KR101974220B1 - Codon-Optimized Microbes for Improving the Content of Free Fatty Acids and Preparation Method thereof - Google Patents

Abstract

The present invention relates to a method for producing (a) a nucleotide sequence encoding an acyl- [acyl carrier protein] thioesterase derived from Streptococcus pyogenes in Escherichia coli Codon-optimizing to be suitable for expression to obtain the nucleotide sequence of the first sequence of Sequence Listing; (b) inserting a codon optimized nucleotide sequence of the acyl- [acyl transfer protein] thioesterase into an expression vector to prepare a recombinant vector; And (c) transforming the recombinant vector into Escherichia coli to produce recombinant Escherichia coli. The present invention further provides a method for producing an Escherichia coli strain expressing an acyl- [acyl transfer protein] thioesterase. The present invention increases the production of C12: 0 and C14: 0 saturated fatty acids and C18: 1 unsaturated fatty acids in the fatty acids produced from E. coli by 200-400% compared to the wild type, It is expected to be mass-produced, eco-friendly, and economically powerful.

Description

[0001] The present invention relates to a codon-optimized strain for improving free fatty acid content and a method for producing the same,

The present invention relates to a codon-optimized strain for improving the free fatty acid content and a method for producing the same.

Although interest in bioethanol, biodiesel, biogas and butanol represented by bioenergy is increasing, all of the types of bioenergy mentioned can be used as a fuel for power generation or transport, but some disadvantages of practical application and production methods , There is a growing interest in hydrocarbon-based compounds as a new renewable energy resource. Biodiesel using hydrocarbon compounds that are currently being commercialized is produced by chemical methods. Interest in recombinant strains capable of producing long chain fatty acids as metabolic products by biological methods is also increasing.

Escherichia coli is an important microorganism used for the production of bioenergy, including biochemicals, because it is fast growing and easy to genetically manipulate. However, very little fatty acid is the main raw material of biodiesel contained in E. coli. In particular, free fatty acid produced in cytosol is hardly produced. Recently, a number of studies have been conducted to increase fatty acids by gene transfer of plants to increase free fatty acid production (Zhang, Metabolic Engineering 13 (2011) 713722).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a recombinant microorganism for enhancing fatty acid productivity stably and effectively. As a result, the nucleotide sequence encoding the acyl- [acyl transport protein] thioesterase ( OPsp14 ) involved in the last stage of the fatty acid biosynthesis pathway of Streptococcus pyogenes was codon optimized -optimization), thereby confirming that the fatty acid production is increased in the case of the transformed E. coli transformed.

Accordingly, an object of the present invention is to provide a method for producing an acyl- [acyl transfer protein] thioesterase-expressing E. coli.

Another object of the present invention is to provide an Escherichia coli for expressing acyl- [acyl transfer protein] thioesterase.

It is still another object of the present invention to provide a method for producing fatty acids.

Another object of the present invention is to provide a nucleic acid molecule having a nucleotide sequence of the first sequence of the sequence listing.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a nucleic acid comprising the steps of: (a) amplifying a nucleotide sequence encoding an acyl- [acyl carrier protein] thioesterase derived from Streptococcus pyogenes Is codon-optimized to be suitable for expression in Escherichia coli to obtain the nucleotide sequence of the first sequence of Sequence Listing; (b) inserting a codon optimized nucleotide sequence of the acyl- [acyl transfer protein] thioesterase into an expression vector to prepare a recombinant vector; And (c) transforming the recombinant vector into Escherichia coli to produce a transformed Escherichia coli. The present invention also provides a method for producing the transformed Escherichia coli for expressing acyl- [acyltransfer protein] thioesterase.

The present inventors have sought to develop a recombinant microorganism for enhancing fatty acid productivity stably and effectively. As a result, the nucleotide sequence encoding the acyl- [acyl transport protein] thioesterase ( OPsp14 ) involved in the last stage of the fatty acid biosynthesis pathway of Streptococcus pyogenes was codon optimized -optimization), and the increase in fatty acid production was confirmed in the case of the transformed E. coli transformed.

The method for producing Escherichia coli of the present invention will be described in detail for each step.

Step (a): codon optimization (codon - optimization) Step

In order to produce the Escherichia coli of the present invention, the nucleotide sequence encoding the acyl- [acyl carrier protein] thioesterase of Streptococcus pyogenes is referred to as Escherichia coli , Lt; RTI ID = 0.0 > codon-optimization < / RTI >

The main characteristic of the present invention is that the acyl- [acyl transfer protein] thioesterase to be expressed in Escherichia coli in the present invention has a codon optimized nucleotide. As shown in FIG. 1, the acyl- [acyl transfer protein] thioesterase is involved in the final stage of the fatty acid biosynthetic pathway and has a reaction for hydrolyzing the fatty acyl-acyl carrier proteins .

