KR20230037293A - Melanin Biosynthetic Enzyme Derived from Flavobacterium kingsejongi and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel melanin biosynthetic enzyme derived from Flavobacterium kingsejongi, and more specifically, 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi, It relates to a recombinant microorganism into which a gene encoding the same is introduced and a method for producing melanin using the same.
Using 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi according to the present invention, melanin can be produced in high yield, and the coding thereof Since melanin can be efficiently mass-produced by introducing the gene into a microorganism capable of producing 4-hydroxyphenylpyruvate (4-HPP), it is industrially useful.

Description

[Melanin Biosynthetic Enzyme Derived from Flavobacterium kingsejongi and Uses Thereof]

The present invention relates to a novel melanin biosynthetic enzyme derived from Flavobacterium kingsejongi, and more specifically, 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi, It relates to a recombinant microorganism into which a gene encoding the same is introduced and a method for producing melanin using the same.

Natural biopolymers can be used to produce many valuable products such as therapeutics, cosmetics, food ingredients, biochemicals and biofuels. In particular, bacterial biopolymers can be used as biomaterials for a wide range of applications (M.F. Moradali, B.H. Rehm, Bacterial biopolymers: from pathogenesis to advanced materials, Nat. Rev. Microbiol. 18(4) (2020) 195-210.) . Melanin is a group of heteropolymeric pigments synthesized by various prokaryotes and eukaryotes. In microbes, melanin has several beneficial roles, including protection from UV radiation, heavy metal toxicity, oxidative stress, and temperature extremes.

Microbial melanins are heterologous, classified into four main types according to their chemical structure and biosynthetic pathway: eumelanin, pheomelanin, pyomelanin, and allomelanin (S. Singh, et al., Microbial melanin: Recent advances in biosynthesis, extraction, characterization, and applications, Biotechnol. Adv. (2021) 107773.). Three of these types (eumelanin, pheomelanin and pyomelanin) are involved in sequential enzymatic actions (e.g. activity of tyrosinase, laccase or 4-hydroxyphenylpyruvate deoxygenase (HPPD)) and autocatalysis. It is biosynthesized from the same precursor, L-tyrosine (Fig. 1). Black-brown eumelanin (or DOPA-melanin) is synthesized from L-tyrosine, which is oxidatively transformed to L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosinase. L-DOPA is further oxidized to L-dopaquinone by tyrosinase. The resulting L-dopaquinone is cyclized, oxidized and polymerized to produce eumelanin. Orange-yellow pheomelanin is also synthesized through the oxidation of L-tyrosine to L-dopaquinone by tyrosinase. L-dopaquinone is then cysteinylated to cysteinyl DOPA, which is further oxidized, cyclized and polymerized to form pheomelanin. A third class, phyomelanin, is synthesized from L-tyrosine via another pathway, the homogentisate pathway, where L-tyrosine is deaminated by tyrosine aminotransferase to form 4-hydroxyphenylpyruvate (4-HPP). ) to form Then, 4-HPP is further oxidized by HPPD to homogentisate, which is oxidized and polymerized to produce pyomelanin (S. Ahmad, et al., Identification of a Gene Involved in the Negative Regulation of Pyomelanin Production in Ralstonia solanacearum, J. Microbiol.Biotechnol.27(9) (2017) 1692-1700.).

Meanwhile, microbial fermentation is attracting attention as a promising method for the industrial synthesis of melanin because it is environmentally friendly and relatively easy to scale up. Melanin has a number of uses, including: It can be used for physiologically active substances such as antioxidants, sunscreens, and antibacterial agents, and can also be used for functional biomaterials that act as sensitizers for dye-sensitized solar cells and scaffold polymers for metal nanoparticles. Therefore, a systematic study to evaluate the viability of microbial fermentation as a melanin supply method for various industrial applications is required to be performed.

Currently, research on the production and physiological role of phyomelanin is focused on well-known microorganisms such as Pseudomonas aeruginosa, Streptomyces avermitilis, and Aspergillus fumigatus (A. Rodriguez-Rojas, et al., Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection, Microbiology 155(4) (2009) 1050-1057.), and the regulation of melanin production and biosynthesis in Flavobacterium kingsejongi strains has not been reported.

Under this technical background, the present inventors have made diligent efforts to efficiently produce melanin usable for various industrial uses, and as a result, a novel 4-hydroxyphenylpyruol synthesizing pheomelanin in a Flavobacterium kingsejongi strain. Bait deoxygenase (4-hydroxyphenylpyruvate dioxygenase, HPPD) was discovered, and when the gene encoding the enzyme is introduced into a microorganism capable of producing 4-hydroxyphenylpyruvate (4-HPP), melanin can be produced in large quantities. It was confirmed that it could be produced and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be

An object of the present invention is to provide a novel melanin biosynthesis enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi.

Another object of the present invention is to provide a gene encoding the enzyme, a recombinant vector containing the gene, and a recombinant microorganism having the recombinant vector introduced into a host cell.

Another object of the present invention is to provide a method for producing melanin using the recombinant microorganism or Flavobacterium kingsejong.

In order to achieve the above object, the present invention is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi represented by the amino acid sequence of SEQ ID NO: 1 provides

The present invention also provides a gene encoding the enzyme, a recombinant vector containing the gene, and a recombinant microorganism having the gene or recombinant vector introduced into a host cell.

The present invention also comprises the steps of biosynthesizing melanin by culturing the recombinant microorganism; And it provides a method for producing melanin comprising the step of recovering the biosynthesized melanin.

The present invention also includes the steps of biosynthesizing melanin by culturing Flavobacterium kingsejongi; And it provides a method for producing melanin comprising the step of recovering the biosynthesized melanin.

Using 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi according to the present invention, melanin can be produced in high yield, and the coding thereof Since melanin can be efficiently mass-produced by introducing the gene into a microorganism capable of producing 4-hydroxyphenylpyruvate (4-HPP), it is industrially useful.

), 잔류 티로신 농도는 빨간색 사각형(

)으로 표시된다. 도 5B에서는 설정된 시간 간격으로 배양액에서 상등액을 수집하고 육안으로 검사했다. 도 5C에서는 회분식 발효 동안 배양액에 존재하는 멜라닌을 정량하여 F. kingsejongi 멜라닌 생성의 동역학을 모니터링하였다. 도 5D는 발효 배양 5, 19, 50 및 99시간에 수집된 F. kingsejongi 세포에서 HPPD를 코딩하는 hpd 유전자의 RT-PCR 분석을 도시한다. tuf 유전자를 참고로 사용했다.
도 6은 HPPD를 코딩하는 F. kingsejongi hpd 유전자를 발현하는 멜라닌 생성 재조합 대장균(E. coli)의 생물 반응기 회분식 발효를 도시한다. 도 6A에서는 600 nm(OD₆₀₀)에서 광학 밀도를 측정하고 배양액에 존재하는 포도당 및 티로신의 농도를 정량화하여 세포 성장의 동역학 및 포도당 및 티로신 소비 속도를 모니터링했다. OD₆₀₀은 채워진 원(●), 잔류 포도당 농도는 녹색 삼각형(

