KR20240147949A - Recombinant corynebacterium glutamicum producing 1,5-pentanediol and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant Corynebacterium glutamicum strain producing 1,5-pentanediol from glucose and to a use thereof. Specifically, the recombinant strain according to the present invention has the advantage of not only excellent production efficiency and production capacity of 1,5-pentanediol, but also excellent production capacity of 5-hydroxyvalerate, which is a key precursor for 1,5-pentanediol production. In addition, since no by-products are generated during the production process, the problem of reduced yield due to side reaction products can be solved.

Description

RECOMBINANT CORYNEBACTERIUM GLUTAMICUM PRODUCING 1,5-PENTANEDIOL AND USES THEREOF

This application claims the benefit of priority based on Korean Patent Application No. 10-2023-0043218, filed on March 31, 2023 with the Korean Intellectual Property Office, the contents of which are incorporated herein by reference in their entirety.

The present invention relates to a recombinant Corynebacterium glutamicum strain producing 1,5-pentanediol and its use.

1,5-Pentanediol is in high demand worldwide because it can be used as an additive in general products such as cosmetics, or as an intermediate or monomer for producing various chemical substances in the fine chemical field.

1,5-Pentanediol (1,5-PDO) can be industrially produced from petroleum products, but there is a problem of limited supply of C5 petroleum-based feedstock, and when produced by chemical reaction, there are problems such as low yield due to side products, high energy consumption, and high temperature and high pressure conditions. Accordingly, biotechnological production of 1,5-pentanediol has emerged as a promising low-cost and sustainable alternative to chemical synthesis.

Recently, the production of 1,5-PDO via 5-aminovalerate (5-AVA) using Escherichia coli has been reported (Wang et al., 2020; Cen et al., 2021). There are two 1,5-PDO biosynthetic metabolic pathways reported here, a CoA-dependent metabolic pathway and a CoA-independent metabolic pathway from lysine to the key precursor 5-hydroxyvaleric acid (hereinafter referred to as 5HV). However, the production using the above metabolic pathway remains at a level of less than 1 g/L, and there is a problem of excessive accumulation of by-products such as lysine, 5-AVA, and glutaric acid. In addition, the production of 5HV, a key precursor for 1,5-PDO production, is also evaluated to be low at 1.04 g/L (Cen et al., 2021).

Wang et al., PNAS, 2020, 117, 32, 19159-19167 Cen et al., ACS Synth. Biol. 2021, 10, 1, 192-203 Sohn et al., ACS Sustainable Chem. Eng. 2021, 9, 6, 2523-2533

The purpose of the present invention is to provide a recombinant strain for producing 1,5-pentanediol, which has excellent 1,5-pentanediol production ability and efficiency while producing less by-products.

Another object of the present invention is to provide a method for producing 1,5-pentanediol from glucose using the recombinant strain.

According to one aspect of the present invention, a recombinant strain for producing 1,5-pentanediol is provided, which is transformed with a vector comprising a gene for converting lysine into 5-aminovalerate and a gene for converting 5-aminovalerate into 5-hydroxyvalerate, and a gene encoding an enzyme for converting 5-hydroxyvalerate into 5-hydroxyvalerate and/or a gene encoding an enzyme for converting 5-hydroxyvalerate into 1,5-pentanediol.

According to one embodiment of the present invention, the recombinant strain of the present invention may be Corynebacterium glutamicum , for example, may be a recombinant Corynebacterium glutamicum strain PKC. Specifically, the recombinant strain of the present invention may be a Corynebacterium glutamicum strain deposited under the accession number KCTC 19163P (hereinafter referred to as “5HV-PKC” or “PKC-5HV”).

The recombinant strain according to the present invention is designed to produce 1,5-pentanediol according to a metabolic pathway using lysine (see FIG. 1). However, according to the conventional technology, there was a problem in that a large amount of lysine was observed as a byproduct in the production of 1,5-pentanediol using microorganisms. In the present invention, in order to prevent the leakage of the produced lysine and to efficiently convert it into 5AVA (5-aminovalerate), a gene for converting lysine into 5-aminovalerate was inserted into the strain. Preferably, the lysE gene in the strain may be deleted, and a gene for converting lysine into 5AVA may be inserted into the corresponding site.

