 . A Clostridium strain modified for enhanced butanol production, said Clostridium strain comprising
a modification to the native cat1 gene, said modification preventing expression of a functional cat1 gene product; and an exogenous sequence encoding
i) an aldehyde dehydrogenase;
ii) a bifunctional aldehyde/alcohol dehydrogenase; or
iii) an aldehyde dehydrogenase and an alcohol dehydrogenase.

2 . The Clostridium strain of claim 1 wherein said Clostridium cat1 gene is modified by the insertion of said exogenous sequence into the cat1 gene rendering the cat1 gene incapable of expressing a functional gene product.

3 . The Clostridium strain of claim 2 wherein said exogenous sequences comprises a bifunctional alcohol/aldehyde dehydrogenase gene selected from the group consisting of adhE1 and adhE2

4 . The Clostridium strain of claim 3 wherein said modified strain, when cultured at a temperature of less than 30° C. using glucose as a carbon source, produces at least 20 g/L of butanol after 72 hours of culture.

5 . A Clostridium strain modified for enhanced butanol production, said Clostridium strain comprising
an exogenous gene encoding for aldehyde dehydrogenase activity, and a modified native Clostridium cat1 gene, wherein said modification prevents expression of a functional cat1 gene product, further wherein said modified strain, when cultured at a temperature of less than 30° C. using glucose as a carbon source, produces at least 20 g/L of butanol after 72 hours of culture.

6 . The strain of claim 5 wherein said exogenous gene is inserted into the cat1 gene rendering the cat1 gene incapable of expressing a functional gene product.

7 . The strain of claim 6 wherein said exogenous gene is an adhE gene having at least 95% sequence identity to SEQ ID NO: 133 or SEQ ID NO: 134.

8 . The strain of claim 1 wherein the strain is the Clostridium tyrobutyricum strain deposited with Agriculture Research Culture Collection (NRRL) and assigned accession no. NRRL B-67519.

9 . A vector for introducing modifications into a target genomic site of bacteria via a CRISPR-Cas complex, wherein said target genomic site is a contiguous nucleic acid sequence comprising a first protospacer sequence, a first upstream sequence and a first downstream sequence, said vector comprising
a synthetic CRISPR array; an inducible promoter operably linked to said synthetic CRISPR array; and a first homology arm polylinker site; wherein
said synthetic CRISPR array comprises
a first and second direct repeat, wherein said first and second direct repeat have greater than 95% sequence identity to one another and are orientated relative to each other as direct repeats; and a first spacer polylinker site, wherein the first spacer polylinker site is located between the first and second direct repeat; and a CRISPR terminator sequence located after said second direct repeat.

10 . The vector of claim 9 wherein
said first and second direct repeat independently comprise a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2; and
said CRISPR terminator sequence comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3.

11 . The vector of claim 10 wherein the inducible promoter is a lactose inducible promoter.

12 . The vector of claim 11 further comprising a native Clostridium tyrobutyricum Cas encoding sequence.

13 . The vector of claim 12 wherein said native Clostridium tyrobutyricum Cas encoding sequence is operably linked to an inducible promoter.

14 . The vector of claim 11 further comprising elements for introducing modifications into a first and second target genomic site of bacteria via a CRISPR-Cas complex, wherein said first target genomic site is a contiguous nucleic acid sequence comprising a first protospacer sequence, a first upstream sequence and first downstream sequence, and said second target genomic site is a contiguous nucleic acid sequence comprising a second protospacer sequence, a second upstream sequence and second downstream sequence, said vector further comprising
a second homology arm polylinker site; and
said synthetic CRISPR array further comprises
a third direct repeat, wherein said third direct repeat comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2 and is orientated as a direct repeat relative to the first and second direct repeats; and
a second spacer polylinker site, wherein the second spacer polylinker site located between the second and third direct repeat, wherein said CRISPR terminator sequence is located after said third direct repeat.

15 . The vector of claim 11 wherein
a first spacer sequence of 20 to 50 nucleotides is inserted into said first spacer polylinker site; and
a first and second homology arm sequence are inserted into said first homology arm polylinker site, wherein said first homology arm sequence comprises a nucleotide sequence sharing at least about 90% sequence identity to said first upstream sequence, and the second homology arm comprises a nucleotide sequence sharing at least about 90% sequence identity to said first downstream sequence.

16 . The vector of claim 14 wherein
a first spacer sequence of 20 to 50 nucleotides is inserted into said first spacer polylinker site;
a second spacer sequence of 20 to 50 nucleotides is inserted into said second spacer polylinker site;
a first and second homology arm sequence are inserted into said first homology arm polylinker site, wherein said first homology arm sequence comprises a nucleotide sequence sharing at least about 90% sequence identity to said first upstream sequence, and the second homology arm comprises a nucleotide sequence sharing at least about 90% sequence identity to said first downstream sequence; and
a third and fourth homology arm sequence are inserted into said second homology arm polylinker site, wherein said third homology arm sequence comprises a nucleotide sequence sharing at least about 90% sequence identity to said second upstream sequence, and the fourth homology arm comprises a nucleotide sequence sharing at least about 90% sequence identity to said second downstream sequence.

17 . A method of producing butanol, said method comprising the steps of
culturing the Clostridium strain of claim 1 under conditions suitable for growth of the strain; and recovering the butanol produce by said cell.

18 . The method of claim 17 wherein the strain is cultured at a temperature selected from the range of about 20° C. to about 30° C.

19 . A method of modifying a target site of a bacterial cell genome, said method comprising
transforming said bacterial cell with the vector of claim 11 and selecting for transformants comprising said vector; inducing the expression of said CRISPR array; and identifying recombinant bacteria having a modification to said target site of the genome.

20 . The method of claim 19 wherein the target site is the cat1 gene and the first spacer sequence comprises the sequence

(SEQ ID NO: 4)

CTTGTAGAAGATGGATCAACCCTACAACTTGGTA.

Assignees

• Univ Auburn

Inventors

• Wang Yi
• Zhang Jie

Classifications

• C12N1/205

  Bacterial isolates · CPC title

• C12R2001/145

  Clostridium · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12P7/16

  Butanols · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title