), 잔류 티로신 농도는 빨간색 사각형(

)으로 표시된다. 도 6B에서는 설정된 시간 간격으로 배양액에서 상등액을 수집하고 육안으로 검사했다. 도 6C에서는 회분식 발효 동안 배양액에 존재하는 멜라닌을 정량화하여 재조합 대장균에 의한 멜라닌 생성의 동역학을 모니터링했다.
도 7은 F. kingsejongi의 흑갈색 색소 침착을 도시한다. 도 7A는 25℃에서 LB 한천 플레이트에서 성장한 F. kingsejongi의 콜로니를 도시한다. 도 7B는 25℃에서 LB 배지에서 배양한 F. kingsejongi 배양액의 시간 경과 모니터링을 도시한다. 도 7C는 티로신, 코직산 및 티로신/코직산이 첨가된 LB 한천 플레이트에서 성장한 F. kingsejongi의 색소 침착의 생리학적 변화를 도시한다. 멜라닌을 생성하는 Streptomyces avermitilis와 색소 침착되지 않은 E. coli를 각각 양성 및 음성 대조군으로 사용했다.
도 8은 추정 HPPD를 발현하는 대장균(E. coli)을 함유하는 배양액에서 정제된 멜라닌의 구조 분을 도시한다. 추정 F. kingsejongi HPPD 를 발현하는 E. coli 에서 정제된 UV-vis 스펙트럼(도 8A), FT-IR 스펙트럼(도 8B), ¹H NMR 스펙트럼(도 8C)을 도시한다.
도 9는 F. kingsejongi에서 HPPD를 발현하는 E. coli의 흑갈색 색소 침착 억제를 도시한다. tyrosine 또는 tyrosine + sulcotrione이 첨가된 LB 한천 플레이트에서 성장한 F. kingsejongi HPPD를 발현하는 재조합 E. coli의 색소 침착 변화를 도시한다. 멜라닌을 생성하는 F. kingsejongi 및 무색소 E. coli를 각각 양성 및 음성 대조군으로 사용하였다.
도 10은 재조합 대장균(E. coli)에서 과발현된 6ХHis-tagged F. kingsejongi HPPD의 정제를 도시한다. 도 10A는 GE

KTA FPLC™ 고속 단백질 액체 크로마토그래피(FPLC) 시스템에서 결합 단백질 용출의 크로마토그램을 도시한다. 피크 아래의 빨간색 화살표는 FPLC에서 풀링된 용리액 분획을 나타낸다. 도 10B는 정제 단계에서 단백질의 SDS-PAGE 분석을 도시한다.
도 11은 선택된 Flavobacterium HPPD 중 F. kingsejongi HPPD의 아미노산 서열에 따른 계통발생적 위치를 도시한다. 트리는 (A) neighbor-joining method, (B) maximum likelihood method 및 (C) 산술 평균을 이용한 unweighted pair group method 을 사용하여 생성되었다. 노드의 백분율은 1,000개 재샘플로 부트스트랩을 기반으로 한 신뢰도 수준을 나타낸다. Figure 1 shows the biosynthetic circuit of three types of microbial melanins (eumelanin, pheomelanin and pyomelanin) from the precursor L-tyrosine.
Figure 2 shows the structural analysis of melanin purified from F. kingsejongi culture medium. Figure 2A shows the UV-vis spectrum analysis of F. kingsejongi melanin and commercial eumelanin (control), Figure 2B shows the FT-IR spectrum of F. kingsejongi melanin and commercial eumelanin (control), and Figure 2C shows The ¹ H NMR spectrum of F. kingsejongi melanin is shown, and Figure 2D shows ^{the 1} H NMR spectrum of commercial melanin (control).
Figure 3 shows the heterologous expression of eight putative proteins of F. kingsejongi involved in melanin synthesis in E. coli and the organization of genes including the homogentisate pathway of F. kingsejongi. Figure 3A depicts E. coli cultures expressing eight F. kingsejongi candidate genes after growing for 2 days at 30°C on LB agar plates supplemented with 1 g/L tyrosine, plate sections designated as follows: 1, tryptophan 2,3-dioxygenase; 2, 4-hydroxyphenylpyruvate dioxygenase (HPPD); 3, homogentisate 1,2-dioxygenase; 4, 3-hydroxyanthranilate 3,4-dioxygenase; 5, phytanoyl-CoA dioxygenase; 6, phytanoyl-CoA dioxygenase; 7, putative dioxygenase; 8, hypothetical protein; C, empty plasmid (negative control). 3B shows time course monitoring of E. coli cultures expressing putative HPPD. Figure 3C shows the organization of genes encoding homogentisate pathway enzymes and related enzymes in F. kingsejongi.
Figure 4 shows the in vitro activity of purified 6xHis-tagged HPPD and native HPPD in crude protein extracts. Referring to Figure 4A, HPPD activity was investigated using 4-HPP as a substrate for purified 6xHis-tagged HPPD. The red box represents the observed activity of purified 6xHis-tagged HPPD. Red and blue arrows indicate peaks corresponding to homogentisate (product) and 4-HPP (substrate), respectively. Referring to Figure 4B, tyrosinase activity was investigated using tyrosine as a substrate for purified 6xHis-tagged HPPD. Referring to Figure 4C, HPPD activity was investigated using 4-HPP as a substrate for native HPPD in crude protein extracts. The red box represents the observed activity of native HPPD. Red and blue arrows indicate peaks corresponding to homogentisate (product) and 4-HPP (substrate), respectively. Referring to Figure 4D, tyrosinase activity was investigated using tyrosine as a substrate for native HPPD in crude protein extracts. Figure 4E shows HPLC chromatograms of commercial standards: 4-HPP, homogentisate (HGA), L-DOPA and tyrosine.
Figure 5 shows batch fermentation of melaninogenic F. kingsejongi in a 5-L bioreactor. In Figure 5A, the kinetics of cell growth and the rate of glucose and tyrosine consumption were monitored by measuring the optical density at 600 nm (OD ₆₀₀ ) and quantifying the concentrations of glucose and tyrosine present in the culture medium. OD ₆₀₀ is a filled circle (●), residual glucose concentration is a green triangle (

), and the residual tyrosine concentration is the red square (

) is displayed. In Figure 5B, the supernatant was collected from the culture at set time intervals and inspected visually. In FIG. 5C , the kinetics of F. kingsejongi melanogenesis were monitored by quantifying the melanin present in the broth during batch fermentation. Figure 5D shows RT-PCR analysis of the hpd gene encoding HPPD in F. kingsejongi cells collected at 5, 19, 50 and 99 hours of fermentation culture. The tuf gene was used as a reference.
6 shows bioreactor batch fermentation of melanin-producing recombinant E. coli expressing the F. kingsejongi hpd gene encoding HPPD. In Figure 6A, the kinetics of cell growth and the rates of glucose and tyrosine consumption were monitored by measuring the optical density at 600 nm (OD ₆₀₀ ) and quantifying the concentrations of glucose and tyrosine present in the culture medium. OD ₆₀₀ is a filled circle (●), residual glucose concentration is a green triangle (