Accordingly, according to one embodiment of the present invention, the recombinant strain may be one in which the gabD gene and the lysE gene are deleted. According to one embodiment, the gabD gene may comprise the sequence of SEQ ID NO: 1. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence described in SEQ ID NO: 1. According to one embodiment, the lysE gene may comprise the sequence of SEQ ID NO: 2. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence described in SEQ ID NO: 2.

The above homology refers to the degree of relationship between two given amino acid sequences or base sequences, and can be determined by a known method, such as a method using a known computer algorithm or a method of comparing sequences by a Southern hybridization experiment.

According to one embodiment of the present invention, the gene that converts the lysine into 5-aminovalerate may be a gene derived from Pseudomonas putida . According to one embodiment of the present invention, the gene that converts the lysine into 5-aminovalerate may comprise the sequence of SEQ ID NO: 3. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence described in SEQ ID NO: 3.

In addition, according to the present invention, in order to prevent the production of glutaric acid produced as a by-product and to efficiently convert 5AVA into 5HV (5-hydroxyvalerate), a gene for converting 5-aminovalerate into 5-hydroxyvalerate (5HV) is inserted into the strain. According to one embodiment of the present invention, the recombinant strain of the present invention may preferably be one in which the gabD3 gene in the strain is deleted and a gene for converting 5AVA into 5HV is inserted. Accordingly, according to one embodiment of the present invention, the recombinant strain may be one in which the gabD3 gene is deleted.

In one embodiment, the gene converting 5AVA into 5HV may include a gene derived from Pseudomonas putida and a gene derived from E. coli YahK. In another embodiment, the gene converting 5AVA into 5HV may include a sequence of SEQ ID NO: 5. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence set forth in SEQ ID NO: 5.

According to the present invention, a recombinant strain for producing 1,5-pentanediol may be transformed with a vector including, in addition to the introduction of the genes described above, a gene encoding an enzyme that converts 5-hydroxyvalerate into 5-hydroxyvaleraldehyde and/or a gene encoding an enzyme that converts 5-hydroxyvaleraldehyde into 1,5-pentanediol.

The term "vector" in the present invention means an expression vector, which means a genetic construct including essential regulatory elements such as a promoter so that a target gene can be expressed in a suitable host cell. Examples of the vector include a plasmid vector, a cosmid vector, a bacteriophage vector, and the like. According to one embodiment of the present invention, the vector may be a plasmid vector.

In one embodiment, a vector comprising a gene encoding an enzyme that converts 5-hydroxyvalerate into 5-hydroxyvaleraldehyde and/or a gene encoding an enzyme that converts 5-hydroxyvaleraldehyde into 1,5-pentanediol may be composed of a first vector comprising a gene encoding an enzyme that converts 5-hydroxyvalerate into 5-hydroxyvaleraldehyde and a second vector comprising a gene encoding an enzyme that converts 5-hydroxyvaleraldehyde into 1,5-pentanediol.

In another embodiment, the recombinant strain may be further transformed with a third vector comprising, in addition to the transformation described above, a gene for converting lysine to 5-aminovalerate and/or a gene for converting 5-aminovalerate to 5-hydroxyvalerate.

In one embodiment, the gene encoding the enzyme that converts the 5-hydroxyvalerate into 5-hydroxyvaleraldehyde may be a gene encoding carboxylic acid reductase (CAR). In one embodiment, the gene encoding the CAR may include the sequence of SEQ ID NO: 16. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence set forth in SEQ ID NO: 16.

In one embodiment, the gene encoding the enzyme that converts the 5-hydroxyvaleraldehyde into 1,5-pentanediol may be a YahD gene, specifically, an E. coli YahD gene. The gene encoding the enzyme that converts the 5-hydroxyvaleraldehyde into 1,5-pentanediol may comprise the sequence of SEQ ID NO: 21. Considering the degeneracy of the genetic code, the gene may be a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95%, 98%, or 99% homology with the base sequence set forth in SEQ ID NO: 21.

According to one embodiment of the present invention, the recombinant strain of the present invention may have the accession number KCTC 19163P.

According to another aspect of the present invention, a method for producing 1,5-pentanediol is provided, comprising the step of culturing the aforementioned recombinant Corynebacterium glutamicum strain to obtain a culture.

The method for culturing the recombinant Corynebacterium glutamicum strain of the present invention may be any conventional method used for culturing a host. Any method commonly used for culturing microorganisms, such as batch, fluidized batch, continuous culture, or reactor type, may be used as the culturing method. As a medium for culturing the recombinant Corynebacterium glutamicum strain, a complete medium or a synthetic medium, such as LB medium or NB medium, may be exemplified.

