Patent Application
20200407730
The present invention relates to a promoter system inducing expression of 3-hydroxypropionic acid (3-HP) and a method for biological production of 3-HP. 3-hydroxypropionic acid (3-HP) is an important synthetic immediate used in various chemical processes, and is used as a raw material for producing acrylic acid, acrylamide, 1,3-propanediol, malonic acid, and the like. 3-HP is also used for synthesis of a biodegradable polymer. Biological production of 3-HP using glycerol has been successfully accomplished through genetic engineering of key enzymes required for 3-HP production pathways in various bacteria. In detail, production of 3-HP has been confirmed in bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas denitrificans, and the like, by (over)expression of glycerol dehydratase which is a coenzyme B12-dependent enzyme, DhaB reactivase which is a glycerol dehydratase reactivase, and aldehyde dehydrogenase which is a NAD+-dependent enzyme, and the like through genetic engineering. Some recombinant strains including E. coli W DUBGK were able to produce more than 40 g/L of 3-HP for 48 hours, but had a difficulty in increasing 3-HP production beyond that. It was particularly observed that, as fermentation time for the production of 3-HP continues, problems that enzymes, such as glycerol dehydratase and aldehyde dehydrogenase, become unstable or lose activity occur. In this regard, one important reason why the activity of glycerol dehydratase disappears is due to a mechanism called suicidal inactivation. In such a mechanism, coenzyme B12 which is a coenzyme of glycerol dehydratase is irreversible damaged during a dehydration reaction from glycerol to 3-hydroxypropionaldehyde (3-HPA), and such a deactivation reaction is promoted in the presence of oxygen. In recent years, to ease deactivation-based on such a mechanism above, mutant glycerol dehydratase has been developed according to site-directed mutagenesis. Several mutant enzymes have been found to have improved enzyme stability, but enzyme activity thereof has been observed to be significantly reduced when compared to conventional enzymes.
Another reason for the problems above is due to toxicity of 3-HPA which is a highly reactive intermediate. When glycerol dehydratase or aldehyde dehydrogenase is present with 3-HPA, activity of glycerol dehydratase or aldehyde dehydrogenase decreases depending on a concentration of 3-HPA. Aldehydes are known to react with amino acid residues, such as ε-amino acid (NH3+), a sulfhydryl group (—C—SH), and an imidazole group that are present in lysine, cysteine, and histidine, respectively. Efforts have been made to improve the stability of many enzymes in the presence of aldehydes using site-directed mutagenesis and random mutagenesis, but have been limitedly succeeded.
An interesting alternative to solving the problems that the enzymes become unstable is continuous synthesis of new enzymes having activity during the whole period of cell culture. Theoretically, if new enzymes can be supplied as much as the enzymes that become unstable, the enzyme activity of the enzymes in cells can be kept constant. In particular, it is necessary to continuously express enzymes at the time when the growth of cells in the latter half of fermentation slows down and the total metabolic activity of the cells is decreased due to high concentrations of 3-HP.
The present invention provides an inducible promoter for 3-hydroxypropionic acid (3-HP) or a derivative thereof, the inducible promoter including a binding site to a LysR protein that is reactive to 3-HP or a derivative thereof.
The present invention also provides a recombinant gene expression cassette reactive to 3-HP or a derivative thereof, the recombinant gene expression cassette including a lysR gene encoding a LysR protein that is reactive to 3-HP or a derivative thereof, a promoter comprising a binding site to a LysR protein, and a gene encoding a target expression protein.
To achieve the technical problems above, the present invention provides an inducible promoter for 3-hydroxypropionic acid (3-HP) or a derivative thereof, the inducible promoter including a binding site to a LysR protein that is reactive to 3-HP or a derivative thereof.
In addition, the present invention relates to a recombinant expression vector including the inducible promoter for 3-HP or a derivative thereof, a recombinant microorganism transformed by the recombinant expression vector, and a method of producing 3-HP, the method including culturing the recombinant microorganism.
In addition, the present invention provides a recombinant gene expression cassette reactive to 3-HP or a derivative thereof, the recombinant gene expression cassette including a lysR gene encoding a LysR protein that is reactive to 3-HP or a derivative thereof, a promoter including a binding site to a LysR protein, and a gene encoding a target expression protein.
In addition, the present invention provides a recombinant expression vector including the recombinant gene expression cassette that is reactive to 3-HP or a derivative thereof, a recombinant microorganism transformed with the recombinant expression vector, a recombinant microorganism including the recombinant gene expression cassette, which is reactive to 3-HP or a derivative thereof, inserted in a chromosome of a host cell, and a method of producing a target expression protein, the method including culturing the recombinant microorganism.
Accordingly, in terms of efficiently maintaining expression of a 3-hydroxypropionic acid (3-HP) production enzyme, the inventors of the present invention have discovered a specific gene transcription promoter system, which induces the expression of 3-HP, in various microorganisms, and then, examined genetic and biochemical characteristics of the promoter system. The promoter system is an unusual system that has never been reported in documents yet, and consists of a transcription activating protein of 3-HP and a DNA sequence specifically binding to the transcription activating protein. The inventors of the present invention developed a recombinant strain capable of producing 3-HP at a high concentration from glycerol by overexpressing DhaB, GdrAB, and KGSADH by using the promoter system, thereby completing the present invention.
The present invention provides an inducible promoter for 3-HP or a derivative thereof, the inducible promoter including a binding site to a LysR protein which is reactive to 3-HP or a derivative thereof.
In addition, the present invention provides a recombinant expression vector including the inducible promoter for 3-HP or a derivative thereof. Preferably, the recombinant expression vector may further include a gene encoding a foreign protein linked to the inducible promoter for 3-HP or a derivative thereof. More preferably, the foreign protein may be glycerol dehydratase (DhaB), DhaB reactivase (GdrAB), or α-ketoglutaric semialdehyde dehydrogenase (KGSADH), but embodiments of the present disclosure are not limited thereto.
In addition, the present invention provides a recombinant microorganism transformed with the recombinant expression vector. Preferably, the recombinant microorganism may have producibility of 3-HP. More preferably, the recombinant microorganism may be Pseudomonas denitrificans, and more preferably, may be a stain of Pseudomonas denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhI from which 3hpdh, 3hibdh, and mmsadh genes relating to degradation of 3-HP are deleted in a strain of P. denitrificans, but embodiments of the present disclosure are not limited thereto.
In addition, the present invention provides a method of producing 3-HP, the method including culturing the recombinant microorganism.
In addition, the present invention provides a recombinant gene expression cassette reactive to 3-HP or a derivative thereof, the recombinant gene expression cassette including a lysR gene encoding a LysR protein that is reactive to 3-HP or a derivative thereof, a promoter comprising a binding site to a LysR protein, and a gene encoding a target expression protein.
In addition, the present invention provides a recombinant expression vector including the recombinant gene expression cassette that is reactive to 3-HP or a derivative thereof, and a recombinant microorganism transformed with the recombinant expression vector.
In addition, the present invention provides a recombinant microorganism including the recombinant gene expression cassette that is reactive to 3-HP or a derivative thereof inserted in a chromosome of a host cell. It will be obvious to one of ordinary skill in the art that, even if the recombinant gene expression cassette is inserted into the genome of the host cell, the same effect as the case where the recombinant vector is introduced into a host cell is made.