), and the residual tyrosine concentration is the red square (

) is displayed. In Figure 6B, the supernatant was collected from the culture at set time intervals and inspected visually. In Figure 6C, the kinetics of melanogenesis by recombinant E. coli were monitored by quantifying the melanin present in the culture during batch fermentation.
7 shows dark brown pigmentation of F. kingsejongi. 7A shows colonies of F. kingsejongi grown on LB agar plates at 25°C. 7B shows time course monitoring of F. kingsejongi cultures grown in LB medium at 25°C. 7C shows the physiological changes in pigmentation of F. kingsejongi grown on LB agar plates supplemented with tyrosine, kojic acid and tyrosine/kojic acid. Streptomyces avermitilis, which produces melanin, and non-pigmented E. coli were used as positive and negative controls, respectively.
8 shows the structure of melanin purified from a culture medium containing E. coli expressing putative HPPD. The UV-vis spectrum (FIG. 8A), FT-IR spectrum (FIG. 8B), and ¹ H NMR spectrum (FIG. 8C) purified from E. coli expressing putative F. kingsejongi HPPD are shown.
Figure 9 shows inhibition of dark brown pigmentation of E. coli expressing HPPD in F. kingsejongi. Pigmentation changes of recombinant E. coli expressing F. kingsejongi HPPD grown on LB agar plates supplemented with tyrosine or tyrosine + sulcotrione are shown. F. kingsejongi and achromatic E. coli, which produce melanin, were used as positive and negative controls, respectively.
Figure 10 shows the purification of 6ХHis-tagged F. kingsejongi HPPD overexpressed in recombinant E. coli . 10A shows GE

A chromatogram of bound protein elution on a KTA FPLC™ Fast Protein Liquid Chromatography (FPLC) system is shown. The red arrows below the peak indicate the pooled eluent fractions in FPLC. 10B shows SDS-PAGE analysis of proteins in the purification step.
Figure 11 shows the phylogenetic position according to the amino acid sequence of F. kingsejongi HPPD among selected Flavobacterium HPPD. Trees were generated using (A) neighbor-joining method, (B) maximum likelihood method, and (C) unweighted pair group method using arithmetic mean. The percentage of nodes represents the confidence level based on bootstrapping with 1,000 resamples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In one embodiment of the present invention, a novel melanin-producing F. kingsejongi strain produces dark brown pyomelanin via the homogentisic acid pathway, with a titer of 6.07 ± 0.32 g/L and a conversion yield of 60% (0.6 ± 0.03 g melanin per g tyrosine), it was confirmed that a large amount of melanin was produced with a productivity of 0.03 g/L·h. In addition, the recombinant E. coli strain expressing F. kingsejongi HPPD had a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per g tyrosine), and a yield of 0.04 g/L h. It was confirmed that melanin was produced with productivity. Through this, it was confirmed that both strains can be used as platform strains for large-scale bacterial phyomelanin production.

Accordingly, in one aspect, the present invention relates to 4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi represented by the amino acid sequence of SEQ ID NO: 1. it's about

In the present invention, the amino acid sequence of SEQ ID NO: 1 is as follows:

MSKEVKSVEYGLEKIFEGAQDFLPLLGTDYVEFYVGNAKQAAHFYKTAFGYQSLAYAGLETGVRDRASYVLKQDKIRLVLTTPLNEDSPINEHLKKHGDGVKVAALWVEDARSAFEETTKRGARPFMEPTVEKDEFGEVVRSGIYTYGETVHIFVERKNYNGVFLPGYKEWKSDYNPAPTGLKYIDHMVGNVGWGEMNTWVKWYEDVMGFVNFLSFDDKQINTEYSALMSKVMSNGNGRIKFPINEPAEGKKKSQIEEYLEFYGGPGIQHVAIATDDIIKTVSELKSRGVEFLSAPPRSYYEAIPSRLGAHMDMMKEDLNEIEKLAIMVDADEEGYLLQIFTKPVQDRPTLFFEIIQRMGARGFGAGNFKALFESIEREQEKRGTL(서열번호 1).

The term "4-hydroxyphenylpyruvate dioxygenase (HPPD)" of the present invention is an enzyme also known as α-ketoisocaproate dioxygenase (KIC dioxygenase). , refers to an oxidase that catalyzes the conversion of 4-hydroxyphenylpyruvate (4-HPP) into homogentisate during the catabolism of tyrosine.

In another aspect, the present invention relates to a gene encoding the 4-hydroxyphenylpyruvate dioxygenase (HPPD).

In the present invention, the gene may be characterized in that it is represented by the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of SEQ ID NO: 2 is as follows:

atgtcaaaagaagtaaaatcagtagaatacggattagagaaaatatttgaaggcgcacaggatttcctgccgcttttaggaacagattatgtagagttttatgttggtaatgccaaacaggcagcgcatttttataaaacagcattcggctaccagtcacttgcttatgcaggtttggaaacgggtgtacgtgacagggcttcctatgtattgaaacaggataaaatacgtttggtactgactacgccattaaatgaggactctccaatcaacgagcaccttaaaaaacatggtgatggggtaaaagtagcagcgctttgggttgaagatgccagaagtgctttcgaagagaccactaaaagaggagcaaggccttttatggagccaacggttgaaaaagatgaatttggagaagtagtacgttccggaatctacacctatggggaaacggtacatatttttgtagaaagaaaaaattacaatggtgtgtttcttccgggctacaaagaatggaagtctgattataaccccgctccaacaggattaaaatacatcgaccacatggtggggaatgtaggctggggtgaaatgaatacttgggtaaaatggtatgaagatgtgatggggttcgtgaatttcctttcttttgatgacaaacaaatcaatacggagtattctgctttgatgagtaaggtaatgtccaatgggaatggaagaatcaagtttccaatcaatgaaccggcagaaggaaagaaaaaatcacagattgaggaatatttggagttttatggcggccctggtatacagcacgttgctatagcgacagatgatattatcaaaacggtttctgaattgaaatcccgtggagtagaattcctttctgcaccaccgagatcgtattatgaagcaattccatcccgtttaggagctcatatggatatgatgaaagaagacctgaacgaaattgaaaaactggcgattatggtagatgccgatgaag aaggatacctgcttcagatttttaccaaaccagttcaggacaggccaacccttttctttgaaattatccagagaatgggcgccagaggttttggagccggaaactttaaagctttatttgagtctattgaacgcgaacaagaaaaaagaggaacgttataa (SEQ ID NO: 2).

In another aspect, the present invention relates to a recombinant vector containing the gene represented by the nucleotide sequence of SEQ ID NO: 2.

In another aspect, the present invention relates to a recombinant microorganism in which the gene represented by the nucleotide sequence of SEQ ID NO: 2 or a recombinant vector containing the gene is introduced into a host cell.