Carbon sources are necessary for the growth of microorganisms, and examples thereof include sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; lower alcohols such as ethanol, propanol, and butanol; polyhydric alcohols such as glycerol; organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, and gluconic acid; and fatty acids such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid. According to one embodiment of the present invention, in the new method of obtaining 1,5-pentanediol of the present invention, the cultivation can be performed on a substrate containing sugars, specifically, glucose.

According to one aspect of the present invention, a composition for producing 1,5-butanediol comprising the recombinant strain of the present invention is provided. The composition of the present invention may include, for example, a polypeptide, broth, cell lysate, or the like suitable for producing 1,5-pentanediol by culturing the strain.

The recombinant strain for producing 1,5-pentanediol according to the present invention has the advantage of excellent production ability of 5-hydroxyvalerate, which is a key precursor for 1,5-pentanediol production. In addition, the recombinant strain of the present invention is excellent in production efficiency and production ability of not only the precursor but also 1,5-pentanediol, and since it generates little by-products during the production process, it can solve the problem of reduced yield due to side reaction products.

Figure 1 is a metabolic pathway for producing 1,5-pentanediol according to the present invention.
Figure 2 is a graph showing the results of measuring the productivity of precursor 5HV according to Experimental Example 1 of the present invention.
Figure 3 (a) is a graph showing the results of CAR screening for 5HV and 15PDO production of the 5HVx1 strain.
Figure 3 (b) is a graph showing the CAR screening results for 5HV and 15PDO production of the 5HVx2 strain.
Figure 4 is a graph showing the 1,5-PDO productivity over time by batch fermentation of 5HVx1-CAR11.
Figure 5 is a graph showing the 1,5-PDO productivity over time by batch fermentation of 5HVx2-CAR11.
Figure 6 is a graph showing the 1,5-PDO productivity over time by fed-batch culture of 5HVx1-CAR11.
Figure 7 is a graph showing the 1,5-PDO productivity over time by fed-batch culture of 5HVx2-CAR11.

Hereinafter, the present invention will be described in more detail through examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the examples described herein, and may be embodied in other forms. The examples introduced herein are provided so that the idea of the present invention can be sufficiently conveyed to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

[Manufacturing Example] Production of a recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol

(1) Corynebacterium glutamicum PKC △ gab Manufacturing of D

Corynebacterium glutamicum PKC was prepared as a strain. The gabD gene (SEQ ID NO: 1) was deleted from the strain (Production of Corynebacterium glutamicum PKC △ gab D) according to the method described in the above (Non-patent Document 3: Sohn et al., 2021).

(2) Production of PKC-5AVA strain

The lysE gene in Corynebacterium, which is responsible for lysine export, was deleted in the above Corynebacterium glutamicum PKC △ gab D, and H30DavBHisA (lysine 5-AVA conversion module; H30 promoter- Pseudomonas putida davB _his A gene-terminator) was inserted into the corresponding site, thereby creating Corynebacterium glutamicum PKC △ gab D △ lysE :: H30 DavB _His A strain (PKC-5AVA strain).

Specifically, genetic manipulation of C. glutamicum was performed using the vector pK19mobsacB for chromosomal integration (NEB). To construct a pK19mobsacB-based vector for LysE gene deletion and H30DavBHisA insertion, 500 bp of the homologous region upstream and downstream of the lysE gene were first cloned into HindIII/PstI and BamHI/EcoRI sites, respectively: pK19mobsacBLysE. The primer sequences used here are shown in Table 1 below.

Sequence (5'-3') pK19mobsacBLysEf-F (SEQ ID NO: 22) aagcttagagggttcccgcgcc pK19mobsacBLysEf-R (SEQ ID NO: 23) ctgcaggtggattttcgccgctg pK19mobsacBLysEb-F (SEQ ID NO: 24) ggatccgacctgtaatgaagatttccat pK19mobsacBLysEb-R (SEQ ID NO: 25) gaattctagcttcacgggttaccgc

Afterwards, for insertion of the H30DavBHisA operon, H30DavBHisA was cloned into the XbaI site between lysE upstream (500 bp) and lysE downstream (500 bp) using the primers listed in the table below.