In the present invention, as a method of inserting the recombinant gene expression cassette to a chromosome of a host cell, any gene engineering method known in the art may be used. In one embodiment, a method using a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentivirus vector, or a non-viral vector may be used.
In addition, the present invention provides a method of producing a target expression protein, the method including culturing the recombinant microorganism. Preferably, the culturing of the recombinant microorganism may further include adding for 3-HP.
Preferably, the LysR protein or the promoter may be derived from a microorganism with 3-HP degradability. More preferably, the LysR protein or the promoter may be Achromobacter denitrificans, Acidovorax avenae subsp., Acidovorax sp., Acinetobacter baumannii, Aeromonas hydrophilia, Agrobacterium sp., Alcaligenes faecalis, Alcanivorax hongdengensis, Alicycliphilus denitrificans, Alteromonas marina, Amycolatopsis sp., Anaeromyxobacter dehalogenans, Azospirillum brasilense, Azotobacter vinelandii, Beijerinckia indica, Bordetella avium, Bradyrhizobium japonicum, Burkholderia ambifaria, Catenulispora acidiphilia, Caulobacter sp., Castellaniella defragrans, Chromobacterium violaceum, Collimonas arenae, Comamonas testosteroni, Corynebacterium vitaeruminis, Cupriavidus necator, Curvibacter gracilus, Delftia acidovorans, Ferrimonas balearica, Glaciecola nitratireducens, Gordonia bronchialis, Hahella chijuensis, Halomonas elongata, Hirschia litorea, Idiomarina sp., Janthinobacterium lividum, Kitasatospora setae, Kutzneria albida, Methylobacterium sp., Methylocystis sp., Novosphingobium sp., Oceanimonas smirnovii, Paracoccus sp., Parvibaculum lavamentivorans, Phenylobacterium kunshanensis, Photobacterium gaetbuleda, Polynucleobacter necessarius asymbioticus, Pseudoalteromonas carrageenovora, Pseudogulbenkiania sp., Pseudomonas denitrifcans ATCC13867, P. knackmussii, P. protegens, P. fluorescens, Pseudoxanthomonas spadix, Psychrobacter phenylpyruvicus, Ralstonia oxalatica, Rhodomicrobium vannielli, Segniliparus rotundus, Shewanella oneidensis, Simiduia agarovorans, Sinorhizobium meliloti, Sphingobium chlorophenolicum, Sphingomonas wittichii, Sphingopyxis alaskensis, Stenotrophomonas maltophilia, Streptomyces nodosus, Tatlockia micdadei, Thalassospira xiamenensis, Variovorax paradoxus, Verminephrobacter eiseniae, Vibrio furnissii, Xanthobacter autotrophicus, Xanthomonas campestri, and Xanthomonas oryzae, but embodiments of the present disclosure.
Preferably, the LysR protein may have an N-terminal domain having a helix-turn-helix structure and binding to DNA, a C-terminal domain binding to 3-HP or a derivative thereof, and a C-terminal domain contributing to stabilization of a LysR protein dimer, but embodiments of the disclosure are not limited thereto.
More preferably, the N-terminal domain having a helix-turn-helix structure and binding to DNA may include an amino acid sequence represented by SEQ ID NO: 1 or 2, the C-terminal domain binding to 3-HP or a derivative thereof may include an amino acid sequence represented by SEQ ID NO: 3, and the C-terminal domain contributing to stabilization of a LysR protein dimer may include an amino acid sequence represented by SEQ ID NO: 4, but embodiments of the present disclosure are not limited thereto.
“X” or “Xaa” described in SEQ ID NOs: 1 to 4 does not refer to particular amino acid, meaning that any amino acid may be used. More preferably, the LysR protein may be a LysR protein having Genebank ID represented by Table 4 and 5, but embodiments of the disclosure are not limited thereto.
Preferably, the binding site to the LysR may include two LysR protein dimers that are bonded to each other, and may include a base sequence selected from SEQ ID NOs: 5 to 43, wherein an inverted repeat sequence and another inverted repeat sequence paired with therewith may be repeated twice in the binding site to a LysR protein, wherein the inverted repeat sequence may consist of a base sequence represented by one selected from SEQ ID NOs: 5 to 43, but embodiments of the disclosure are not limited thereto.
More preferably, the binding site to a LysR protein may consist of a base sequence represented by SEQ ID NO: 44 or 45.
“n” described in the SEQ ID NOs: 5 to 43 does not refer to a particular base, meaning the any base may be used.
The SEQ ID NO: 44 or 45 may be a promoter base sequence derived from Pseudomonas denitrificans ATCC13867.
Preferably, the derivative may be 3-hydroxyisobutyrate (3HIB) or 3-hydroxybutyrate (3-HB), but embodiments of the disclosure are not limited thereto.
The term “vector” as used herein refers to a self-replicating DNA molecule that is used to carry the clone gene (or other fragments of the clone DNA).
The term “expression vector” as used herein refers to a recombinant DNA molecule including a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked to a particular host organism. The expression vector may include at least one selective marker. The marker may be a nucleic acid sequence having characteristics that can be generally selected by a chemical method, and may include all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples of the marker include antibiotic resistance genes, such as ampicillin, kanamycin, G418, Bleomycin, hygromycin, and chloramphenicol, but embodiments of the present disclosure are not limited thereto. Such a marker can be appropriately selected from one of ordinary skill in the art.
Hereinafter, the present inventive concept will be described below in further detail through Examples and Comparative Examples. However, such Examples are for illustrative purposes only and do not limit the scope of the present inventive concept.
1. Materials
A number of strains including Achromobacter denitrificans and Acinetobacter baumannii were obtained from the Korean Culture Center of Microorganisms (KCCM). A number of strains including Acidovorax avenae subsp. and Agrobacterium sp. were purchased from the Korean Collection for Type Cultures (KCTC). A number of strains including Alicycliphilus denitrificans and Anaeromyxobacter dehalogenans were obtained from DSM in Germany. A number of strains including Aeromonas hydrophilia and Pseudomonas denitrificans ATCC13867 purchased from ATCC in the United States. A primer was synthesized by COSMO GENETECH (Seoul, Korea). 3-HP was purchased from Tokyo Kasei Kogyo (TCI America, Portland, Oreg.) in Japan. An enzyme extract (Cat. 212750) and tripton (Cat. 211705) were purchased from Difco (Becton Dickinson; Franklin Lakes, N.J.). All chemicals and enzymes not mentioned were purchased from Sigma Aldrich (St. Louis, Mo.).
2. 3-HP Production of Proliferating Cells and 3-HP Degradation in Dormant Cells
Shake flask experiments were carried out in a 250 mL non-baffled Erlenmeyer flask in a volume of 30 mL in shaking incubator at a temperature of 37° C. and a stirring speed of 200 rpm. Experiments on 3-HP production by P. denitrificans were performed under conditions where an M9 medium having a modified volume of 30 mL was added to a 250 mL non-baffled Erlenmeyer flask for culturing in a shaking incubator at a temperature of 37° C. at a stirring speed of 200 rpm. Here, compositions of the modified M9 medium for culturing a strain included 100 mM phosphate buffer (pH 7.0), 0.25 g/L of MgSO4 7H2O, 1.0 g/L of NaCl, 1.0 g/L of NH4Cl, and 3-HP 25 mM.