In the present invention, the host cell is not limited as long as it has the ability to produce 4-hydroxyphenylpyruvate (4-HPP), but is preferably E. coli, more preferably E. coli. coli TOP10 or E. coli BL21 (DE3), but is not limited thereto.

In the present invention, the recombinant microorganism may be characterized in that it produces melanin (melanin).

In the present invention, the melanin may be characterized in that pyomelanin.

In the present invention, "vector" means a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply latent genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself. As the plasmid is currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) an origin of replication to ensure efficient replication, including several hundred plasmid vectors per host cell, and (b) a host cell transformed with the plasmid vector to be selected for selection. and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and the foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

As the vector used in the present invention, expression vectors known in the art may be used, and preferably pUCM, pUCM_TDO, pUCM_HPPD, pUCM_HGD, pUCM_HAAO, pUCM_PHYH, pUCM_PHYH_HypoP, pUCM_PutaD, pUCM_HypoP, pET21α(+) or pET21α(+)_HPPD (Table 2), but is not limited thereto.

After ligation, the vector must be transformed into an appropriate host cell. In the present invention, the preferred host cell is a prokaryotic cell. Suitable prokaryotic host cells include E.coli XL-1Blue (Stratagene), E.coliDH5α, E.coli JM101, E.coli K12, E.coli W3110, E.coli X1776, E.coli BL21 and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the above E.coli , strains of the genus Agrobacterium such as Agrobacterium A4, bacilli such as Bacillus subtilis, Salmonella typhimurium or Serratia marcescens ), and various strains of the genus Pseudomonas can be used as host cells.

In the present invention, a method for introducing a recombinant vector into a cell may use a method known in the art, and preferably a transformation method. Here, 'transformation' means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or by completion of chromosomal integration. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be performed by selecting an appropriate standard technique according to the host cell as known in the art. These methods include heat shock method, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE (diethylaminoethyl) ) -Dextran method, cationic liposome method, and lithium acetate-DMSO method may be included, but is not limited thereto.

As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are included in a single recombinant vector that includes a bacterial selectable marker and an origin of replication.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function equally well for the same expression system. However, those skilled in the art can make appropriate selections among various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, in selecting a vector, consideration must be given to the host, since the vector must replicate within it. The vector's copy number, ability to control copy number, and expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.

In another aspect of the present invention, biosynthesis of melanin by culturing the recombinant microorganism; And it relates to a method for producing melanin comprising the step of recovering the biosynthesized melanin.

In the present invention, the melanin may be characterized in that pyomelanin.

In the present invention, in the step of culturing the recombinant microorganism, it is possible to increase the titer of melanin produced by adding a metal such as Cu, Fe, or Mg to the culture medium.

In another aspect of the present invention, biosynthesis of melanin by culturing Flavobacterium kingsejongi; And it relates to a method for producing melanin comprising the step of recovering the biosynthesized melanin.

In the present invention, the melanin may be characterized in that pyomelanin.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Characteristics of dark brown pigment produced from F. kingsejongi

F. kingsejongi produced dark brown pigment in both LB agar and LB culture medium. The pigment was characterized as melanin using a previously reported characterization method. The solubility of the purified pigment obtained from F. kingsejongi was very similar to commercially available melanin (Table 1), suggesting that it belongs to one of the melanin classes.

Next, to test the hypothesis that F. kingsejongi has a melanin biosynthesis pathway, (1) 1 g/L tyrosine (known as a melanin biosynthesis promoter and precursor), (2) 100 mg/L kojic acid (known as a melanin biosynthesis inhibitor), ( 3) The pigmentation of F. kingsejongi grown on LB agar supplemented with both tyrosine (1 g/L) and kojic acid (100 mg/L) was monitored. Similar to melanin-producing S. ermitilis (positive control), F. kingsejongi significantly increased pigmentation intensity on the tyrosine/LB agar plate, but showed significant pigmentation on both the kojic acid/LB agar plate and the tyrosine/kojic acid/LB agar plate. is seriously reduced. Escherichia coli (negative control) cultured on these three types of plates showed no pigmentation (Fig. 7). Thus, F. kingsejongi was identified as having a melanin biosynthetic pathway.

Melanin solubility test Test Melanin Purified melanin
( F. kingsejongi ) Synthetic melanin
(Sigma-Aldrich, No. M8631) Solubility Water - - 1-N NaOH + + Ethanol - - Acetone - - Chloroform - - Benzene - - Phenol + + Precipitation 1 N HCl + + 1% FeCl ₃ + + Decolorization 30% H ₂ O ₂ + +

Example 2. Chemical Characteristics of Dark Brown Pigments Produced from F. kingsejongi

When the chemical properties of the purified pigment were compared with standard commercially available eumelanin, the dark brown pigment produced by F. kingsejongi was considered to be melanin. Commercial eumelanin and F. kingsejongi pigment showed very similar broadband UV-vis spectra with lmax at 221 nm (Fig. 2A). This unique lmax at 221 nm is in good agreement with the general characteristics of melanin structure. FT-IR spectroscopic analysis further revealed that functional groups characterizing melanin were present in F. kingsejongi pigments (Fig. 2B). First, there was a characteristic band of about 3272 cm-1 corresponding to the stretching vibrations of the -OH group and -NH group. Second, there were separate peaks at 1707, 1621, and 1516 cm-1 corresponding to the aromatic ring, and third. , there was a peak at 1500 to 1350 cm-1 due to the bending vibration of the -NH group and the stretching vibration of the CN group of the indole unit. Fourth, the stretching vibration of the phenol-OH group was detected at 1,219 cm-1. Compared with commercially available eumelanin, F. kingsejongi melanin showed characteristic peaks at 2967-2874 cm-1, 1081-1040 cm-1, and 873 cm-1, which are asymmetric stretching vibrations of _CH2 , symmetric contraction vibrations of COC, - This can be attributed to the out-of-plane refractive vibration of CH. Finally, ¹ H NMR spectrum analysis of F. kingsejongi melanin (Fig. 2C) showed a broad peak of ~7.0 ppm corresponding to aromatic protons of indole and pyrrole units, and a broad peak of 8.4 ppm corresponding to -NCH- aromatic ring group protons. showed sharp peaks. The ¹ H NMR spectrum of commercial melanin (Fig. 2D) also showed a characteristic sharp peak at 8.4 ppm.

Example 3. Identification and heterologous expression of the putative phyomelanin synthesis gene of F. kingsejongi

After confirming that F. kingsejongi synthesizes melanin, this melanin biosynthetic pathway (Fig. 1) was further characterized for molecular studies and metabolic engineering purposes. Therefore, a putative tyrosinase candidate was found in the genome of F. kingsejongi using the amino acid sequence of S. lincolnensis tyrosinase, but no tyrosinase candidate was found.