Sequence pCES208H30-F (SEQ ID NO: 26) tctagacacctgcagactaaagggaacaaaagctg pCES208H30-R (SEQ ID NO: 27) tctagagtcggatccggtccacctacaacaaagct

For cloning, PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan). 1 pM of oligonucleotides and 10 ng of template DNA were added to a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and 25 to 30 cycles were performed at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan).

The cloned vector was transformed into E. coli DH5a (HIT Competent cells ^TM , Cat No. RH618), spread on LB agar plates containing 50 ㎍/㎖ of kanamycin, and cultured at 37℃ for 24 hours. The colonies formed were isolated to confirm whether the insert was exactly present in the vector, and the vector was isolated using the Wizard Genomic DNA Purification Kit (Promega, USA). The same conditions were used in subsequent studies.

(3) Preparation of PKC-5HA strain

To block the production of glutaric acid and efficiently convert 5-aminovalerate (5AVA) to 5-(hydroxyvalerate (5HV), the gabD3 gene, which mediates the same reaction as the gabD gene present in Corynebacterium, was targeted and deleted, while inserting the H30DavTYahK (5AVA → 5HV conversion module; H30 promoter- P. putida DavT & E. coli YahK-terminator) operon, developing the C. glutamicum PKC △ gabD △ lysE :: H30DavBHisA △ gabD3::H30DavTYahK strain (PKC-5HV strain). Genetic manipulation of C. glutamicum was performed using the chromosomal integration vector pK19mobsacB (NEB, Manufactured by).

To construct a pK19mobsacB-based vector for GabD3 gene deletion and H30DavTYahK insertion, 500 bp of the homologous regions upstream and downstream of the gabD3 gene were first amplified by overlap PCR to produce a 1000 bp PCR product, which was then cloned into HindIII/EcoRI sites: pK19mobsacBcg0067. The primer sequences used here are listed in the table below.

Sequence pK19mobsacBCg0067f-F (SEQ ID NO: 28) aagcttagtgaccatcggcggagtgttcg pK19mobsacBCg0067f-R (SEQ ID NO: 29) ggggcagatgtctagagatgcgttattttccttcac pK19mobsacBCg0067b-F (SEQ ID NO: 30) cgcatctctagacatctgcccctttacaaatcc pK19mobsacBCg0067b-R (SEQ ID NO: 31) gaattccctgccatccagtcggcatac

Afterwards, for insertion of the H30DavTYahK operon, H30DavTYahK was cloned into the XbaI site between gabD3 upstream (500 bp) and gabD3 downstream (500 bp) using the primers listed in the table below.

Sequence pCES208H30-F (SEQ ID NO: 32) tctagacacctgcagactaaagggaacaaaagctg pCES208H30-R (SEQ ID NO: 33) tctagagtcggatccggtccacctacaacaaagct

(4) Screening of various Carboxylic Acid Reductase (CAR) for conversion of 5HV to 15PDO and construction of recombinant strains for 15PDO production

A total of 14 CARs (SEQ ID NOs: 6 to 19) were screened for the conversion of 5-hydroxyvalerate → 5-hydroxyvaleraldehyde. The conversion of 5-hydroxyvaleraldehyde → 15PDO was accomplished via the yqhD gene from E. coli (SEQ ID NO: 21).

Plasmids were constructed as follows for the expression of 14 CARs and Sfp [ Bacillus subtils pptase, which acts as a phosphopantetheinyl transferase to activate the apoenzyme CAR through phosphopantetheinylation, SEQ ID NO: 20] and Yqh D ( E. coli, alcohol dehydrogenase , SEQ ID NO: 21).