Experiments on the dormant cells were carried out to investigate 3-HP degradation in a total of 69 microorganisms including P. denitrificans, and bacteria used in the experiments are shown in Table 1. To prepare active cells, 3-HP was added to a nutrient-enriched medium specified for each strain, and then, cultured in a for 250 mL non-baffled erlenmeyer flask at a volume of 50 mL. Strain culture was performed at a temperature of 37° C., and when OD600 of cells reached about 1 to 1.5, cells were harvested by centrifugation at a speed of 5,000 rpm for 10 minutes. The precipitated cells were washed with 100 mM phosphate buffer (pH 7.0), and resuspended with 252 mmol/L of 3-HP in the same buffer. The above-mentioned cell harvesting, washing and resuspending procedures were performed prior to the 3-HP degradation experiments. Samples were taken periodically to investigate the concentration of 3-HP.
Achromobacter
denitrificans
Acidovorax
avenaesubsp.
Acidovorax sp
Acinetobacter
baumannii
Aeromonas
hydrophilia
Agrobacterium sp.
Alcaligenes
faecalis
Alcanivorax
hongdengensis
Alicychphilus
denitrificans
Alteromonas
marina
Amycolatopsis sp.
Anaeromyxo-
bacter
dehalogenans
Azospirillum
brasilense
Azotobacter
vinelandii
Beijerinckia
indica
Bordetella
avium
Bradyrhizobium
japonicum
Burkholderia
ambifaria
Catenulispora
acidiphilia
Caulobacter sp.
Castellaniella
defragrans
Chromobacterium
violaceum
Collimonas
arenae
Comamonas
testosteroni
Corynebacterium
vitaeruminis
Cupriavidus
necator
Curvibacter
gracilus
Delftia
acidovorans
Ferrimonas
balearica
Glaciecola
nitratireducens
Gordonia
bronchialis
Hahella
chijuensis
Halomonas
elongata
Hirschia
litorea
Idiomarina sp.
Janthino-
bacterium
lividum
Kitasatospora
setae
Kutzneria
albida
Methylo-
bacterium sp.
Methylocystis sp.
Novo-
sphingobium sp.
Oceanimonas
smirnovii
Paracoccus sp.
Parvibaculum
lavamentivorans
Phenylo-
bacterium
kunshanensis
Photobacterium
gaetbuleda
Polynucleo-
bacter
necessarius
asymbioticus
Pseudo-
alteromonas
carrageenovora
Pseudo-
gulbenkiania
Pseudomonas
denitrificans
Pseudomonas
knackmussii
Pseudomonas
protegens
Pseudomonas
fluorescens
Pseudo-
xanthomonas
spadix
Psychrobacter
phenylpyruvicus
Ralstonia
oxalatica
Rhodomicrobium
vannielli
Segniliparus
rotundus
Shewanella
oneidensis
Simiduia
agarovorans
Sinorhizobium
meliloti
Sphingobium
chloro-
phenolicum
Sphingomonas
wittichii
Sphingopyxis
alaskensis
Stenotro-
phomonas
maltophilia
Streptomyces
nodosus
Tatlockia
micdadei
Thalassospira
xiamenensis
Variovorax
paradoxus
Vermine-
phrobacter
eiseniae
Vibrio
furnissii
Xanthobacter
autotrophicus
Xanthomonas
campestri
Xanthomonas
oryzae
3. RNA Extraction and Reverse Transcription Polymerase Chain Reaction
An M9 medium was used for culture of a P. denitrificans strain (ATCC 13567), and a nutrient medium specified for each strain was used for culture of other microorganisms shown in Table 1. Then, when examining the effect of 3-HP, 25 mM of 3-HP was added to the media provided herein. all the strains were cultured in a shaking incubator at a temperature of 37° C. at a stirring speed of 200 rpm under an aerobic condition, and cells were harvested when the cultured cells reached an exponential growth phase. After the cells were harvested in an amount of about 5×108, centrifugation was performed thereon at 5,000 g for 10 minutes. Then, 500 μl of RNA later solution (Ambion, UK) was immediately added to the precipitated cells, and then, the mixed solution was resuspended. RNA was extracted by using a total RNA isolation kit (Macherey-Nagel, Germany). 1 μg of total RNA was used for synthesis of 20 μl first-strand cDNA, and a SuperScript III first-strand synthesis system provided by Invitrogen was used for synthesis of cDNA.
A reverse transcription polymerase chain reaction was performed by using a SYBR green step with a One Real Time PCR system (Applied Biosystems, USA). In a reaction solution (20 μL) for the reverse transcription polymerase chain reaction, 300 ng of cDNA, 10 μL 2× Power SYBR Green PCR Master Mix (Applied Biosystems, UK), 5 pmol of forward and reverse primers, and DEPC treated water were contained. Conditions for the reverse transcription polymerase chain reaction were determined as follows: denaturation, 1 cycle of 95° C. for 30 seconds; amplification, 40 cycles of 95° C. for 15 seconds, 62° C. for 30 sections, and 72° C. for 30 seconds. Prior to performing the reverse transcription polymerase chain reaction, PCR was performed to confirm the effect of the primers used in the experiments for measurement of mRNA levels, and relative quantification for mRNA levels was calculated according to the ΔΔCT method.
4. Gene Cloning of LysR Protein, Protein Production, and Separation-Purification
In the case of P. denitrificans, two operons, i.e., 3HPDH (hereinafter, referred to as C3 system) and 3HIBDH-IV (hereinafter, referred to as C4 system), that are involved in the 3-HP degradation were present, as well as LysR proteins, such as C3-LysR and C4-LysR, that regulate transcription of the operons. Among the proteins, protein production was attempted for C4-LysR. E. coli BL21 (DE3) was used as a host and E. coli strain Top10 was used for cloning and maintaining of plasmids. C4 LysR genes were amplified in the P. denitrificans genome by PCR, cloned into a pET30b(+) plasmid, and E. coli strain Top10 was added thereto to confirm sequences, and then, E. coli BL21 (DE3) was added thereto. For protein purification, a His tag was labeled at a C-terminus site. To express the LysR protein in an active soluble form, the LysR protein was co-expressed along with several chaperone plasmids, such as pG-KJE8, pGRO7, pG-TF2, and pTF-16. For use as a medium, an LB medium appropriately supplemented with kanamycin, chloramphenicol, L-arabinose, or the like was used and cultured under aerobic conditions. When the cell concentration reached OD of 0.6, 0.1 mM of IPTG was added to induce production of the LysR protein. For water-soluble expression of the LysR protein, various culture conditions were examined, and eventually, the LysR protein was cultured at a temperature of 25° C. at a speed of 150 rpm for 10 hours. The cultured cells were obtained by centrifugation, washed with 100 mM (pH 7) phosphate buffer, resuspended in binding buffer, and then, disrupted by French Press. Afterwards, the resulting cells were subjected to centrifugation again, and solid and undisrupted cells were removed while the solution was purified by using a Ni-affinity column. followed by being stored in a 20% glycerol solution at a temperature of 80° C.
5. Measurement of Electrophoretic Mobility Shift Assay (EMSA) for Analyzing Protein-DNA Binding Under In Vitro Conditions
To study binding between the separated C4-LysR gene and a promoter region thereof under in vitro conditions, a DNA fragment of the promoter region was synthesized (see
6. Analysis Method
The cell concentration was measured by using a double beam spectrophotometer (Lambda 20, Perkin-Elmer, Norwalk, Conn.) with a cuvette having a length of 10 mm. The 3-HP concentration was measured by using a high performance liquid chromatography (HPLC) (Raj et al, 2008). For preparation of a sample, a sample taken for HPLC analysis was centrifuged at 10,000×g for 10 minutes to remove cell precipitates, and a tuffryn membrane filter (Acrodisc; Pall Life Sciences, Port Washington, N.Y.). A column used for HPLC analysis had a size of 300 mm×7.8 mm Aminex HPX-87H (Bio-Rad, USA) and 2.5 mmol/L of H2SO4 was used as a mobile phase at a temperature of 65° C.