Next, the search was extended to putative dioxygens responsible for melanin synthesis. Candidate genes that may be involved in melanin biosynthesis are tryptophan 2,3-dioxygenase, HPPD, homogentisate 1,2-dioxygenase, 3-hydroxyanthranilate 3,4-dioxygenase, phytanoyl-CoA dioxygenase, phytanoyl-CoA dioxygenase, a putative dioxygenase, and It is a hypothetical protein. Experiments were performed using recombinant E. coli expressing eight genes each and an empty plasmid (control), but only the E. coli strain expressing the putative HPPD was supplemented with 1 g/L tyrosine and incubated at 30 °C. Brown colonies formed on LB agar plates maintained on (Fig. 3A).

In the LB medium supplemented with 1 g/L of tyrosine, HPPD-expressing coliforms secreted a brown pigment, and the culture medium turned dark brown after 24 hours (FIG. 3B). When the brown pigment of Escherichia coli expressing putative HPPD was analyzed by UV-vis, FT-IR spectroscopy, and ¹ H NMR spectroscopy, it was found to be structurally very similar to melanin of F. kingsejongi (FIG. 8). Also, pigmentation of both F. kingsejongi and HPPD-expressing recombinant Escherichia coli was severely reduced in a medium supplemented with sulcotrione, a well-known HPPD inhibitor (FIG. 9). It was demonstrated that HPPD has a phyomelanin biosynthesis activity in non-blackish Escherichia coli. These results suggest that putative HPPD catalyzes the biosynthesis of pyomelanin from tyrosine in F. kingsejongi. Since phyomelanin is produced by HPPD activity in the homogentisate pathway, genome mining was performed to identify genes involved in the homogentisate pathway in the genome of F. kingsejongi. Genome mining revealed that HPPD is present in the genome of F. kingsejongi along with two genes encoding other homogentisate pathway enzymes (homogentisate 1,2-dioxygenase and fumarylacetoacetase) (Fig. 3C).

Example 4. In vitro activity of 6ХHis-tagged HPPD and natural HPPD purified from crude protein extract

In order to confirm that the observed in vivo activity was due to putative HPPD, in vitro analysis of HPPD activity was performed using both purified 6ХHis-tagged HPPD (FIG. 10) and natural HPPD contained in the crude protein extract of E. coli. Tyrosine and 4-HPP were used as substrates to determine tyrosinase-like activity and HPPD-like activity, respectively. Therefore, four reaction conditions were tested: (1) 6ХHis-tagged HPPD + 4-HPP, (2) 6ХHis-tagged HPPD + tyrosine, (3) Native HPPD + 4-HPP, and (4) Native HPPD + tyrosine. HPPD-like activity was demonstrated by a new peak corresponding to homogentisate (a phyomelanin biosynthetic intermediate; FIG. 1) in the HPLC chromatograms (Figures 4A and 4C) in both reaction mixtures supplemented with 4-HPP as substrate (Conditions 1 and 4C). 3) was observed only. No tyrosinase-like activity was observed in the reaction mixture containing tyrosine even when a longer incubation time was applied (Fig. 4B and 4D). Thus, in vitro HPPD activity was observed but not tyrosinase activity, which means that the putative HPPD of F. kingsejongi actually belongs to one of the HPPD enzyme families. In particular, as a result of phylogenetic analysis based on the amino acid sequence, F. kingsejongi HPPD was in a clade separate from the main clade containing HPPD in other Flavobacterium strains (FIG. 11).

Example 5. Bioreactor batch fermentation of F. kingsejongi

Key variables to consider when developing a microbial process for commercialization of bacterial melanin include titer, conversion yield, and productivity. The choice between wild-type or natural mutant strains and metabolically engineered strains should also be considered. Therefore, the kinetics of growth and melanogenesis of F. kingsejeongi were investigated by performing batch fermentation in a 5 L fermentor containing TB medium supplemented with 20 g/L glucose and 10 g/L tyrosine. F. kingsejongi showed a two-step adaptive growth pattern at 40-50 hours and reached an OD600 of 30 ± 1.2 at 200 hours (Fig. 5A). Glucose in the medium was completely consumed at 40 hours, after which tyrosine began to be used, and tyrosine was completely consumed at 195 hours. After 50 hours, the color of the culture medium changed significantly with the start of tyrosine intake (Fig. 5B). The melanin titer increased from 0.17 ± 0.02 g/L at 72 hours to 6.07 ± 0.32 g/L at 200 hours (Fig. 5C). The productivity and conversion yield of melanin were 0.03 g/L per hour and 0.6 ± 0.03 g (~60%) per 1 g of tyrosine, respectively. For comparison with other melanin-producing bacteria, S. kathirae can produce up to 1.76 g/L melanin in suboptimal medium and up to 13.7 g/L melanin in optimal medium. Most other melanogenic bacteria, such as Pseudomonas, Streptomyces, and Bacillus strains, can produce 0.1 to 7.6 g/L of melanin in both suboptimal and optimal media.

Therefore, although the titer of melanin produced by F. kingsejongi is far below 13.7 g/L, it is judged that there is considerable potential to increase titer and productivity through understanding and manipulation of melanin biosynthesis regulation and network based on comparative genomics. . In particular, melanin and dark culture color were detected after 50–74 h incubation, but in RT-PCR, the hpd gene encoding HPPD was detected at both the glucose consumption (5 h and 19 h assays) and tyrosine consumption (50 h and 99 h assays) stages. It was found to be constitutively transcribed in (Fig. 5D). Thus, the observed two-step adaptive growth pattern, which reduces melanin conversion yield and titre, can be avoided by optimizing the fed-batch fermentation of F. kingsejongi strains expressing HPPD. Moreover, inactivation of the hmg gene (homogentisate 1,2-dioxygenase), which further oxidizes homogentisate to fumarate and acetoacetate, can increase the titer of melanin produced in F. kingsejongi. Non-genetic interventions can also be applied to increase melanin titre. For example, adding metals such as Cu, Fe, and Mg to the culture medium can act as a stress simulator to increase the titer of melanin.

Example 6. Bioreactor Batch Fermentation of Recombinant E. coli Expressing HPPD

In addition to wild-type melanogenic bacteria, recombinant Escherichia coli has shown potential as a melanin producer. In a recent study, HPPD-expressing E. coli recombinants produced 0.21 g/L and 0.31 g/L of melanin in P. aeruginosa and Ralstonia pickettii , respectively. The effect of batch fermentation in a bioreactor was tested using a recombinant E. coli strain constitutively expressing F. kingsejongi HPPD to study the kinetics of growth and melanogenesis as in F. kingsejongi. Similar to F. kingsejongi, the recombinant E. coli strain showed a diauxic growth pattern between 11 and 23 hours and reached an OD600 of 35 ± 3.2 at 95 hours (Fig. 6A). Glucose was completely consumed at 17 hours, and tyrosine started to be used at 11 hours and was completely consumed at 41 hours. The color of the culture solution changed significantly at 17 hours (Fig. 6B), at which time the melanin titer was 0.12 ± 0.03 g/L, and increased continuously to 3.76 ± 0.30 g/L at 95 hours (Fig. 6C). Productivity and conversion yield were 0.38 ± 0.03 g (~38%) per 1 g of 0.04 g/L melanin and tyrosine per hour, respectively. The melanin produced by recombinant E. coli was 38% lower than that of F. kingsejongi, but the culture time occupied about half of the time required for F. kingsejongi, resulting in high melanin productivity (200 hours were required for F. kingsejongi, and 200 hours were required for recombinant E. coli). requires 95 hours). Thus, F. kingsejongi produced 0.03 g/L melanin per hour, whereas recombinant E. coli produced 0.04 g/L melanin per hour.