Plasmid Related Information pCES208H30-MCS Sohn et al., ACS Sustainable Chem. Eng. 2021, 9, 6, 2523-2533 pBL712H30-MCS Sohn et al., ACS Sustainable Chem. Eng. 2021, 9, 6, 2523-2533 pCES208H30DavTYahKB _His A pCES208 derivative; P _H30 , P. putida KT2440 davTB _His A, E. coli yahK ; km ^r pCES208H30CAR1pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (I7FIG7), B. subtils pptase ; km ^r pCES208H30CAR2pptase pCES208 derivative; P _H30 , Mycobacterium abscessus CAR (B1MCR9), B. subtils pptase ; km ^r pCES208H30CAR3pptase pCES208 derivative; P _H30 , Mycobacterium abscessus CAR (B1MLD7), B. subtils pptase ; km ^r pCES208H30CAR4pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (A0R484), B. subtils pptase ; km ^r pCES208H30CAR5pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (B1MDX4), B. subtils pptase ; km ^r pCES208H30CAR6pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (A0QWI7), B. subtils pptase ; km ^r pCES208H30CAR7pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (B1MCS0), B. subtils pptase ; km ^r pCES208H30CAR8pptase pCES208 derivative; P _H30 , Mycobacterium smegmatis CAR (I7GER2), B. subtils pptase ; km ^r pCES208H30CAR9pptase pCES208 derivative; P _H30 , Mycobacterium marinum CAR (B2HN69), B. subtils pptase ; km ^r pCES208H30CAR10pptase pCES208 derivative; P _H30 , Mycobacterium marinum CAR (B2HE95), B. subtils pptase ; km ^r pCES208H30CAR11pptase pCES208 derivative; P _H30 , Mycobacterium paratuberculosis K-10 CAR (Q741P9), B. subtils pptase ; km ^r pCES208H30CAR12pptase pCES208 derivative; P _H30 , Nocardia brasiliensis ATCC19296 CAR (A0A034UK40), B. subtils pptase ; km ^r pCES208H30CAR13pptase pCES208 derivative; P _H30 , Nocardia brasiliensis ATCC19296 CAR (A0A034UFC8), B. subtils pptase ; km ^r pCES208H30CAR14pptase pCES208 derivative; P _H30 , Mycobacterium phlei CAR, B. subtils pptase; km ^r pBL712H30YqhD pBL712 derivative; P _H30 , E. coli yqhD ; Sp ^r pBL712H30CAR1pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (I7FIG7), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR2pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium abscessus CAR (B1MCR9), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR3pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium abscessus CAR (B1MLD7), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR4pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (A0R484), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR5pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (B1MDX4), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR6pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (A0QWI7), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR7pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (B1MCS0), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR8pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium smegmatis CAR (I7GER2), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR9pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium marinum CAR (B2HN69), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR10pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium marinum CAR (B2HE95), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR11pptaseyqhD pBL712 derivative; P _H30 , Mycobacterium paratuberculosis K-10 CAR (Q741P9), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR12pptaseyqhD pBL712 derivative; P _H30 , Nocardia brasiliensis ATCC19296 CAR (A0A034UK40), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR13pptaseyqhD pBL712 derivative; P _H30 , Nocardia brasiliensis ATCC19296 CAR (A0A034UFC8), B. subtils pptase, E. coli yqhD ; Sp ^r pBL712H30CAR14pptaseyhhD pBL712 derivative; P _H30 , Mycobacterium phlei CAR, B. subtils pptase, E. coli yqhD ; Sp ^r

At this time, pCES208H30CAR~ plasmid and pBL712H30YqhD were cloned using restriction enzyme sites by amplifying genes with the following primers, and pBL712H30CAR~ plasmid was produced using the Gibson Assembly method using the following primers (sequence numbers 34 to 97 in order in Table 6).