7. Results
(1) Screening of 3-HP Inducible Promoter in P. denitrificans
3-HP is a carbon compound rarely present in natural environments, and there are few reports on its use as a carbon substrate or its biological degradation. However, recently, the inventors of the present invention found that P. denitrificans degraded 3-HP rapidly in both a growth phase and a non-growth phase. During the growth phase of the cells, P. denitrificans was able to be use 3-HP as a sole carbon source and an energy source (
The expression of the genes, i.e., 3hpdh, 3hibdhIV, and mmsadh, was similar with 3-HP in size, but the possibility of being amplified by other compounds having different structures from the genes above was also examined (
(2) Analysis of 3-HP Inducible Gene Expression System
LysR-type transcriptional regulators (LTTRs) are known as transcriptional activators that regulate the same catabolic pathway as the aromatic compounds. In general, genes encoding the LTTRs are located in front of a population of genes involved in degradation of aromatic compounds and regulate the compound degradation. To identify the 3-HP degradation pathway, the gene structure analysis of operons related to 3HPDH and 3HIBDH-IV of P. denitrificans was performed. As a result, it was confirmed that the LTTRs were located in a similar gene sequence in the front part of the 3-HP degrading genes (
More detailed analysis of the C4 LysR inducible promoter present in P. denitrificans was performed. O1 and O2 operators between a LysR gene and a mmsadh gene were each located at positions −58 and −9 relative to a transcription start site of the mmsadh gene, and each had an inverted repeat (
The effect of the O1 and O2 operators on biosynthesis of the C4-LysR protein and the mmsadh expression was examined by using a green fluorescent protein (GFP) as a reporter (Table 2). In Table 2, the effect of the C4-LysR protein and the O1 and O2 operators on the expression of the C4-LysR and the mmsadh. The relative size of the GFP was shown relative to the wild type in the absence of 3-HP. the C4-LysR protein was either not present or was produced by a constitutive promoter, and a plasmid was prepared to control the expression of the GFP upon the existing promoter having the O1 and O2 operators. Then, the experiment was carried out by inserting the prepared plasmid into a host of P. denitrificans from which genes between the C4-LysR and the mmsadh was deleted. Accordingly, the expression of the GFP was suppressed by the C4-LysR protein itself. That is, when the C4-LysR is expressed, the GFP expression was reduced by more than 10-fold. In addition, the expression regulation was also constant regardless of the presence of 3-HP. In this regard, it was confirmed that the transcription of C4-LysR mRNA was negatively regulated by the C4-LysR protein. This experiment was repeated by using a promoter that randomized the O1 and O2 operators to eliminate the symmetrical dyad. As a result, in the case where the O1 or O2 operator site was randomized, the GFP expression was not regulated by the C4-LysR protein, referring that the C4-LysR protein in the P. denitrificans strain was negatively regulated by the strain itself.
Meanwhile, the effect of the O1 and O2 operators on the mmsadh gene expression was examined in a similar manner. That is, the C4-LysR protein was constitutively expressed, and a plasmid was prepared so that GFP can be located after a promoter having O1 and O2 promoters. Here, an O1 or O2 operator was mutated so that the symmetrical dyad of an O1 or O2 site was randomized. As a result, when the O1 or O2 site was mutated, the phenomenon of up-regulation of transcription by 3-HP disappeared. That is, it means that the O1 and O2 operators are all essential sites for up-regulation of the expression by 3-HP. Thus, it has been found that the promoter of the present invention is a promoter requiring the presence of the O1 and O2 operators.
1GFP was used as a reporter protein, and that is, a plasmid to which a GFP gene was inserted at a C4-LysR position or a mmsadh position was used.
23-HP was added in a concentration of 25 mM.
3When O1 and O2 were randomized, C4-LysR used a promoter expressed weakly but constitutively.
Meanwhile, the LTTRs protein was known to consist of an N-terminal which is a DNA binding domain (helix-turn-helix motif), a C-terminal which is a substrate binding domain, and a linker connecting the N-terminal and the C-terminal. The LysR protein forms a homodimer to bind RS and AS, and when an effector molecule (3-HP in the case of the present invention) binds each of the LysR proteins, due to protein-protein interaction between two LTTR dimmers the LTTR forms a tetramer, which in turn causes a structural change in DAN associated with the LysR protein. A derivative specifically binding to the LTTR tetramer is known to cause a structural changes of the LysR protein, and subsequently alter the structure of the promoter region DNA, thereby ultimately helping binding of a RNA polymerase to the promoter (
When examining the structure of the C4-LysR protein which has been emphasized in the present invention, it was also resulted that the C4-LysR protein consists of an N-terminal which is a DNA binding domain (helix-turn-helix motif), a C-terminal which is a substrate binding domain, and a linker connecting the N-terminal and the C-terminal (
To confirm that the LysR protein is a transcriptional regulator, genes encoding the C3 and C4 LysR proteins were removed from P. denitrificans chromosomes, and then, transcription induction of transcriptionally regulated genes (mmsadh, 3hibdhIV, and 3hpdh) was examined. First, when the C4 LysR gene was removed, regardless of the presence of 3-HP, the expression of the mmsadh and 3hibdhIV genes was low, and addition of 3-HP did not increase the expression. When the C3 LysR gene was removed, the expression of the 3hpdh gene was not amplified by addition of 3-HP. However, when C3 LysR or C4 LysR gene was expressed again by using a plasmid in strains from which the C3 LysR or C4 LysR gene was removed (complementation experiment), the gene expression was amplified and restored to the same level as that of the wild type strain upon 3-HP. In this regard, it was confirmed that the C3 LysR protein and the C4 LysR protein were each a transcriptional regulatory protein that regulates the expression of the mmsadh, 3hibdhIV, and 3hpdh in cells.