By optimizing the medium and fermentation method and improving the strain, there is a possibility that the melanin titer, productivity, conversion yield, etc. of fermentation using recombinant E. coli can be further increased. Altogether, the two strains can serve as models to explain the regulation of the melanin biosynthetic pathway and its network with other cellular pathways and to understand the cellular response of melanin-producing bacteria to environmental changes, including nutrient deprivation and other stresses.

Example 7. Materials and Methods

Example 7-1. culture conditions

F. kingsejongi (KCTC 42908 ^T ) and S. avermitilis (KCTC 9063 ^T ) were grown in Luria-Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L; shaking at 250 rpm) and LB It was cultured on an agar plate at 25°C under aerobic conditions. E. coli strain TOP10 was grown at 30° C. in LB medium (shaking at 250 rpm) flasks and LB agar plates containing 100 μg/mL ampicillin.

Example 7-2. Melanin synthesis inhibition test

To investigate melanin synthesis in F. kingsejongi, (1) 1 g/L tyrosine (Sigma-Aldrich, St. Louis, MO, USA), (2) 100 mg/L kojic acid (Sigma-Aldrich), or (3) 1 g/L Grow the cells for 16 days at 25 °C on LB agar plates supplemented with L tyrosine + 100 mg/L kojic acid. As a positive and negative control group, S. avermitilis (KCTC 9063 ^T ), which produces melanin, and E. coli TOP10, which does not produce melanin, were cultured (25°C for 7 days for S. avermitilis and 6 days for E. coli). 37°C for days).

Example 7-3. Extraction and analysis of melanin

F. kingsejongi and E. coli expressing HPPD were flask-cultured in LB medium supplemented with 1 g/L tyrosine, and the cell-free culture medium was recovered, adjusted to pH 2.0 by adding 1-N HCl, and maintained at 25°C for 7 days. politicized Next, the culture solution was boiled in a water bath for 1 hour and then centrifuged at 10,000 Хg for 15 minutes. The collected black pellet was washed 3 times with 0.1-N HCl and soaked in anhydrous EtOH. EtOH-soaked melanin pellets were reacted in a boiling water bath for 10 min and then stored at 25 °C for 1 day. Finally, the melanin pellets were washed twice with anhydrous EtOH, air-dried, and weighed on an electronic balance. Three independent measurements were made and the average was calculated.

Example 7-4. Characteristics of melanin extracted from culture medium

Melanin pigments purified from F. kingsejongi, S. avermitilis and E.coli and commercial eumelanin samples were analyzed for 200-1100 nm spectra using a JASCO V-770 UV-visible spectrometer (JASCO, Oklahoma City, OK, USA). did. Samples were measured in quartz cuvettes at room temperature. Fourier transform infrared (FT-IR) spectra of the four melanins were measured with a Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA) in the attenuated total reflection (ATR) mode using a diamond crystal. Melanin-KBr disk samples for FT-IR spectra were prepared by mixing melanin powder with KBr powder after drying in a vacuum oven at 30 °C. Proton nuclear magnetic resonance (1H NMR) spectrum was measured at room temperature using a 600 MHz NMR spectrometer (JEOL, Tokyo, Japan). For 1 H NMR measurement, 10 mg of each melanin powder was dissolved in 0.75 mL of 0.5 M sodium deuterium aqueous solution and sonicated for 15 minutes.

Example 7-5. Genome mining and heterologous expression of proteins involved in melanin biosynthesis

A standalone basic local alignment and search tool program package (BLAST+ v.2.31; http://www.ncbi.nlm.nih.gov/) was used to encode the F. kingsejongi genome that may be involved in melanin biosynthesis. gene was searched. The putative tyrosinase was searched by running the blastp program with default parameters against the BLAST+ local protein database for the F. kingsejongi genome (GenBank accession number CP020919). This database was searched for an amino acid sequence similar to tyrosinase (P55023.2) from S. lincolnensis. Gene sequences encoding HPPD proteins were extracted from genome annotation files in GFF format, which were generated by running the locally installed version of the NCBI Prokaryotic Genome Annotation Pipeline. Eight candidate genes were identified: (1) tryptophan 2,3-dioxygenase (744 bp), (2) 4-hydroxyphenylpyruvate dioxygenase (1161 bp), (3) homogentisate 1,2-dioxygenase (936 bp), (4) 3-hydroxyanthranilate 3,4-dioxygenase (540 bp), (5) phytanoyl-CoA dioxygenase (546 bp), (6) phytanoyl-CoA dioxygenase (738 bp), (7) a putative dioxygenase (738 bp), (8) ) a hypothetical protein (834 bp). Candidate genes were amplified from gDNA with gene-specific primers and then cloned into a constitutive expression vector plasmid (pUCM) to generate eight expression plasmids (Table 2). Eight plasmids were individually transformed into E. coli, and the transformed cells were grown at 30 °C for 2 days on LB agar supplemented with 1 g/L tyrosine.

Strains and plasmids used Strains and plasmids Relevant properties Source or reference strains E. coli TOP10 F mcr A Δ( mrr-hsd RMS -mcr BC) φ80 lac ZΔM15 Δ lac X74 rec A1 ara D139 Δ( ara-leu )7697 gal U gal K rps L (StrR) end A1 nup G Invitrogen E. coli BL21 (DE3) F ompT gal dcm lon hsdSB(r _B m _B ) λ ( DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+] K-12 ( λS ) NEB Flavobacterium kingsejongi KCTC ^42908T Streptomyces avermitilis KCTC ^9063T Plasmids pUCM Cloning vector modified from pUC19; constitutive lac promoter, Ap Kim et al., 2010 pUCM_TDO Constitutively expressing putative tryptophan 2,3-dioxygenase from F. kingsejongi , Ap the present invention pUCM_HPPD Constitutively expressing putative 4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi , Ap the present invention pUCM_HGD Constitutively expressing putative homogentisate 1,2-dioxygenase from F. kingsejongi , Ap the present invention pUCM_HAAO Constitutively expressing 3-hydroxyanthranilate 3,4-dioxygenase from F. kingsejongi , Ap the present invention pUCM_PHYH Constitutively expressing putative phytanoyl-CoA dioxygenase from F. kingsejongi , Ap the present invention pUCM_PHYH_HypoP Constitutively expressing putative phytanoyl-CoA dioxygenase, hypothetical protein (dioxygenase) from F. kingsejongi , Ap the present invention pUCM_PutaD Constitutively expressing putative dioxygenase from F. kingsejongi , Ap the present invention pUCM_HypoP Constitutively expressing putative hypothetical protein (dioxygenase) from F. kingsejongi , Ap the present invention pET21α(+) f1 ori, T7 promoter, C-terminal His-tag sequence, AmpR Novagen pET21α(+)_HPPD f1 ori, T7 promoter, inducible expression of His6-tagged HPPD, AmpR the present invention