CAR1-F-BamHI aaaaa ggatcc atgtgggacatgctcttc CAR1
(I7FIG7) CAR1-R-SpeI aaaaa actagt ttacaacaggccgaccttc CAR1-F-Gibson gcaggagtatattggaattcatgtgggacatgctcttc CAR1-R-Gibson cgggtaccgagctcgaattcttacaacaggccgaccttc CAR2-F-SpeI aaaaa actagt atgaccgtgaccaacgaaa CAR2
(B1MCR9) CAR2-R-SbfI aaaaa cctgcagg ttataggagtccgagctg CAR2-F-Gibson gcaggagtatattggaattcatgaccgtgaccaacgaaa CAR2-R-Gibson cgggtaccgagctcgaattcttataggagtccgagctg CAR3-F-BamHI aaaaa ggatcc atgactgaaacgatctcc CAR3
(B1MLD7) CAR3-R-SpeI aaaaa actagt ttacaccaggcccaacag CAR3-F-Gibson gcaggagtatattggaattcatgactgaaacgatctcc CAR3-R-Gibson cgggtaccgagctcgaattcttacaccaggcccaacag CAR4-F-SpeI aaaaa actagt atgaccagcgatgttcac CAR4
(A0R484) CAR4-R-SbfI aaaaa cctgcagg ttagatcagaccgaactc CAR4-F-Gibson gcaggagtatattggaattcatgaccagcgatgttcac CAR4-R-Gibson cgggtaccaggctcgaattcttagatcagaccgaactc CAR5-F-SpeI aaaaa actagt atgacggctggtgcggcg CAR5
(B1MDX4) CAR5-R-SbfI aaaaa cctgcagg ttacagcagcctgtgtgc CAR5-F-Gibson gcaggagtatattggaattcatgacggctggtgcggcg CAR5-R-Gibson cgggtaccgagctcgaattcttacagcagcctgtgtgc CAR6-F-SpeI aaaaa actagt atgacgatcgaaacgcgc CAR6
(A0QWI7) CAR6-R-SbfI aaaaa cctgcagg ttacagcaatccgagcatc CAR6-F-Gibson gcaggagtatattg gaattc atgacgatcgaaacgcgc CAR6-R-Gibson cgggtaccgagctcgaattcttacagcaatccgagcatc CAR7-F-BamHI aaaaa ggatcc atgacgatcgacgccacc CAR7
(B1MCS0) CAR7-R-SpeI aaaaa actagt tagagtaacccgagctg CAR7-F-Gibson gcaggagtatattggaattcatgacgatcgacgccacc CAR7-R-Gibson cgggtaccgagctcgaattcttagagtaacccgagctg CAR8-F-SpeI aaaaa actagt atgcaccagctcacggtc CAR8
(I7GER2) CAR8-R-SbfI aaaaa cctgcagg ttagatcagaccgaactc CAR8-F-Gibson gcaggagtatattggaattcatgcaccagctcacggtc CAR8-R-Gibson cgggtaccgagctcgaattcttagatcagaccgaactc CAR9-F-BamHI aaaaa ggatcc atgtcgccaatcacgcgtg CAR9
(B2HN69) CAR9-R-SpeI aaaaa actagt ttagagcaggccgagtag CAR9-F-Gibson gcaggagtatattggaattcatgtcgccaatcacgcgtg CAR9-R-Gibson cgggtaccgagctcgaattcttagagcaggccgagtag CAR10-F-BamHI aaaaa ggatcc atgtcaattacctgtgtg CAR10
(B2HE95) CAR10-R-SpeI aaaaa actagt ttaagccaggccgagaag CAR10-F-Gibson gcaggagtatattggaattcatgtcaattacctgtgtg CAR10-R-Gibson cgggtaccgagctcgaattcttaagccaggccgagaag CAR11-F-SpeI aaaaa actagt atgtcgactgccaccccatg CAR11
(Q741P9) CAR11-R-SbfI aaaaa cctgcagg ttagagcagcccgagcag CAR11-F-Gibson gcaggagtatattggaattcatgtcgactgccaccccatg CAR11-R-Gibson cgggtaccgagctcgaattcttagagcagcccgagcag CAR12-F-SpeI aaaaa actagt atggagcgcaaggcggaag CAR12
(A0A034UK40) CAR12-R-SbfI aaaaa cctgcagg ttacagcaggtttcgcaat CAR12-F-Gibson gcaggagtatattggaattcatggagcgcaaggcggaag CAR12-R-Gibson cgggtaccgagctcgaattcttacagcaggtttcgcaat CAR13-F-BamHI aaaaaggatccatgacagatgtagaggtag CAR13
(A0A034UFC8) CAR13-R-SpeI aaaaaactagtttacagtccgaggtgctccag CAR13-F-Gibson gcaggagtatattggaattcatgacagatgtagaggtag CAR13-R-Gibson cgggtaccgagctcgaattcttacagtccgaggtgctccag CAR14-F-SpeI aaaaaactagtatggcatcagaatcccgtg CAR 14
(WP_003889896.1)
CAR14-R-SbfI aaaaacctgcaggttacagcccgagcagccgcagatcg CAR14-F-Gibson gcaggagtatattggaattcatggcatcagaattccgtg CAR14-R-Gibson cgggtaccgagctcgaattcttacagcccgagcagccgcagatcg Pptase-F-SbfI aaaaa cctgcagg tctagataactttaagaaggagatatacatgaagaatttacggaattt Pptase