(3) In Vitro Examination of Binding Characteristics of C4-LysR Protein and O1 and O2 Operator Sites
To examine in vitro characteristics of the C4-LysR protein, the C4-LysR protein having a histidine tag at the C-terminal was produced in E. coli and purified therefrom. First, the His tag was labeled at the C-terminal and the N-terminal, and then, the above-mentioned complementation experiment was carried out. As a result, both cases showed the same performance as the wild type LysR having no His tag. Accordingly, among two recombinant LysR proteins, only the C-His tagged LysR was subjected to biochemical experiment. The recombinant LysR was mostly expressed in an insoluble form in E. coli. Here, detailed optimization experiment for the expression conditions (temperature, pH, medium composition, IPTG concentration, etc) was also carried out. In addition, the influence of the various chaperon proteins also carried out. As a result, a sufficient amount of water-soluble C4-LysR was able to be produced from E. coli at a comparatively low temperature of 25° C. under common conditions with 0.1 M IPTG, an LB medium, and GroEL-ES chaperon (
The binding between the genetically recombinant C4-LysR and the promoter DNA through EMSA experiments (
When F12 had higher affinity than F12M or F1M2, it means that the binding of O1 and O2 of the LsyR is cooperative. That is, when the LysR protein binds to the O1 site having high affinity, the binding to the O2 site is promoted. The results of the EMSA experiments showed that F12 always had low mobility, compared to F12M or F1M2. In addition, at a low concentration of the LysR, the bane having only one shift was shown, meaning that there is always more LysR proteins bound to F12. That is, when the LysR binds to F12, it means that the LysR binds to both O1 and O2 sites. F12 having higher affinity than F12M or F1M2 and having lower mobility than F12M or F1M2 shows the fact that the binding of the LysR protein to the O1 and O2 sites in F12 was cooperative. From these results of the EMSA experiments, the important properties of the 3-HP inducible promoter can be summarized as follows: (i) the promoter is characterized by having two or more inverted repeat sequence pairs, each consisting of 9 bases, and providing a binding site of the LysR protein; (ii) the LysR protein is able to bind to the promoter regardless of the binding with an inducer molecule, but the improvement in transcription efficiency is only shown by the LysR protein associated with an inducer molecule, wherein the inducer molecule may be 3-HP or 3-HIB and 3-HB that are structurally similar with 3-HP; (iii) the promoter provides a site where two LysR protein dimmers are bound, wherein the binding is cooperative each other; (iv) the promoter provides a structure that can interact with the LysR protein when the RNA polymerase binds thereto; and (v) the promoter includes the O1 and O2 operators, wherein each operator consists of 9 bases and has well conserved inverted repeat sequences.
(4) Virtual Search of 3-HP Inducible Gene Expression System and Analysis of Characteristics of the Expression System
To find out a new 3-HP inducible gene expression system, putative LysR regulatory genes and mmsadh, 3hipdh, 3hpdh, and the like were screened from various microorganisms, based on the gene homology of P. denitrificans. The BlastP similarity search results show that similar 3-HP inducible gene expression systems are present in various microorganisms, and among well-known microorganisms, the presence of a population of genes assumed to be 3HIBDH (C4 system) and 3HPDH(C3 system) was discovered (
In a total more than 150 microorganisms, the 3-HP inducible gene expression systems were discovered, and depending on the presence of the C3-LysR and the C4-LysR and the gene arrangement characteristics, the gene expression systems were divided into a total of 16 groups. Among the groups, 9 groups had both C4 the C3 systems, and 7 groups had only the C4 system. A group having only the C3 system was not discovered. In addition, the case of the C3 system, a gene encoding the LysR protein and a gene regulating the expression thereof by the LysR protein were all characterized in that the direction of transcription was opposite. In the case of the C4 system, a gene encoding the LysR protein and a gene regulating the expression thereby of the LysR were mostly characterized in that the direction of transcription was opposite. However, in the microorganisms belonging to Groups 15 and Group 16, the direction of transcription was the same.
The characteristics of the promoter sequences that are reactive to the C3 and C4 LysR proteins were analyzed. In the same manner as in P. denitrificans, two tandem operator sites (designated as O1 and O2) were present. Two operators had a dyad symmetry, wherein each of the inverted repeats consists of 9 bases. Here, the distance between the centers of the dyad symmetry was 50 bases, so that when the LysR protein binds to the O1 and O2 operator sites, the LysR protein was spaced to bind in the same direction. In addition, the 9 bases within the inverted repeat sequence was well conserved in many microorganisms.
The palindromic binding site to the LysR that is reactive to 3-HP was conserved in various microorganisms. However, depending on species, only the O1 operator, i.e., a primary/repression binding site (PBS/RBS), was conserved (Table 3). In addition, in all species, PBS including the conserved T-N11/12-A motif and having high affinity was present adjacent to positions −65 and −75 relative to a transcription start site (TSS). That is, the O2 operator, i.e., a secondary/activation binding site (SBS/ABS) motif, had low sequence conservation, so that in silico prediction of the ABS motif is complex and difficult. The RBS and ABS sites were each reported to play a key role in autogenic inhibition and activation. Despite having a common function as proteins that are reactive to 3-HP, the 3-HP-LysR protein had low sequence similarity among other genus and had high sequence similarity within the same genus. Therefore, it is not logically wrong that the DNA sequence of the operator region to which the LysR protein binds is different among different genus. Here, transcription factors (promoters; −10 and −35 regions) were predicted by using BPROM and BDGP tools.
The 9 base sequences shown in Table 3 refer to the conserved regions corresponding to the binding sites to the LysR that is reactive to 3-HP in each genus, wherein the capital letters are bases that appear to be conserved in all target subjects.
Achromobacter
Acidovorax
Acinetobacter
Advenella
Aeromonas
Alcaligenes
Alcanivorax
Alicycliphilus
Anaeromyxobacter
Azospirillum
Azotobacter
Beijerinckia
Bordetella
Bradyrhizobium
Brucella
Burkholderia
Corynebacterium
Cupriavidus
Delftia
Ferrimonas
Glaciecola
Gordonia
Halomonas
Janthinobacterium
Marinobacter
Methylocystis
Phenylobaculum
Pseudomonas
Ralstonia
Shewanella
Sinorhizobium
Sphingobium
Stenotrophomonas
Tistrella
Variovorax
Verminephrobacter
Vibrio
Xanthobacter
Xanthomonas
Achromobacter sp.
Acidovorax
avenae subsp.
Acidovorax sp.
Acinetobacter
baumannii
Aeromonas
hydrophilia
Agrobacterium sp.
Alcaligenes faecalis
Alcanivorax
hongdengensis
Alicycliphilus
denitrificans
Alteromonas
marina
Anaeromyxobacter
dehalogenans
Azospirillum
brasilensse
Azotobacter
vinelandii
Beijerinckia
indica
Bordetella avium
Bradyrhizobium
japonicum
Burkholderia
ambifaria
Catenulispora
acidiphila
Caulobacter sp.
Castellaniella
defragrans
Chromobacterium
violaceum
Collimonas arenae
Comamonas
testosteroni
Corynebacterium
vitaeruminis
Cupriavidus
necator
Carvibacter gracilus
Delftia
acidovorans
Ferrimonas
balearica
Glaciecola
nitratireducens
Gordonia
bronchialis
Hahella chejuensis
Halomonas elongata
Hirschia sp.
Idiomarina sp.
Janthinobacterium
lividum
Kitasatospora setae
Kutzneria albida
Methylobacterium
Methylocystis sp.
Novosphingobium
Oceanimonas
smirnovii
Paracoccus sp.
Parvibaculum
lavamentivorans
Phenylobacterium
koreense
Photobacterium
gaetbuleda
Polynucleobacter
necessarius
asymbioticus
Pseudoalteromonas
carrageenovora
Pseudogulbenkiania
Pseudomonas
denitrificans
Pseudomonas
knackmussii
Pseudomonas
protegens
Pseudomonas
fluorescens
Pseudoxanthomonas
spadix
Psychrobacter
phenylpyruvicus
Ralstonia oxalatica
Rhodomicrobium
vannielli
Segniliparus
rotundus
Shewanella
oneidensis
Simiduia
agarivorans
Sinorhizobium
meliloti
Sphingobium
chlorophenolicum
Sphingomonas
wittichi
Sphingopyxis
alaskensis
Stenotrophomonas
maltophilia
Tatlockia micdadei
Thalassospira
xiamenensis
Variovorax
paradoxus
Verminephrobacter
eiseniae
Vibrio furnissii
Xanthobacter
autotrophicus
Xanthomonas
campestri
Xanthomonas oryzae
Achromobacter sp.