Example 7-6. Expression and purification of F. kingsejongi HPPD-encoding genes using E. coli

KTA 고속 단백질 액체 크로마토그래피 시스템(GE Healthcare Column, GE Healthcare)에 넣고 50 mM Tris-HCl 완충액(150 mM NaCl, pH 8.0)으로 평형화했다. 결합 단백질은 0~250 mM 이미다졸 사이의 선형 구배 프로파일과 함께 1 mL/min의 유량으로 용출되었다. 6ХHis tagged HPPD를 포함한 fraction을 모은 뒤, PD10 탈염 컬럼(GE Healthcare)으로 탈염되고 Amicon Ultra-15 원심 필터(밀리포어, 벌링턴, MA, 미국)에 농축되며 최종적으로 20 mM Tris-HCl 버퍼(50 mM NaCl, 1mM dithiothreitol, 0.1mM phenylmethylsulfonyl fluoride; ph 8.0)으로 교환되었다. 각 단계의 검체는 Coomassie brilliant blue 얼룩을 사용하여 10%(w/v) SDS-PAGE(황산나트륨 도데실 폴리아크릴아미드 겔 전기영동)로 분석하였다. Bradford 방법을 사용하여 정제된 6ХHis-tagg HPPD의 농도는 0.4 μg/μL로 측정되었다. 정제된 6ХHis tagged HPPD는 -80 °C에서 50% (v/v) 글리세롤에 저장되었다.The hpd gene encoding HPPD in F. kingsejongi was amplified from genomic DNA by PCR with gene-specific primers (Table 3). Then, the PCR sample was cloned into the induction vector pET21α(+) to construct pET21α(+)_HPPD expressing 6ХHis tagged HPPD (Table 2). E. coli BL21(DE3) containing either pET21α(+) or pET21α(+)_HPPD was cultured in 50 mL LB medium at 30 °C (shaking at 250 rpm), supplemented with 100 μg/ml ampicillin. When the recombinant BL21(DE3) culture reached an optical density of 0.6-0.8 at 600 nm (OD600), 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture medium for protein induction. , and then cultured the cells for 3 hours. Then, the cells were collected by centrifugation (3800 rpm, 20 minutes, 4 °C), washed twice with 50 mM Tris-HCl buffer (150 mM NaCl, pH 8.0), and then resuspended in 20 mL of the same buffer. The resuspended cells were lysed with 30% sonication for 5 min on ice (15 pulse cycles, 5 sec on, 10 sec off), and the supernatant containing soluble protein was centrifuged (4000 rpm, 4 °C, 30 °C). minutes) was separated from cell debris. The supernatant was transferred to a GE equipped with a HisTrap FF affinity chromatography column (GE Healthcare, Chicago, IL, USA).

It was placed in a KTA high-speed protein liquid chromatography system (GE Healthcare Column, GE Healthcare) and equilibrated with 50 mM Tris-HCl buffer (150 mM NaCl, pH 8.0). The bound protein was eluted at a flow rate of 1 mL/min with a linear gradient profile between 0 and 250 mM imidazole. After collecting the fraction containing 6ХHis-tagged HPPD, it was desalted with a PD10 desalting column (GE Healthcare), concentrated on an Amicon Ultra-15 centrifugal filter (Millipore, Burlington, MA, USA), and finally mixed with 20 mM Tris-HCl buffer (50 mM NaCl, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride; pH 8.0). Samples from each step were analyzed by 10% (w/v) SDS-PAGE (sodium dodecyl polyacrylamide gel electrophoresis) using Coomassie brilliant blue stain. The concentration of 6ХHis-tag HPPD purified using the Bradford method was determined to be 0.4 μg/μL. Purified 6ХHis tagged HPPD was stored in 50% (v/v) glycerol at -80 °C.

Primers used (capital letters indicate restriction enzyme sites, underlines indicate ribosome binding sequences) Gene Primer sequences Enzyme site sequence number Tryptophan 2,3-dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa atgtaccatcaggttaatgaa-3′ Xba I 3 R: 5′-aaggaaaaaaGCGGCCGCttaaacgtttttttcttctccc-3′ Not I 4 4-Hydroxyphenylpyruvate dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa atgtcaaaagaagtaaaatcagt-3′ Xba I 5 R: 5′-aaggaaaaaaGCGGCCGCttataacgttcctctttttctt-3′ Not I 6 Homogentisate 1,2-dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa atgctgaaaggttttgaactaa-3′ Xba I 7 R: 5′-aaggaaaaaaAGCGGCCGCttactctacccaggacttat-3′ Not I 8 3-Hydroxyanthranilate 3,4-dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa atggcaatagcaaaaccgtt-3′ Xba I 9 R: 5′-aaggaaaaaaGCGGCCGCttatctttttagggcataccg-3′ Not I 10 Phytanoyl-CoA dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa atggatcactataaagaacaag-3′ Xba I 11 R: 5′-aaggaaaaaaGCGGCCGCttagacaacctgtcccc-3′ Not I 12 Phytanoyl-CoA dioxygenase, hypothetical protein F: 5′-gcTCTAGAgc aggaggattacaaa atggatcactataaagaacaag-3′ Xba I 13 R: 5′-aaggaaaaaaGCGGCCGCtcagcgttgaaaaacctcc-3′ Not I 14 Putative dioxygenase F: 5′-gcTCTAGAgc aggaggattacaaa ctacacgttatgaaaaggaaa-3′ Xba I 15 R: 5′-aaggaaaaaaGCGGCCGCttatccattcactttaatagtgt-3′ Not I 16 Hypothetical proteins F: 5′-gcTCTAGAgc aggaggattacaaa atggcaacactgcagcc-3′ Xba I 17 R: 5′-aaggaaaaaaGCGGCCGCttaaccgaattttaccgatgt-3′ Not I 18