As used herein, the term " codon-optimization " is one method of increasing protein production. When an exogenous protein is expressed in a host, the nucleotide sequence encoding the exogenous protein is codon-optimized for maximum expression, since the nucleotide sequence is not composed of the codons frequently used by the host. The codon optimization is basically to convert codons for which the frequency of expression of the desired nucleic acid sequence is not high to the host in a manner that reflects the frequency of codon expression in the host. http://www.kazusa.or.jp/codon/ presents codon frequencies for all living organisms.

Preferably, the codon optimized nucleotide sequence of the acyl- [acyl transfer protein] thioesterase is SEQ ID NO: 1 sequence.

The nucleotide sequence used in the present invention is interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence in addition to the above-mentioned sequence. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art to obtain a minimum of 80% Homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv . Appl . Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48: 443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc . Acids Res . 16: 10881-90 (1988); Huang et al., Comp. Appl . BioSci . 8: 155-65 (1992) and Pearson et al., Meth . Mol . Biol . 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Step (b): Preparation of recombinant vector

Then, a codon-optimized nucleotide sequence of the acyl- [acyl transfer protein] thioesterase is inserted into an expression vector to prepare a recombinant vector.

As used herein, the term " expression vector " is a linear or circular DNA molecule consisting of a fragment encoding a polypeptide of interest operably linked to an additional fragment provided in the transcription of the expression vector. Such additional fragments include promoter and termination coding sequences. The expression vector also includes at least one replication origin, at least one selection marker, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain both elements.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) This document is incorporated herein by reference.

Preferably, the expression vector of step (b) of the present invention is operatively linked to a promoter sequence, a nucleotide sequence encoding a codon-optimized acyl- [acyl transfer protein] thioesterase, and a transcription terminator sequence . The nucleotide sequence of the codon-optimized acyl- [acyl transfer protein] thioesterase of the present invention may be operatively linked to an expression control sequence and inserted into an expression vector. As used herein, 'operatively coupled' means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. Also, an " expression control sequence " means a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

The vector of the present invention can typically be constructed as a vector for expression. When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as trc promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ^λ promoter, pR ^λ Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of decoding, and a transcription / translation termination sequence. Promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) when E. coli (e.g. HB101, BL21, ) And the left promoter of phage lambda (pL ^lambda promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)). Preferably, the promoter used in the vector of the present invention is the trc promoter and the termination sequence is the rrrB terminator.

The vector injected into the host cell may be expressed in host cells, in which case a large amount of acetylenic reductase is obtained. For example, when the expression vector comprises the trc promoter, the host cell may be treated with IPTG (Isopropylthio-β-D-Galactoside) to induce gene expression.

Meanwhile, vectors that can be used in the present invention include plasmids such as pTrc99A, pSTV28, pSC101, pGV1106, pCYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61 (e.g.,? gt4? B,? -charon,?? z1 and M13) or viruses (e.g., SV40 and the like) Preferably pTrc99A is used.

On the other hand, the vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art and includes, for example, ampicillin, gentamycin, carbinicillin, chloramphenicol, streptomycin, kanamycin, There is a resistance gene for mitine and tetracycline. Preferably, ampicillin is used.

The expression vector of the present invention includes a promoter sequence and a nucleotide sequence of a gene to be expressed (structural gene), and the sequences are preferably linked in the order of 5'-3 '.

In the expression vector of the present invention, it is preferable that only the nucleotide sequence containing the RBS (ribosomal bindingsite) and the portion essential for the expression of the enzyme is inserted into the sequence so that the genes have the minimum length including the overexpression function of the enzyme, It is preferable in terms of reducing the metabolic burden.

Step (c): Preparation of transformed E. coli

Finally, the recombinant vector is transformed into E. coli to prepare a transformed E. coli.

The recombinant vector into which the nucleotide sequence of the codon-optimized acyl- [acyl transfer protein] thioesterase of step (b) is inserted is then introduced into Escherichia coli. The method of carrying the vector of the present invention into E. coli is CaCl ₂ (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 16: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic Acids Res., 16: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol. 6127-6145 (1988)), and preferably step (c) of the present invention is carried out by electroporation.

Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art, such as E. coli MG1655, E. coli W3110, E. coli JM109, E. coli Bacillus sp. Strains such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marsea And enterobacteria and strains such as various Pseudomonas species. Preferably, the host cell of the invention is E. coli MG1655.

The genotype of E. coli MG1655 is F (Fertility Factor) and lambda phage negative and has the characteristic that rph-1 (one base is deleted and more than 15 codons immediately before are frameshift). The E. coli MG1655 has a growth rate that is 85-90% lower than that of the rph-positive strain in the minimal medium and has a reduced expression of orotate phosphoribosyltransferase (PyrE) involved in pyrimidine biosynthesis Respectively. It can be purchased through ATCC (American Type Culture Collection, USA).