Acidovorax
avenae
Acidovorax sp.
Acinetobacter
baumannii
Alcaligenes
faecalis
Alcanivorax
hongdengensis
Alicychphilus
denitrificans
Alteromonas
marina
Azospirillum
brasilense
Bordetella
avium
Bradyrhizobium
japonicum
Burkholderia
ambifaria
Castellaniella
defragrans
Chromobacterium
violaceum
Collimonas
arenae
Comamonas
testosteroni
Cupriavidus
necator
Carvibacter
gracilus
Delftia
acidovorans
Glaciecola
nitratireducens
Gordonia
bronchialis
Halomonas
elongata
Idiomarina sp.
Janthinobacterium
lividum
Parvibaculum
lavamentivorans
Polynucleobacter
necessarius
asymbioticus
Pseudogulbenkiania
Pseudomonas
denitrificans
Pseudomonas
knackmussii
Pseudomonas
protegens
Pseudomonas
fluorescens
Pseudoxanthomonas
spadix
Psychrobacter
phenylpyruvicus
Segnihparus
rotundus
Sinorhizobium
meliloti
Sphingobium
chlorophenolicum
Stenotrophomonas
maltophilia
Variovorax
paradoxus
Verminephrobacter
eiseniae
Vibrio
furnissii
Xanthobacter
autotrophicus
Xanthomonas
campestris
The analysis of the LysR protein was performed in the same manner. As a result BLAST search for the C4 LysR and C3 LysR sequences from the non-redundant NCBI database, it was confirmed that there were 126 and 132 sequences that were homologous to the DNA-binding helix-turn-helix region.
To further examine protein-ligand interaction between LysR and 3-HP, homology modeling and docking experiments were carried out. First, a structure available from the PDB database was used, and as a result of comparing sequence similarity of C4-LysR and C3-LysR in P. denitrificans using the structure, the sequence similarity of 35% or less was shown (PDB ID: 3SZP, 24% identical). Therefore, the modeling of the C4-LysR and the C3-LysR was carried out according to a threading method using MUSTER and LOMET server. As a result, the predicted C4-LysR and C3-LysR models were purified and verified using RAMPAGE, and it was confirmed by Ramachandran plot that 98% of the amino acid residues were in the appropriate region. Then, the active sites to which 3-HP binds in C4-LysR and C3-LysR were predicted by using COACH. Here, the valid models and the residues at the predicted active sites were used to perform docking experiments in the Maestro program on SCHRODINGER™. Target proteins (C4-LysR and C3-LysR) and a ligand (3-HP) were each examined by using Protein Preparation Wizard and LigPrep Wizard. To produce a grid box, a receptor grid generation tool was used, and ligand docking was performed in the produced grid box by using standard precision (SP) and eXtra precision (XP) docking settings. Consequently, the excellent docking pose was shown when having a docking score of 5.01 for C4-LysR and a docking score of 3.74 for C3-LysR. It was also confirmed that C4-LysR and C3-LysR had interaction with 3-HP and several molecules. Among amino acid residues of C4-LysR, it was examined that Asp-159, Thr-160, Pro-237, and Phe-239 had hydrogen bonding with 3-HP, and ARG24 had hydrophobic interaction with 3-HP (
(5) 3-HP Degradation and Expression of 3-HP Inducible Genes by Microorganisms Having 3-HP Inducible Genes
According to the gene structure analysis, the 3-HP degradation pathway was found to be present in various microorganisms. To evaluate the 3-HP degradation ability of various microorganisms, cells were suspended in a 100 mM phosphate solution containing 25 mmol/L of 3-HP and allowed to degrade 3-HP for 24 hours (Table 6). As a result, there was a difference in 3-HP degrading rates depending on the microorganisms, but all of the microorganisms were found to effectively degrade 3-HP. The transcription levels of the 3-HP degrading genes (3hpdh, 3hibdh, and mmsadh) were evaluated depending on the presence of 3-HP (Table 7). As shown in Table 7, 3-HP increased the expression of the 3hpdh, 3hibdh, and mmsadh genes by 6-fold, 14-fold, and 16-fold, respectively in the microorganisms. Such results refer that the 3-HP inducible systems are common in various microorganisms. Meanwhile, in comparison with P. denitrificans, the rate of transcription increase in other microorganisms was about 10 times lower than that of P. denitrificans, probably due to differences in culture conditions. That is, except for P. denitrificans, to improve the growth of other microorganisms, the microorganisms were cultured in a medium supplemented with a large amount of complex nitrogen source, but in this case, in addition to 3-HP, amino acids included in the complex nitrogen source or degradation products of the amino acids activated the transcription of 3hpdh, 3hibdh, and mmsadh at a certain point under conditions where 3-HP was not present so that the amount of the transcription was able to be highly maintained even in the absence of 3-HP.
Achromobacter
denitrificans
Acidovorax
avenae subsp.
Acidovorax sp.
Acinetobacter
baumannii
Aeromonas
hydrophilia
Agrobacterium sp.
Alcaligenes
faecalis
Alcanivorax
hongdengensis
Alicychphilus
denitrificans
Alteromonas
marina
Amycolatopsis sp.
Anaeromyxobacter
dehalogenans
Azospirillum
brasilensse
Azotobacter
vinelandii
Beijerinckia
indica
Bordetella
avium
Bradyrhizobium
japonicum
Burkholderia
ambifaria
Catenulispora
acidiphilia
Caulobacter sp.
Castellaniella
defragrans
Chromobacterium
violaceum
Collimonas
arenae
Comamonas
testosteroni
Corynebacterium
vitaeruminis
Cupriavidus
necator
Curvibacter
gracilus
Delftia
acidovorans
Ferrimonas
balearica
Glaciecola
nitratireducens
Gordonia
bronchialis
Hahella
chijuensis
Halomonas
elongata
Hirschia
litorea
Idiomarina sp.
Janthinobacterium
lividum
Kitasatospora
setae
Kutzneria
albida
Methylobacterium sp.
Methylocystis sp.
Novosphingobium sp.
Oceanimonas
smirnovii
Paracoccus sp.
Parvibaculum
lavamentivorans
Phenylobacterium
kunshanensis
Photobacterium
gaetbuleda
Polynucleobacter
necessarius
asymbioticus
Pseudoalteromonas
carrageenovora
Pseudogulbenkiania sp.
Pseudomonas
denitrificans ATCC13867
Pseudomonas
knackmussii
Pseudomonas
protegens
Pseudomonas
fluorescens
Pseudoxanthomonas
spadix
Psychrobacter
phenylpyruvicus
Ralstonia
oxalatica
Rhodomicrobium
vannielli
Segnihparus
rotundus
Shewanella
oneidensis
Simiduia
agarovorans
Sinorhizobium
meliloti
Sphingobium
chlorophenolicum
Sphingomonas
wittichii
Sphingopyxis
alaskensis
Stenotrophomonas
maltophilia
Streptomyces
nodosus
Tatlockia
micdadei
Thalassospira
xiamenensis
Variovorax
paradoxus
Verminephrobacter
eiseniae
Vibrio
furnissii
Kanthobacter
autotrophicus
Kanthomonas
campestri
Kanthomonas
oryzae
aThe amount of 3-HP degraded was calculated between 0 and 24 h.
