# US 2019/0169591 A1 · Expression system for psicose epimerase and production for psicose using the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169591 A1 - Document ID: US20190169591A1 - Status: Published application - Kind: A1 - Application number: 16/274,418 - Filed: 2019-02-13 - Published: 2019-06-06 - Earliest claimed: 2014-10-30 - Assignees: Samyang Corporation - Applicants: Samyang Corporation - Inventors: Jin Sol Heo; Hye Jung Kim; Min Jeong Kim; Jeong Yoon Choi; Chong Jin Park; Kang Pyo Lee - Web page: https://patentgrep.com/patent/US20190169591A1 ## CPC - C12N 9/90: Isomerases (5.) - C12N 15/67: General methods for enhancing the expression - C12N 15/77: for Corynebacterium; for Brevibacterium - C12Y 501/03: acting on carbohydrates and derivatives (5.1.3) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract A gene expression cassette capable of producing psicose at high yield with high stability, a GRAS (Generally recognized as safe) microorganism, a method of producing the enzyme by using the GRAS microorganism, and a method of producing the psicose by using the GRAS microorganism and enzyme are provided. ## Claims ### Claim 1 A gene expression cassette, producing a psicose epimerase in Corynebacterium sp., and comprising: a nucleotide sequence encoding the psicose epimerase; and a regulating sequence being operably connected to the nucleotide sequence in the upstream regulating the expression of the nucleotide sequence in Corynebacterium sp, and comprising a promoter, a ribosome binding site (RBS) sequence and a first spacer sequence in the direction of 5′ to 3′, wherein the promoter includes the nucleotide sequence of SEQ ID NO: 1, the ribosome binding site (RBS) sequence is a nucleotide sequence in a size of 7 to 20 bases including the nucleotide sequence of SEQ ID NO: 2, and the first spacer sequence is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 4 to SEQ ID NO: 6. ### Claim 2 (depends on 1) The gene expression cassette according to claim 1, wherein the regulating sequence further comprises a second RBS sequence which is connected to 3′-end of the first spacer directly or via a linker sequence in a length of 5 to 100 bases, and wherein the second RBS sequence is a nucleotide sequence in a size of 7 to 20 bases including the nucleotide sequence of SEQ ID NO: 2. ### Claim 3 (depends on 2) The gene expression cassette according to claim 2, wherein the linker sequence is a nucleotide sequence in a size of 42 to 100 bp which includes the nucleotide sequence of SEQ ID NO: 12. ### Claim 4 (depends on 1) The gene expression cassette according to claim 1, wherein the regulating sequence further comprises a second spacer sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 11, wherein the second spacer is connected to 3′-end of the second RBS. ### Claim 5 (depends on 3) The gene expression cassette according to claim 3, wherein the regulating sequence further comprises a second spacer sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 11, wherein the second spacer is connected to 3′-end of the second RBS. ### Claim 6 (depends on 2) The gene expression cassette according to claim 2, wherein the regulating sequence comprise a nucleotide sequence selected from the group consisting of the sequences of SEQ ID NO: 13 to SEQ ID NO: 16. ### Claim 7 (depends on 3) The gene expression cassette according to claim 3, wherein the regulating sequence comprise a nucleotide sequence selected from the group consisting of the sequences of SEQ ID NO: 13 to SEQ ID NO: 16, and the linker sequence of SEQ ID NO: 12. ### Claim 8 (depends on 1) The gene expression cassette according to claim 1, wherein the regulating sequence comprises the promoter nucleotide sequence of SEQ ID NO: 1, the RBS nucleotide sequence of SEQ ID NO: 2, the first spacer sequence selected from the group consisting of the sequences of SEQ ID NO: 4 to SEQ ID NO: 6, the second RBS nucleotide sequence of SEQ ID NO: 2, and the second spacer sequence selected from the group consisting of the sequences of SEQ ID NO: 7 to SEQ ID NO: 11. ### Claim 9 (depends on 1) The gene expression cassette according to claim 1, wherein the Corynebacterium sp. is at least one selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium efficiens. ### Claim 10 (depends on 1) The gene expression cassette according to claim 1, wherein the psicose epimerase is derived from Clostridiun scidens, Treponema primitia, Ensifer adhaerens or Ruminococcus torques. ### Claim 11 (depends on 10) The gene expression cassette according to claim 10, wherein the psicose epimerase is an amino acid sequence selected from the group consisting of nucleotides of SEQ ID NO: 33 to SEQ ID NO: 36. ### Claim 12 (depends on 11) The gene expression cassette according to claim 11, wherein the nucleotide sequence encoding the psicose epimerase is a nucleotide sequence selected from the group consisting of nucleotides of SEQ ID NO: 37 to SEQ ID NO: 44. ### Claim 13 (depends on 1) A vector comprising an expression cassette of claim 1. ### Claim 14 (depends on 13) The vector according to claim 13, wherein the regulating sequence comprises a nucleotide sequence selected from the group consisting of the sequences of SEQ ID NO: 13 to SEQ ID NO: 16. ### Claim 15 (depends on 13) The vector according to claim 13, wherein the regulating sequence comprises a nucleotide sequence selected from the group consisting of the sequences of SEQ ID NO: 18 to SEQ ID NO: 32. ### Claim 16 (depends on 13) The vector according to claim 13, wherein the vector further comprises at least one sequence selected from the group consisting of a replication origin, leader sequence, a selection marker, a cloning site, and a restriction enzyme recognition site. ### Claim 17 (depends on 1) A recombinant Corynebacterium sp. host cell comprising a gene expression cassette of claim 1, or being transformed by a gene expression cassette of claim 1. ### Claim 18 (depends on 17) The recombinant Corynebacterium sp. host cell according to claim 17, wherein the Corynebacterium sp. is at least one selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium efficiens. ### Claim 19 (depends on 1) A composition for producing a psicose epimerase, comprising at least one selected from the group consisting of an psicose epimerase obtained by using the recombinant Corynebacterium sp., a recombinant cell, a culture of the recombinant cell, a lysate of the recombinant cell and an extract of cell culture or cell lysate, where the recombinant Corynebacterium sp. is transformed by a gene expression cassette of claim 1 or a vector comprising a gene expression cassette of claim 1. ### Claim 20 (depends on 13) The vector according to claim 13, wherein the regulating sequence further comprises a second RBS sequence which is connected to 3′-end of the first spacer directly or via a linker sequence in a length of 5 to 100 bases, wherein the second RBS sequence is a nucleotide sequence in a size of 7 to 20 bases including the nucleotide sequence of SEQ ID NO: 2.