According to another aspect of the present invention, there is provided an Escherichia coli for expressing acyl- [acyltransfer protein] thioesterase produced by the method for producing Escherichia coli for expressing acyl- [acyltransfer protein] thioesterase of the present invention.

According to another aspect of the present invention, the present invention provides a process for preparing a fatty acid comprising the steps of:

 (a) culturing the above-mentioned acyl- [acyl transfer protein] thioesterase-expressing E. coli in a medium to biosynthesize a fatty acid; And

(b) obtaining said biosynthetic fatty acid.

In the fatty acid producing method of the present invention, the medium of step (a) may be cultured under LB medium or minimal medium.

According to a preferred embodiment of the present invention, the Escherichia coli expressing the acyl- [acyl transfer protein] thioesterase of step (a) is cultured in a minimal medium. Preferably, the minimal medium comprises glucose, sodium sulfate, tryptone, ammonium chloride, dipotassium phosphate, sodium phosphate, (NH ₄ ) ₂ -H-citrate, magnesium sulfate and thiamine. More preferably, the culture medium of step (a) is _{1-8 g / L Na 2 SO 4} , 1-10 g / L _{tryptone, 0.1-3 g / L NH 4 Cl} , 2-13 g / LK 2 HPO ₄ , 0.1-4.0 g / L NaH ₂ PO ₄ H ₂ O, 8-16 g / L (NH ₄ ) ₂ -H-citrate, 0.05-0.50 g / L MgSO ₄ and 0.005-0.100 g / , More preferably 2-7 g / L Na ₂ SO ₄ , 2-8 g / L tryptone, 0.4-2.3 g / L NH ₄ Cl, 4-11 g / LK ₂ HPO ₄ , including _{0.5-3.0 g / L NaH 2 PO 4} H 2 O, 10-14 g / L (NH 4) 2 -H- citrate, 0.1-0.4 g / L MgSO _4, and 0.007-0.08 g / L thiamine and , Most preferably 3-5 g / L Na ₂ SO ₄ , 4-6 g / L tryptone, 0.7-1.9 g / L NH ₄ Cl, 6-9 g / LK ₂ HPO ₄ , 1.2-2.5 g / L NaH ₂ PO ₄ H ₂ O, 11-13 g / L (NH ₄ ) ₂ -H-citrate, 0.2-0.3 g / L MgSO ₄ and 0.01-0.06 g / L thiamine.

The fatty acid produced by the fatty acid production method of the present invention has a yield of 1.5-4.0 times higher than that of wild-type E. coli.

According to a preferred embodiment of the present invention, the fatty acid prepared by the fatty acid production method of the present invention shows a 2-fold increase in the fatty acid after 8 hours from the addition of the IPTG, compared with the wild type strain (FIG. 11).

Preferably, the fatty acids produced by the fatty acid production process of the present invention are saturated fatty acids of C12: 0 and C14: 0 and unsaturated fatty acids of C18: 1.

Since the method for producing a fatty acid of the present invention uses the above-mentioned acyl- [acyltransfer protein] thioesterase-expressing E. coli, the description common to the two is omitted in order to avoid the excessive complexity of the present specification.

In accordance with another aspect of the present invention, the present invention provides a method for treating Streptococcus a nucleic acid molecule having a nucleotide sequence of a codon-optimized sequence listing sequence of Escherichia coli against an acyl- [acyl carrier protein] thioesterase from a pyogenes- derived acyl- to provide.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method capable of promoting the production of fatty acid by over-expressing an exogenous bacterial gene encoding a thioester raise related to the last stage of a fatty acid biosynthetic pathway by codon-optimization to be suitable for expression in E. coli .

(b) The present invention provides a method for producing an acyl- [acyl transfer protein] thioesterase-expressing E. coli.

(c) The present invention increases the production of C12: 0 and C14: 0 saturated fatty acids and C18: 1 unsaturated fatty acids in the fatty acids produced from E. coli by 200-400% compared to the wild type.

(d) The present invention provides a way for the developed strains to increase the production of fatty acids efficiently through changes in the composition of the medium.

(e) The present invention is expected to mass produce fatty acid production that will be the basis of bio-energy production, and to be eco-friendly as well as economic economically.