Achromobacter denitrificans
Acidovorax avenae subsp.
Acidovorax sp.
Acinetobacter baumannii
Aeromonas hydrophilia
Agrobacterium sp.
Alcaligenes faecalis
Alcanivorax hongdengensis
Alicychphilus denitrificans
Alteromonas marina
Amycolatopsis sp.
Anaeromyxobacter dehalogenans
Azospirillum brasilensse
Azotobacter vinelandii
Beijerinckia indica
Bordetella avium
Bradyrhizobium japonicum
Burkholderia ambifaria
Catenulispora acidiphilia
Caulobacter sp.
Castellaniella defragrans
Chromobacterium violaceum
Collimonas arenae
Comamonas testosteroni
Corynebacterium vitaeruminis
Cupriavidus necator
Curvibacter gracilus
Delftia acidovorans
Ferrimonas balearica
Glaciecola nitratireducens
Gordonia bronchialis
Hahella chijuensis
Halomonas elongata
Hirschia litorea
Idiomarina sp.
Janthinobacterium lividum
Kitasatospora setae
Kutzneria albida
Methylobacterium sp.
Methylocystis sp.
Novosphingobium sp.
Oceanimonas smirnovii
Paracoccus sp.
Parvibaculum lavamentivorans
Phenylobacterium kunshanensis
Photobacterium gaetbuleda
Polynucleobacter necessarius
asymbioticus
Pseudoalteromonas carrgeenovora
Pseudogulbenkiania sp.
Pseudomonas denitrificans
Pseudomonas knackmussii
Pseudomonas protegens
Pseudomonas fluorescens
Pseudoxanthomonas spadix
Psychrobacter phenylpyruvicus
Ralstonia oxalatica
Rhodomicrobium vannielli
Segniliparus rotundus
Shewanella oneidensis
Simiduia agarovorans
Sinorhizobium meliloti
Sphingobium chlorophenolicum
Sphingomonas wittichii
Sphingopyxis alaskensis
Stenotrophomonas maltophilia
Streptomyces nodosus
Tatlockia micdadei
Thalassospira xiamenensis
Variovorax paradoxus
Verminephrobacter eiseniae
Vibrio furnissii
Xanthobacter autotrophicus
Xanthomonas campestri
Xanthomonas oryzae
Analysis of 3-HP inducible promoters was performed on the microorganism above. In the same manner as in P. denitrificans of the previous case, all of the promoters had O1 and O2 operator sequences, and it was confirmed that these sequences had a palindromic structure consisting of 9 bases. Although no further studies on these sequences have been made yet, binding to the LysR protein was expected in the same manner as in P. denitrificans.
In conclusion, to improve the 3-HP production in a biological manner, it is necessary to continuously produce new enzymes with enzyme activity. In the present invention, transcriptional regulators that are reactive to 3-HP and promoters have been screened from microorganisms including P. denitrificans, wherein the transcriptional regulators and promoters consist of LysR proteins and specific gene sequences binding to the LysR proteins. In addition, in the presence of 3-HP, it was found that the LysR family transcriptional regulator up-regulated the expression of the corresponding genes. The molecular modeling and docking experiments showed the presence of important residues for C4-LysR (ARG94, LYS96, GLU137, and ARG24) and C3-LysR (LEU74, THR190, THR28, THR73, VAL150, PRO167, PHE127, and PHE169). Therefore, the 3-HP inducible system is expected to be effectively used to regulate the 3-HP metabolic pathway.
1. Strains, Plasmids, and Experimental Materials
Bacterial species and plasmids used in the present study were shown in Table 8. An E. coli strain was provided from KCTC and a P. denitrificans strain was provided from ATCC. E. coli XL1-Blue was used for plasmid cloning and maintenance. A genome DNA separation kit and a pGEM-T vector were purchased from Promega (Madison, Wis., USA), a high-performance pfx polymerase was purchased from Invitrogen (Seoul, Korea), a DNA modification enzyme was purchased from New England Bio-Labs (Beverly, Mass., USA), and a Miniprep and DNA gel extraction kit was purchased from Qiagen (Mannheim, Germany). In addition, primers were purchased from Cosmogenetech Co. Ltd. (Seoul, Korea), bacto tryptone and yeast extract were purchased from Difco (Becton Dickinson; Franklin Lakes, N.J., USA), and other chemicals and enzymes were purchased from Sigma-Aldrich (St. Louis, Mo., USA).
E.
coli
P.
denitrificans
P.
denitrificans ATCC13867;
P.
dentirificans ATCC13867
P.
dentirificans ATCC13867
2. Development of Δ3HpdhΔ3hibdhIVΔ3hibdhI Deletion Mutant Strain of P. denitrificans
To understand the role of the 3-HP degrading genes, 3hibdhI was removed from the chromosome of P. denitrificans Δ3hpdhΔ3hibdhIV. A target gene was deleted therefrom based on a sacB negative counter-selection system. A sacB-Km cassette was introduced to NdeI and XbaI restriction sites of pQE-80L to prepare a pQSAK plasmid which is to be used for removal of the target gene. The genome DNA of P. denitrificans was used to obtain a DNA fragment including ˜700 bp upstream and downstream of the target gene by PCR. After DNA sequencing thereon, the DNA fragment was cloned into a pGEM-T vector. Afterwards, sub-cloning into the pQSAK plasmid was made again, and then, a mutant strain of P. denitrificans was developed through two rounds of recombination. The mutant strain was re-identified by PCR and sequencing performed thereon. The mutant strains thus obtained was designated as P. denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhI.
3. Plasmid Construction
A gene encoding glycerol dehydratase and reactivation enzyme was amplified by using a pUCPK′/PC3-dhaB-gdrAB, PC4-KGSADH plasmid, and an expression cassette was constructed by cloning a C3 promoter and a C3 terminator onto promoters at the 5′-end and 3′-end of gdrAB and dhab123 genes, respectively. The expression cassette was replicated at XbaI and SacI restriction sites of the pUCPK′/PC3-dhaB-gdrAB, PC4-KGSADH plasmid, and a resulting plasmid was designated as pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH. The pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH plasmid thus obtained was transformed with P. denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhI, and finally, a Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH) plasmid was constructed (
4. Determination of Enzyme Activity
The DhaB activity can be measured by measuring the KGSADH enzyme activity. 1 unit of the DhaB activity is defined as the amount of enzyme required to reduce 1 μmol of NAD+ to NADH for 1 minute. Briefly, first, 20 ul of 26 U/mg NAD+-dependent KGSADH was cultured at a temperature of 37° C. for 5 minutes in 50 mM potassium phosphate buffer (pH 8.0) (total volume of 1 mL) containing 1 mM DTT, 15 uM coenzyme B12, 3 mM MgCl2, and 1.5 mM ATP. Here, KGSADH contained 25% glycerol. A reaction was started by the addition of an appropriate amount of a cell extract containing 1.5 mM NAD+ and DhaB preheated at a temperature of 37° C., and was observed through changes in absorbance of NADH. The KGSADH activity was determined according to the method reported by Dr. Raj by measuring the reduction from NAD+ to NADH at 340. A reaction mixture including 50 mM potassium phosphate buffer (pH 8.0), 1 mM DTT, and an appropriate amount of an enzyme extract was cultured at a temperature of 37° C. for 5 minutes, and a reaction was initiated by the addition of 2.0 mM 3-HPA and 2.0 mM NAD+. The amount of NADH was determined by using 6.22×103 M−1 cm−1 of molar extinction coefficient (Δε340). 1 unit activity of KGSADH is defined as the amount of enzyme required to reduce 1 μmol of NAD+ to NADH for 1 minute. All enzyme activities were measured by using a crude cell extract.