Pptase-R-NdeI aaaaa catatg ttataaaagctcttcgtac Pptase-F-Gibson taaagaggagatatacatatgaagaatttacggaatt Pptase-R-Gibson ttcatatgtatatctcctttataaaagctcttcgtac YqhD-F-EcoRI aaaaa ggatcc aggagatatacatatgaacaactttaatctgcacaccc YqhD YqhD-R-KpnI aaaaa agatct atgtatatctcctttagcgggcggcttcgtatatac YqhD-F-Gibson tataaaggagatatacatatgaacaactttaatctgc YqhD-R-Gibson tagaggatccccgggtaccttagcgggcggcttcgtatatac

The plasmids constructed above were each transformed into the pKC-5HV strain ( C. glutamicum PKC △ gabD △ lysE :: H30DavBHisA △ gabD3:: H30DavTYahK) to construct the strains shown in Table 7 below.

Strain information PKC-5HV (Accession number:
KCTC 19163P) C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK 5HVx1-CAR1 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR1pptase + pBL172H30YqhD 5HVx1-CAR2 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR2pptase + pBL172H30YqhD 5HVx1-CAR3 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR3pptase + pBL172H30YqhD 5HVx1-CAR4 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR4pptase + pBL172H30YqhD 5HVx1-CAR5 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR5pptase + pBL172H30YqhD 5HVx1-CAR6 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR6pptase + pBL172H30YqhD 5HVx1-CAR7 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR7pptase + pBL172H30YqhD 5HVx1-CAR8 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR8pptase + pBL172H30YqhD 5HVx1-CAR9 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR9pptase + pBL172H30YqhD 5HVx1-CAR10 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR10pptase + pBL172H30YqhD 5HVx1-CAR11 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR11pptase + pBL172H30YqhD 5HVx1-CAR12 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR12pptase + pBL172H30YqhD 5HVx1-CAR13 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR13pptase + pBL172H30YqhD 5HVx1-CAR14 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30CAR14pptase + pBL172H30YqhD 5HVx2-CAR1 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR1pptaseYqhD 5HVx2-CAR2 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR2pptaseYqhD 5HVx2-CAR3 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR3pptaseYqhD 5HVx2-CAR4 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR4pptaseYqhD 5HVx2-CAR5 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR5pptaseYqhD 5HVx2-CAR6 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR6pptaseYqhD 5HVx2-CAR7 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR7pptaseYqhD 5HVx2-CAR8 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR8pptaseYqhD 5HVx2-CAR9 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR9pptaseYqhD 5HVx2-CAR10 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR10pptaseYqhD 5HVx2-CAR11 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR11pptaseYqhD 5HVx2-CAR12 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR12pptaseYqhD 5HVx2-CAR13 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR13pptaseYqhD 5HVx2-CAR14 C. glutamicum PKC Δ gabD ΔlysE :: H30DavBHisA Δ gabD3:: H30DavTYahK harboring pCES208H30DavTYahKDavBHisA + pBL172H30CAR14pptaseYqhD

[Experimental Example 1] Measurement of 5HV productivity of PKC-5HA strain

The production capacity of 5-hydroxyvalerate, a key precursor for 1,5-pentanediol production, was measured using the PKC-5HV strain (accession number: KCTC 19163P) manufactured in step (3) of the above manufacturing example.

Under the same conditions as those described in the above Non-Patent Document 3 (Sohn et al., 2021), the PKC-5HV strain produced in Manufacturing Example (3) of the present invention was cultured in a flask for 120 hours. As a result, the strain of the present invention showed a similar level of 5HV production ability to the 5HV-7 strain without the additional introduction of a plasmid-based 5HV production system (pCES208H30DavTBHisA + pBL712H30YahK; Sohn et al., 2021) (see Fig. 2). In particular, the strain of the present invention did not show production of by-products such as lysine, 5AVA, and glutaric acid.

[Experimental Example 2] CAR screening through measurement of 1,5-pentanediol productivity

The production of 15PDO was compared for each recombinant strain produced using the PKC-5HA strain in [Table 7] through 120 h of flask culture (same culture conditions as Sohn et al., 2021) (Fig. 3). As a result, 1.2 g/L and 1.8 g/L of 1,5-PDO were produced in the C. glutamicum 5HVx1-CAR11 strain and the C. glutamicum 5HVx2-CAR11 strain expressing CAR11, respectively.