1, Escherichia coli shows the fatty acid biosynthesis pathway (FAS) from glucose in vivo and shows the biosynthesis pathway of acyl- [acyl carrier protein] thioesterase This is a schematic showing the location.
Figure 2 shows a nucleotide sequence coding for the acyl- [acyl carrier protein] thioesterase (EC 3.1.2.14) of Streptococcus pyogenes and a nucleotide sequence coding for codon optimization ( OPSp14 ) coding for the acyl- [acyltransfer protein] thioesterase of the codon- optimized Streptococcus piojeneris .
3A to 3E show results of electrophoresis on agarose gel after PCR. FIG. 3A shows the result of the amplification of the acyl- [acyl transport protein] thioesterase (EC3.1.2.14) gene ( OPSp14 ) of Streptococcus phyjensis codon-optimized in E. coli at the 753 bp position of the agarose gel The amplified results are shown. 3A is a size marker (Takara, Japan), and columns 2 and 3 show the result that the gene ( OPSp14 ) was amplified at the 753 bp position of the agarose gel. FIG. 3B shows the results obtained by inserting an acyl- [acyl transfer protein] thioesterase into a T-vector and treating with a restriction enzyme. In FIG. 3B, the first column is the size marker and the second column to the fifth column are the results showing that the T-vector and the gene ( OPSp14 ) separated by the restriction enzyme were separated. 3C shows the result of inserting acyl- [acyl transfer protein] thioesterase into pTrc99A and treating with restriction enzyme. 3C is a size marker, and columns 2 to 6 show the result of separation of the pTrc99A vector and the gene ( OPSp14 ) separated by restriction enzymes. Figures 3d and 3e show the transformation of pTrc99A with acyl- [acyl transfer protein] thioesterase inserted into E. coli MG1655 (Figure 3d) and E. coli W3110 (Figure 3e) Show the result of confirming conversion. In FIG. 3D, the first column is a size marker, the second column to fourth column are blank, and the fifth column to the seventh column are PCR-confirmed genes ( OPSp14 ) inserted into E. coli MG1655. Column 1 of FIG. 3e is a marker, columns 2 to 4 are blank, and columns 5 to 7 show that a gene ( OPSp14 ) is inserted into E. coli W3110.
FIG. 4 is a graph showing the relationship between the amount of Escherichia coli ( Escherichia coli) in which an acyl- [acyl transfer protein] thioesterase ( tesA ) The vector map of pTrc99A, an E. coli expression vector, is shown. The promoter of the pTrc99A vector is the trc promoter, and the terminator is the rrnB terminator.
FIG. 5 is a graph showing the effect of the codon-optimized codon-optimized Streptococcus pyogenas on the expression of Escherichia ( E. coli) in which the acyl- [acyltransfer protein] thioesterase (EC3.1.2.14) gene ( OPSp14 ) lt; RTI ID = 0.0 > pTrc99A < / RTI > expression vector. The promoter of the pTrc99A vector is the trc promoter, and the terminator is the rrnB terminator.
6A and 6B show the growth of the wild type and the transformed E. coli developed in the present invention with the pTrc99A public vector in the minimal medium containing 10 g / L of glucose (FIG. 6A) and the LB medium (FIG.
FIGS. 7A and 7B show the consumption of glucose of the wild type and the transformants developed in the present invention containing the pTrc99A public vector in the minimal medium (FIG. 7A) containing 10 g / L of glucose and the LB medium (FIG.
Figures 8a and 8b show the production of free fatty acids of the wild-type and the transformants developed in the present invention with the pTrc99A co-vector in the minimal medium (Figure 8a) containing 10 g / L of glucose and the LB medium (Figure 8b) .
Figures 9a-9f illustrate the development of the developmental strains in minimal media containing 10 g / L of glucose and a yeast extract (Figures 9a, 9c and 9e) and tryptone (Figures 9b, 9d and 9f) Growth properties, glucose consumption and acetic acid concentration are shown by incubation time. (Fig. 9A), glucose consumption (Fig. 9B) and acetic acid concentration (Fig. 9E) in a culture medium containing yeast extract as a nitrogen source. Growth in the medium containing tryptone as a nitrogen source Consumption (Fig. 9d) and acetic acid concentration (Fig. 9e).
10A and 10B show the results of measurement of the total free fatty acids of the wild type and the transformed E. coli of the present invention containing the pTrc99A empty vector in a minimal medium containing tryptone as a nitrogen source. Wild type E. coli MG1655 (FIG. 10A) and E. coli W3110 (FIG. 10B). The unit of the vertical axis of the graph is FAME (Fatty Acid Methyl Esterase) mg / L.
FIG. 11 shows the change in the fatty acid composition of the wildtype containing the pTrc99A co-vector in the minimal medium containing 10 mg / L of glucose and nitrogen source and the free fatty acid of the transformed E. coli according to the present invention. The unit of the vertical axis of the graph is FAME ㎎ / L.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Codon-optimization of acyl- [acyl carrier protein] thioesterase, EC 3.1.2.14)

(OPsp14) encoding the acyl- [acyltransfer protein] thioesterase of Streptococcus pyogenes involved in the last stage of fatty acid biosynthesis is subjected to codon optimization to increase the expression rate in E. coli. (Fig. 1). The nucleotide sequence of the acyl- [acyltransfer protein] thioesterase of Streptococcus piogenus was synthesized as a nucleotide sequence optimized for E. coli expression by commissioning Bionix (Korea). The nucleotide sequence encoding the aconyl- [acyltransfer protein] thioesterase (OPsp14) of the codon-optimized Streptococcus piogenus was cloned into a PGM-T easy vector (Bionix, Korea). The nucleotide sequence encoding the acyl- [acyltransfer protein] thioesterase of the Streptococcus piogenus codon optimized to be highly expressed in E. coli (SEQ ID No. 1) and the acyl-amino acid sequence of the non-codon optimized Streptococcus piogenus [Acyltransfer protein] The nucleotide sequence encoding thioesterase (SEQ ID No. 2) was aligned and shown in FIG.