5. Culture Medium and Culture Conditions
Unless otherwise stated, shaking culture was performed by using a 250 mL non-baffled Erlenmeyer flask containing 20 mL of a culture broth at a speed of 200 rpm at a temperature of 30° C. Here, an M9 culture medium supplemented with, per liter, MgSO4, 0.25 g; NaCl, 1.0 g; NH4Cl, 1.0 g; yeast extract, 1 g; glycerol, 100 mmol; L-glutamate, 5 g; tryptone, 2 g; and glucose 2.5 g was used, and the medium contained 100 mM potassium phosphate buffer (pH 7.0). If necessary, 12 μmol/L of coenzyme B12 was additionally injected, and then, the flask was sealed with an oxygen-permeable sponge plug. For the measure of cell mass, residual substrates, and metabolites, sampling was periodically done, and all shaking culture experiments were repeated three times, wherein a standard deviation of biomass and metabolites was less than 10%. The bioreactor experiments were carried out in a 1-L working volume in a 1.5-L capacity Biotron-LiFlus GM bioreactor (Biotron, Seoul, Korea).
An M9 culture medium for the bioreactor experiments was supplemented with, per liter, MgSO4.H2O, 0.25 g; NaCl, 1.0 g; NH4Cl, 1.0 g; yeast extract, 1 g; L-glutamate, 5 g; tryptone, 2 g; casamino acids, 2 g; glucose 2.5 g, and trace element solution, 10 mL/L, and the medium contained 100 mM of potassium phosphate buffer (pH 7.0). The culturing was performed in a fed-type culturing mode at a temperature of 30° C. while concentrated glycerol (10 M) and 7 mM glucose were periodically injected thereto. Here, pH was maintained to 7.0±0.1 by using 5 N NaOH and 2.5 N HCl. Air was continuously supplied at an agitation speed of 650 rpm at 1 vvm. During the culturing, a medium supplemented with tryptone, 2 g/L; casamino acids, 2 g/L; L-glutamate, 5.0 g/L; and yeast extract, 1 g/L was added every 6 hours to the bioreactor. Samples thereof were regularly analyzed to measure cell mass, residual substrates, and metabolites.
6. Analysis Method
The cell concentration was measured by using a spectrophotometer (Lambda 20, Perkin Elmer; Norwalk, Conn., USA) with a cuvette having a length of 10 mm. 1 unit of absorbance at 600 nm (OD600) was consistent with a dry cell volume of 0.3 g per liter. The protein concentration was analyzed according to a Bradford method with a microtiter plate reader based on bovine serum albumin (1420, Wallac Victor 2; Perkin Elmer). The concentrations of glycerol, 3-HP, and other metabolites were measured by HPLC, wherein the supernatant obtained by centrifugation performed on a culturing sample for 10 minutes at 10,000×g was filtered through a tuffryn-membrane (Acrodisc, Pall Life Sciences), and then, was eluted with by 300 mm×7.8 mm Aminex HPX-87H (Bio-Rad, USA) column using 2.5 mM H2SO4 as a mobile phase at a temperature of 65° C.
7. Results
(1) Shaking Flask Culture of Recombinant Pd Δ3HpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH)
The inventors of the present invention had observed in the previous study that the DhaB activity was significantly reduced due to self-destructive catalytic reaction by DhaB when glycerol conversion occurred. Such reduction may be caused by low expression of GdrAb that reactivates DhaB. In addition, it was also expected that the expression of GdrAB would be improved by sequentially arranging GdrAB and DhaB right below the PC3 promoter. Based on this hypothesis, a pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH plasmid was developed and introduced to a P. denitrificans (hereinafter, referred to as Pd) Δ3hpdhΔ3hibdhIVΔ3hibdhI strain for the production of 3-HP. The effect of changing the arrangement order of GdrAB and DhaB regarding the 3-HP production from glycerol was measured in Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH). The supplying effect of coenzyme B12 was examined by supplying 12 μM of coenzyme B12 at 0 h. Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-dhaB-gdrAB, PC4-KGSADH) was used as a control group. S1-S3 in
It was observed that the addition of cobalt led to the production of 3-HPA and 1,3-PDO at 12 h, but in a control strain, it was observed that 3-HPA (with cobalt) was not accumulated (Table 9). The yield of 3-HP from glycerol was about ˜1, meaning that glycerol supplied thereto was completed used for the production of 3-HP, and the produced 3-HP was not degraded again.
P.
denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhI
(2) Enzyme Activity
Time-dependent in vitro enzyme activity of DhaB and KGSADH was measured (
(3) Bioreactor Culture of DhaB, KGSADH Overexpression Recombinant Pd Δ3HpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH)
A fed-type glycerol-glucose bioreactor operation was performed by using Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-gdrAB-dhaB, PC4-KGSADH) and Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-dhaB-gdrAB, PC4-KGSADH) strains. In the bioreactor experiments, the concentrations of glucose and glycerol were maintained at low levels of 10 mM and 150 mM, respectively. For every 6 hours, glutamate was supplied for cell growth. As a result of the culture, similar cell growth was observed in the two bioreactors. The cell growth decreased in both cultures after 9 hours, but the cell growth continued until the end of the reaction. In bioreactor A, a Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK′/PC3-dhaB-gdrAB, PC4-KGSADH) strain was used (
In bioreactor B, the strain in which the gene order (dhaB and gdrAB) was changed was used, and as a result, the production of 3-HP was improved in the latter half of fermentation. Based on a production rate of 1.3 g/L/h and a yield of 0.95 mol/mol from glycerol, about 63±2 g/L of 3-HP was produced. In comparison with bioreactor A, the 3-HP production was increased by 5%. Although the results were not seen in the enzyme activity assays or flask experiments, the degree of GdrAB expression was significantly important in terms of the 3-HP production.
In conclusion, when the 3-HP production enzymes, i.e., DhaB, GdrAB, and KGSADH, were expressed, P. denitrificans was able to produce 3-HP from glycerol. The recombinant plasmids were developed by using two strong inducible promoters, PC3 and PC4, and Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI from which three genes were deleted was used as a host. To alleviate the degree of inactivation of DhaB, gdrAB was located in front of dhaB, so as to enhance the expression of gdrAB. The enzyme activity analysis and protein expression analysis by SDS-PAGE showed that the activity of DhaB was reduced by such a positional change. However, regardless of the reduced activity of DhaB, the production of 3-HP was improved. As a result of operating the fed-type bioreactor using new recombinant strains, 3-HP was obtained in high concentrations, high production rates, and high yields.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2015-0082593 | Jun 2015 | KR | national |
| 10-2016-0073091 | Jun 2016 | KR | national |
This application is a Division of U.S. patent application Ser. No. 15/735,585 filed on May 24, 2018, which is a National Stage Patent Application of PCT International Patent Application No. PCT/KR2016/006261 filed on Jun. 13, 2016, which claims priority to Korean Patent Application Nos. 10-2016-0073091 filed on Jun. 13, 2012 and 10-2015-0082593 filed on Jun. 11, 2015 which are all hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15735585 | May 2018 | US |
| Child | 17017717 | US |