[Experimental Example 3] Production of 1,5-pentanediol using developed recombinant Corynebacterium (1)

To support higher 1,5-PDO production, batch fermentation of strains 5HVx1-11 and 5HVx2-11 (working volume 1.5 L in a 5 L CNS fermenter, the other conditions are the same as Sohn et al., 2021) was performed. The 5HVx1-CAR11 strain produced 7 g/L of 15PDO (Fig. 4), and the 5HVx2-CAR11 strain produced 9.97 g/L of 15PDO (Fig. 5).

[Experimental Example 4] Production of 1,5-pentanediol using developed recombinant Corynebacterium (2)

Fed batch culture was performed for the 5HVx1-CAR11 strain and the 5HVx2-CAR11 strain, respectively. Specifically, 1.5 L of GC100 medium (30 g of yeast extract, 30 g of ( _NH4 ) _{2S04 ,} 100 g of glucose, 0.5 g _of KH2PO4 _, 0.01 g of MgSO4 _{·7H2O, 0.01 g of MnSO4· 1H2O , 0.5} mg of biotin, and 0.3 mg of thiamine-HCl) was used in a fermenter (bioCNS 5 L) and cultured at 30℃, 600 rpm, DO (Dissolved Oxygen Level) 30%, and pH 6.9. Glucose, (NH ₄ ) ₂ S0 ₄ , and MgSO _4· 7H ₂ O were continuously supplied while controlling the pump flow rate to maintain the glucose concentration at 10 to 20 g/L.

For the 5HVx1-CAR11 strain, 17.83 g/L of 15PDO (yield of 0.13 mol/mol) was produced (Fig. 6), and for the 5HVx2-CAR11 strain, 17.52 g/L of 15PDO (yield of 0.13 mol/mol) was produced (Fig. 7).

Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC19163P 20240213

Attach electronic file of sequence list

Claims (17)

A gene that converts lysine to 5-aminovalerate and a gene that converts 5-aminovalerate to 5-hydroxyvalerate are introduced.
A vector transformed with a gene encoding an enzyme that converts 5-hydroxyvalerate into 5-hydroxyvaleraldehyde and/or a gene encoding an enzyme that converts 5-hydroxyvaleraldehyde into 1,5-pentanediol,
A recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The above vector comprises a first vector including a gene encoding an enzyme that converts 5-hydroxyvalerate into 5-hydroxyvaleraldehyde and a second vector including a gene encoding an enzyme that converts 5-hydroxyvaleraldehyde into 1,5-pentanediol.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The strain is further transformed with a third vector comprising a gene for converting lysine to 5-aminovalerate and/or a gene for converting 5-aminovalerate to 5-hydroxyvalerate.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
Defective gabD and lysE genes,
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
gabD3 gene deletion,
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The gene that converts the above lysine into 5-aminovalerate is a gene derived from Pseudomonas putida .
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In Article 6,
The gene that converts the above lysine into 5-aminovalerate comprises the sequence of sequence number 3.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The gene that converts the above 5-aminovalerate into 5-hydroxyvalerate includes a gene derived from Pseudomonas putida and a gene derived from E. coli YahK.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In Article 8,
The gene that converts the above 5-aminovalerate into 5-hydroxyvalerate comprises the sequence of sequence number 5.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The gene encoding the enzyme that converts the above 5-hydroxyvalerate into 5-hydroxyvaleraldehyde is a gene encoding carboxylic acid reductase (CAR).
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In Article 10,
The gene encoding the enzyme that converts the above 5-hydroxyvalerate into 5-hydroxyvaleraldehyde comprises the sequence of SEQ ID NO: 16.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
The gene encoding the enzyme that converts the above 5-hydroxyvaleraldehyde into 1,5-pentanediol is the YahD gene.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In Article 12,
The gene encoding the enzyme that converts the above 5-hydroxyvaleraldehyde into 1,5-pentanediol comprises the sequence of SEQ ID NO: 21.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In paragraph 4,
The endogenous gabD gene comprises the sequence of SEQ ID NO: 1, and the endogenous lysE gene comprises the sequence of SEQ ID NO: 2.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In paragraph 5,
The endogenous gabD3 gene comprises the sequence of sequence number 4.
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. In the first paragraph,
Deposited under the deposit number KCTC 19163P,
Recombinant Corynebacterium glutamicum strain for producing 1,5-pentanediol. A method for producing 1,5-pentanediol, comprising the step of culturing a recombinant Corynebacterium glutamicum strain of any one of claims 1 to 16 to obtain a culture.

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