Example 2: Preparation of recombinant plasmid

The acyl- [acyl transfer protein] thioesterase-inserted recombinant plasmid of the codon-optimized Streptococcus piogenus synthesized in Example 1 pGEM-T: OPSp14 and E. coli Using MG1655 as a template, OPSp14 and tesA were amplified by PCR using the primers shown in Table 1, respectively. The genes were amplified with RBS (ribosome binding site). The size of OPSp14 is 753 bp , and the size of tesA is 627 bp .

gene Primer sequence Restriction enzyme OPSp14 Forward 5'-GTCGACGGAGAGTATTATGGGCCTGAGT-3 ' SalI Reverse 5'-CTGCAGCTCGAGTTAGTCAATCTCGCTTT-3 ' Pst I tesA Forward 5'-GTCGACACTTCTTAAGATGATGAACTTCAA-3 ' SalI Reverse 5'-AAGCTTCTCGAGTTATGAGTCATGATTTAC-3 ' HindIII

Each PCR reaction was performed under the usual reaction conditions (10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2 mM MgSO ₄ and Taq DNA polymerase (DaKaRa) (Denaturation), 60 ° C / 30 sec (annealing) and 72 ° C / 1 min (denaturation) at 60 ° C / And repeated 30 times under 1 minute (extended) conditions. The final step was 95 ° C / 1 min (denaturation), 60 ° C / 1 min (annealing) and 72 ° C / 5 min (extension) for stable extension.

PCR products were identified by electrophoresis and extracted from the gel (Fig. 3A). These were stabilized in T-vector. The stabilization was performed by ligation of the extracted PCR product with a T-vector to obtain recombinant plasmid- E. coli competent cells ( E. coli AG1 competent cell, stratagene, USA) and then replicated sufficiently to stabilize the gene in the T-vector. Again pTrc99A [Zhang X, Li M, Argawala, San KY. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases. Metabolic Engineering, 13: 713-722 (2011)] with restriction enzymes in a 37 ° C water bath for about 2 hours (FIG. 3b). Each of the DNA fragments was digested and reacted at 16 ° C using a T4 ligase (Dakara, Japan) as described in FIGS. 2 to 3 on a multi-cloning site of the pTrc99A vector, followed by ligation To complete the recombinant plasmids pJS40 and pJS41 (Fig. 3C). At each step, E. coli competent cells ( E. coli AG1 competent cell, stratagene, USA), then the recombinant plasmid was extracted and treated with the restriction enzyme of the original insertion site, and the size of the cloned DNA was electrophoresed on 0.8% agarose gel for comparison Respectively.

Figs. 4 and 5 are diagrams showing a map of a vector named pJS40 ( inclusive of tesA (SEQ ID NO: 3) gene) and pJS41 (including OPSp14 gene).

Example  3: Escherichia coli Transformant production

In the present invention, a transformation method by electroporation was used to stably produce E. coli transformants and increase efficiency.

Two E. coli MG1655 and E. coli W3110 cultures of 30 μl (0.1%), which were cultured for 16 hours, were inoculated into 3 ml of LB (10 When the absorbance of the culture solution reached 0.6 at a wavelength of 600 nm, 3 ml of the culture solution corresponding to the whole culture solution was centrifuged (100 g / L of triptone, 10 g / L of NaCl and 5 g / L of yeast extract) The supernatant and cells were separated by separation (12000 rpm, 1 minute). Since the codon optimization is not targeted to a specific E. coli but to a general species such as E. coli, the activity of codon optimization may vary depending on the species, and thus, two kinds of E. coli were transformed. The cells were washed once with 1 ml of 10% glycerol, and then centrifuged again (12,000 rpm, 1 minute) to separate supernatant and cells. The cells were suspended in 80 [mu] l of 10% glycerol. 1 [mu] l of recombinant plasmid (pJS40 or pJS41) was added to the suspended cells.

80 [mu] l of the liquid containing the above plasmid was transferred to a Gene pulser Xcell (BIO-RAD, USA) packed in an electroporation cuvette (BIO-RAD, Gene pulser cuvette, USA) Electric shock was applied. 1 ml of LB (10 g / L of tryptone, 10 g / L of NaCl and 5 g / L of yeast extract) prepared in advance was added and cultured for 1 hour at 200 rpm and 37 ° C with shaking. The cultured Escherichia coli was cultured in a medium supplemented with ampicillin (50 μg / ml) until a single cell was produced. As a result, recombinant plasmids pJS40 and pJS41 were transformed into E. coli MG1655 with E. coli SGJS40 and E. coli SGJS41 was prepared. The recombinant plasmid pJS41 was also transformed into Escherichia coli ( E. coli W3110) strain and developed E. coli SGJS46 (Table 2).

In FIG. 3D and FIG. 3E, the plasmid was taken from the recombinant strain to confirm that the gene of the recombinant plasmid in E. coli was stably expressed. As a result, it was shown that the plasmid was stably maintained.

Developed strain - E. coli SGJS40 Escherichia coli MG1655 :: pTrc99A :: tesA E. coli SGJS41 Escherichia coli MG1655 :: pTrc99A :: OPSp14 E. coli SGJS46 Escherichia coli W3110 :: pTrc99A :: OPSp14

Example  4: Developed Escherichia coli Badge characteristics  Research

The recombinant strains transformed into Escherichia coli were pre-cultured for 16 hours in LB (containing 50 μg / ml of ampicillin), and the culture was inoculated again into LB (containing 50 μg / ml of ampicillin) -1.0, glycerol concentration was 25%, and stored at -80 ℃ until culture.

The culture of the recombinant strain developed above was carried out by preincubating 30 μl of the stock solution in 3 ml of LB supplemented with 50 μg / ml of ampicillin contained in a 10 ml tube for 16 hours, and then adding it into a 500 ml flask (10 g / L of tryptone, 10 g / L of NaCl and 5 g / L of yeast extract) supplemented with 50 μg / ml of ampicillin and 0.5% of the total culture medium was inoculated at 37 ° C. , And cultured at 180 rpm for 30 hours.

Minimal medium (MM)
Medium composition g / L Na ₂ SO ₄ 4 (NH _{4) 2} SO ₄ 5.4 NH ₄ Cl 1.0 K ₂ HPO ₄ 7.3 NaH ₂ PO ₄ H ₂ O 1.8 (NH _{4) 2} -H-Citrate 12 MgSO ₄ 0.25 Thiamine
0.02

E. coli SGJS40, E. coli and E. coli SGJS41 SGJS46 developed in the present invention were compared with the strains were transformed using the vector pTrc99A vector been in two host strains E. coli MG1655 and E. coli W3110. In addition, the foreign gene was codon optimized for E. coli MG1655 (acetyl -CoA carboxyl cyclase, malonyl -CoA [acyl transfer protein; trans acylase and acyl-acyl thio esterase transfer protein) was transformed a strain (E. coli JES1017, Enzyme and Microbial Technology 49 (2011) 4451) to compare the fatty acid production of the transformed E. coli.

For protein expression, 0.5 mM / ㎖ of IPTG (isopropylthio-β-D-galactoside, Sigma, USA) was added when the absorbance of the culture solution reached 0.3-0.6 at 600 ㎚ wavelength. Lt; / RTI > The growth curves of the E. coli and the comparative E. coli prepared for the purpose of the present invention and the species for comparing the production of fatty acid content by the expression of the initial derivative were confirmed according to the above example (FIG. 6).

In the present invention, E. coli SGJS40, E. coli SGJS41 and E. coli SGJS46, which are transformed in E. coli, showed a slight decrease in growth compared to wild type, as shown in FIG. The reason why there is not much difference is considered that the toxicity or inhibition by gene insertion or overexpression was not large. This is because the coding part of the gene includes the overexpression function of the enzyme so that the ribosomal binding site (RBS) and the nucleotide sequence including the essential part for expression of the enzyme are set to have the minimum length and the metabolism of the host cell It is expected to reduce the burden (metabolic burden) and to develop the sequential linkage of the two genes according to the production pathway.

FIG. 6A shows the growth performance of the developed strain in the minimal medium, and FIG. 6B shows the growth performance in the LB medium.

Example  5: Transfection of Escherichia coli Glucose  Consumption and Acetic Acid Measurements

To measure glucose consumption and acetic acid production in culture medium of recombinant strains, supernatant and cells were separated and stored at -40 ° C after centrifugation (12000 rpm, 10 minutes). The supernatant separated from the culture was purified by 1 ml of each 0.2 쨉 m filter and quantitatively analyzed by high performance liquid chromatography (HPLC) under the conditions below.

subject Condition HPLC model   LC-10AT unit (Youngin Apparatus, Korea) column Aminex HPX-87H (BIO-RAD, USA) Flow rate 0.6 ml / min Injection volume 30 μl Mobile phase 0.01 N sulfuric acid Oven temperature 60 ° C Operating time 30 minutes Detector RI 750F detector

The recombinant strains of the present invention showed that glucose consumption in the minimal medium and LB medium was proportional to the growth rate (FIGS. 7A and 7B).

Example  6: Escherichia coli transformed Intracellular  Free fatty acids free fatty acid ) Measure

In order to measure the fatty acid in the cells after the addition of IPTG in the culture medium cultured under the conditions of Example 4, the fatty acid extraction was carried out as follows. The extraction process was divided into 5 stages. In the first step culture, 5 ml of the culture was centrifuged (4500 rpm, 10 minutes) and the cells were stored at -80 ° C. In the second step, the recombinant cells stored in a saponification step were vortexed for 5 to 10 seconds in 1 ml of solution 1 (45 g of NaOH, 150 ml of MeOH, 150 ml of distilled water). After reacting at 100 ° C for 5 minutes, it was vortexed again for 5-10 seconds, reacted again at 100 ° C for 25 minutes, and then the heat was cooled. In the third step, 2 mL of solution 2 (325 mL of 6 N HCl, 275 mL of MeOH) was added to the methylation stage, vortexed for 5-10 seconds, and reacted at 80 ° C for 10 minutes. Allow the heat to cool down quickly after the reaction is over. The fourth step is the extraction step. Add 1.25 ml of solution 3 (Haxane / Methyl tert-Butyl Ester = 1/1) and shake it up and down for 10 minutes. When the layers are separated, the lower layer is extracted and discarded. The fifth step is to add 3 ml of solution 4 (900 ml of distilled water containing NaOH 10.8 g) to the remaining supernatant of the previous step as a washing step to facilitate the analysis by GC, shake it up and down for 5 minutes, Were extracted and analyzed by GC / MS.

The CG / MS used in the present invention was 5975 series MSD and Agilent 7890A, and HP-5 column (30 m × 0.32 mm, film thickness 0.25 μm was used. Heating to 220 ° C at 5 ° C / min, heating to 250 ° C at 5 ° C / min and 250 ° C for 5 minutes, MS interface temperature of 280 ° C.

Total fatty acid content was higher in the LB medium, but the E. coli developed in the present invention was more stable and more enhanced than the wild type in which the empty vector pTrc99A was inserted in the minimal medium (FIG. 8).

Example  7: Developed Escherichia coli Badge characteristics (Nitrogen source) irradiation

As a result of the measurement of fatty acid in Example 6, it was confirmed that the recombinant strains enhanced the free fatty acid in the minimum medium. However, the increase in the total production in LB medium was much higher. Therefore, LB medium contained two types of nitrogen sources (yeast extract and tryptone). Thus, Table 3 (NH _{4) 2} SO ₄ instead of the culture was added to a yeast extract or tryptone 5 g / L of. The medium was inoculated with 0.5% pre-culture solution and cultured at 37 캜 and 180 rpm until the OD reached 0.3-0.6. After addition of 0.5 mM IPTG, the transformed Escherichia coli was cultured at 30 ° C and 180 rpm.

The growth curve of the transformed E. coli developed by the present invention is shown in Fig. The growth of the transformed E. coli grown in the minimal medium containing yeast extract as a nitrogen source (Fig. 9a) was not significantly different from that of transformed E. coli grown in the minimal medium containing the tryptone (Fig. 9b) as a nitrogen source. However, glucose consumption was inversely related to growth and there were more recombinants grown in media containing tryptone (Fig. 9c and Fig. 9d). It was also confirmed that the acetic acid in the transformed E. coli increased with the incubation time (FIGS. 9E and 9F).

Transformed E. coli cultured on a minimal medium containing the nitrogen source tryptone were measured for free fatty acids over time. All recombinants were found to have an increase in fatty acid according to the incubation time, and the content of free fatty acids in E. coli SGJS41 was increased by about 30% compared with Escherichia coli ( E. coli MG1655) containing a blank vector (Fig. 10A). E. coli W3110 showed a larger amount of fatty acids than wild-type E. coli W3110, showing no increase in the amount of E. coli W3110 (Fig. 10B).

And fatty acid composition was confirmed by time (FIG. 11). The E. coli transformed with the present invention had the saturated fatty acids C12: 0, C14: 0, C16: 0 and C18: 0, and the unsaturated fatty acids C16: 1, C18: 1 and C18: 2. The E. coli SGJS41 and E. coli SGJS46 developed by the present invention showed up to 300% more increase in the C12: 0 and C14: 0 than in the E. coli containing the co-vector, and the unsaturated fatty acid C18: 1 was also significantly increased. It was confirmed that the composition of fatty acids in the cells was changed by overexpression of thioester raising of exotic species by codon optimization in E. coli.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Codon-Optimized Microbes for Improving the Content of Free Fatty          Acids and Preparation Method thereof <130> PN120454 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 753 <212> DNA <213> Codon-optimization Streptococcus pyogenes 3.1.2.14 <400> 1 atgggcctga gttatcagga ggagctcaca cttccgttcg aactgtgtga cgtcaaatca 60 gatataaaac tgcccctgct cctggactat tgcttgatgg tttctggtag acaaagtgcc 120 caactgggac gcagcaacaa caatctgttg gtcgattaca agctggtttg gattgtaact 180 gattatgaaa tcacgattca tcgcttgccg cactttcagg aaacaatcac cattgaaacc 240 aaagctctgt cctacaacaa atttttctgt tatcgccagt tttatatata tgatcaagag 300 gggggactgc ttgtggatat cctggcgtat ttcgctctgc taaatccaga tacgcgaaaa 360 gtggcaacca ttccggagga tctggtagcc ccttttaaga cagattttgt taaaaagctt 420 taccgtgttc cgaaaatgcc gcttctggaa caatcaattg accgtgacta ttacgtgcgt 480 tatttcgata ttgacatgaa tggtcatgta aataatagta aatatttgga ctggatgtac 540 gacgtgctgg ggtgcgcatt tttaaaaacc catcagcctc ttaagatgac tttaaaatat 600 gttaaagaag tcagcccagg cggtcagatt acttcctcgt accatctgga ccagctcaat 660 tcttaccacc agatcacgtc agatggccag ctgaacgcgc aggccatgat cgagtggcgg 720 gcaataaagc aaaccgaaag cgagattgac taa 753 <210> 2 <211> 753 <212> DNA <213> Streptococcus pyogenes 3.1.2.14 <400> 2 atgggattaa gttatcagga ggagttgaca cttccttttg aattatgtga tgtcaaatca 60 gatataaaat tgcccctttt attagactat tgtttgatgg tttctggtag acagtctgcg 120 caattaggac gaagtaacaa caacctttta gtcgattaca agcttgtttg gattgtaacg 180 gattatgaga tcactattca tcgcttgcca cattttcaag aaaccatcac cattgaaaca 240 aaagcccttt cctataataa atttttttgt tatcgccaat tttatattta tgatcaagag 300 gggggtcttt tagtggatat cttagcctat tttgctttgt taaacccaga tacgcgaaaa 360 gtggcaacta ttccagaaga tttagtagcg ccttttaaga ctgattttgt taaaaagtta 420 taccgtgttc ctaaaatgcc tcttttagaa caatcaattg atcgtgatta ttatgtgcgt 480 tattttgata ttgatatgaa tggtcatgtc aacaacagta aatatttaga ttggatgtat 540 gatgtgttgg ggtgtgcgtt tttaaaaacg catcagcctc ttaagatgac tttgaaatat 600 gttaaagaag tctcaccagg cggtcaaatt acttccagtt accatttgga ccaattaaat 660 tcttaccatc aaatcacctc agatgggcag ctgaatgccc aagccatgat tgaatggcga 720 gcgattaaac aaacagaaag cgagatagac tag 753 <210> 3 <211> 627 <212> DNA <213> E. coli MG1655 tesA <400> 3 atgatgaact tcaacaatgt tttccgctgg catttgccct tcctgttcct ggtcctgtta 60 accttccgtg ccgccgcagc ggacacgtta ttgattctgg gtgatagcct gagcgccggg 120 tatcgaatgt ctgccagcgc ggcctggcct gccttgttga atgataagtg gcagagtaaa 180 acgtcggtag ttaatgccag catcagcggc gacacctcgc aacaaggact ggcgcgcctt 240 ccggctctgc tgaaacagca tcagccgcgt tgggtgctgg ttgaactggg cggcaatgac 300 ggtttgcgtg gttttcagcc acagcaaacc gagcaaacgc tgcgccagat tttgcaggat 360 gtcaaagccg ccaacgctga accattgtta atgcaaatac gtctgcctgc aaactatggt 420 cgccgttata atgaagcctt tagcgccatt taccccaaac tcgccaaaga gtttgatgtt 480 ccgctgctgc ccttttttat ggaagaggtc tacctcaagc cacaatggat gcaggatgac 540 ggtattcatc ccaaccgcga cgcccagccg tttattgccg actggatggc gaagcagttg 600 cagcctttag taaatcatga ctcataa 627

Claims (12)

delete delete delete delete delete delete delete A method for producing a fatty acid comprising the steps of:
(a) Escherichia coli transformed with a recombinant vector prepared by inserting a nucleotide sequence encoding an acyl- [acyl carrier protein] thioesterase of the first sequence of the sequence into an expression vector MG1655 (Escherichia coli MG1655) the step of the biosynthesis sodium sulfate, tryptone, ammonium chloride, di-potassium phosphate, sodium phosphate, (NH _{4) 2} -H- citrate, fatty acid by culturing in a culture medium consisting of magnesium sulfate, and thiamine; And
(b) obtaining said biosynthetic fatty acid.
delete 9. The method of claim 8, wherein the method increases the yield for fatty acids by 1.5-4.0 fold compared to wild-type E. coli.
9. The method of claim 8, wherein the fatty acid is a saturated fatty acid of C12: 0 and C14: 0 and an unsaturated fatty acid of C18: 1.
delete

Images