Example 7-7. Measurement of HPPD activity in vitro

In vitro HPPD activity measurements were performed using purified 6ХHis-tagged HPPD or crude protein extract containing HPPD. Purified 6ХHis tagged HPPD (0.4 μg/μL) in 130 μL assay mixture consisting of 100 mM Tris-HCl (pH 7.5), 50 mM ascorate, 2 mM 4-hydrophenylpyruvate to determine the activity of HPPD 8 μg was added. To prepare a crude protein extract containing HPPD, IPTG-induced cells were washed, resuspended in 100 mM Tris-HCl (pH 7.5, 50 mM ascorbate), cell disrupted, and centrifuged. HPPD activity in crude protein extract was tested by adding 2 mM 4-HPP to 100 mM Tris-HCl (pH 7.5, 50 mM ascorbate). Likewise, to determine the extent to which HPPD exhibits tyrosinase-like activity, 6ХHis-tagged HPPD (0.4 μg/μL) purified in a 130-μL assay mixture consisting of 100 mM sodium phosphate (pH 7.0), 0.01 mM CuSO4, and 3 mM tyrosine 8 μg was added. To test the tyrosinase activity of the crude protein extract, it was suspended in 100 mM sodium phosphate (pH 7.0) and 0.01 mM CuSO4, and the reaction was started by adding 3 mM tyrosine. All in vitro assays were performed in a reaction volume of 150 μL at 30 °C and stopped at predetermined time points by the addition of 20% (w/v) trifluoroacetic acid (TFA). After filtering, the in vitro test samples were filtered using a ZORBAX SB-C18 column (ID 4.6 mm, length 250 mm, particle size, 5 μm, Agilent, USA) equipped with a photodiode array detector (Agilent, Santa Clara, CA, USA). Analyzed on a 1200 series HPLC system. The mobile phase (70% water, 30% acetonitrile, 0.1% TFA) was allowed to flow at 1 mL/min for HPPD activity test and 0.5 mL/min for tyrosinase activity test, and the column temperature was maintained at 30°C. Commercial standards (4-HPP, homogentizate, L-DOPA, tyrosine) were purchased from Sigma-Aldrich.

Example 7-8. HPPD mRNA expression level analysis

f.kingsejongi cells grown in LB medium were harvested by centrifugation at mid-log phase and stationary phase (4°C, 13000 rpm). Pelleted cells were resuspended in RNAlater solution (Invitrogen, Waltham MA, USA). A cDNA library was constructed using the Toyobo ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). The cDNA concentrations of the hpd and tuff genes were analyzed using a Rotor-Gene Q PCR machine (QIAGEN, Hilden, Germany). The tuf gene encoding the transform elongation factor Tu was used as a reference gene.

Example 7-9. Phylogenetic analysis of HPPD

The phylogenetic position of F. kingsejongi HPPD was calculated based on the amino acid sequences of F. kingsejongi HPPD and HPPD of other flavobacterial strains. Evolutionary distances between HPPDs were calculated using the Jukes and Cantor model, and phylogenetic trees were generated using the neighbor-joining method, maximum likelihood, and unweighted pair group method using arithmetic mean. The results obtained bootstrap analysis based on 1,000 resampling.

Example 7-10. Batch fermentation of F. kingsejongi in a 5-L bioreactor

To evaluate the melanin productivity of F. kingsejongi in a 5-L jar fermentor, F. kingsejongi was added to a modified 100 mL TB (Treatic Broth) medium (20 g/L glucose, 12 g/L tryptone, 24 g/L Yeast extract, 2.31 g/L KH2PO4, 12.59 g/LK2HPO4) after culture at 25°C under aerobic conditions, when OD600 reached 2-3, 5 with 1.5L modified TB medium supplemented with 10g/L tyrosine -L jar fermentor (BioFlo 320, Eppendorf, Framingham, MA, USA) was inoculated. Fermentation was performed at 25 °C with an air flow rate of 1.5 vol/vol/min (vvvm). The dissolved oxygen (DO) level was maintained at 30% by supplying pure O2 gas or adjusting the agitation to 300-600 rpm. The pH was automatically maintained at 7.0 with 14% (v/v) NH4OH and 2-N HCl.

Example 7-11. Batch fermentation of recombinant E. coli in a 5 L bioreactor

The TB medium was evaluated for its melanin production potential using recombinant Escherichia coli expressing F. kingsejongi HPPD in a 5L jar fermentor. Recombinant E. coli cultures grown at 30 °C in 500 mL flasks containing 100 mL of ampicillin and 100 mL of modified TB medium were inoculated into 5 L jar fermentors containing 1.5 L modified TB medium supplemented with 10 g/L tyrosine and 100 µg/ml. Fermentation was performed at 30 °C with an air flow rate of 1.5 vvm. The DO level was maintained at 30% by supplying pure O2 gas or adjusting the agitation rate from 300 to 600 rpm. The pH was maintained at 7.0 by automatic addition of 14% (v/v) NH4OH and 2-N HCl.

Example 7-12. analysis method

Cell growth was monitored by measuring the OD600 of the cultures using a MAX Plus 384 spectrophotometer (Bio-Rad, Hercules, CA, USA). Glucose concentration in the culture medium was quantified on an Agilent 1260 Infinity HPLC system equipped with an Agilent 1200 refractive index detector, using 4 mM H2SO4 as the mobile phase on an Aminex HPX-87H column (300 Х 7.8 mm; Bio-Rad). The flow rate was 0.7 mL/min and the column temperature was maintained at 50 °C. To quantify the tyrosine concentration, the culture medium was diluted 10-fold with distilled water, the pH was adjusted to 9.0 by adding 1-N NaOH, and the absorbance at 245 nm was read using a MAX Plus384 spectrophotometer.

As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC42908

Entrusted date: 20151112

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Melanin Biosynthetic Enzyme Derived from Flavobacterium kingsejongi and Uses Thereof <130> P20-B175 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 386 <212> PRT <213> artificial sequence <220> <223> 4-hydroxyphenylpyruvate dioxygenase from Flavobacterium kingsejongi <400> 1 Met Ser Lys Glu Val Lys Ser Val Glu Tyr Gly Leu Glu Lys Ile Phe 1 5 10 15 Glu Gly Ala Gln Asp Phe Leu Pro Leu Leu Gly Thr Asp Tyr Val Glu 20 25 30 Phe Tyr Val Gly Asn Ala Lys Gln Ala Ala His Phe Tyr Lys Thr Ala 35 40 45 Phe Gly Tyr Gln Ser Leu Ala Tyr Ala Gly Leu Glu Thr Gly Val Arg 50 55 60 Asp Arg Ala Ser Tyr Val Leu Lys Gln Asp Lys Ile Arg Leu Val Leu 65 70 75 80 Thr Thr Pro Leu Asn Glu Asp Ser Pro Ile Asn Glu His Leu Lys Lys 85 90 95 His Gly Asp Gly Val Lys Val Ala Ala Leu Trp Val Glu Asp Ala Arg 100 105 110 Ser Ala Phe Glu Glu Thr Thr Lys Arg Gly Ala Arg Pro Phe Met Glu 115 120 125 Pro Thr Val Glu Lys Asp Glu Phe Gly Glu Val Val Arg Ser Gly Ile 130 135 140 Tyr Thr Tyr Gly Glu Thr Val His Ile Phe Val Glu Arg Lys Asn Tyr 145 150 155 160 Asn Gly Val Phe Leu Pro Gly Tyr Lys Glu Trp Lys Ser Asp Tyr Asn 165 170 175 Pro Ala Pro Thr Gly Leu Lys Tyr Ile Asp His Met Val Gly Asn Val 180 185 190 Gly Trp Gly Glu Met Asn Thr Trp Val Lys Trp Tyr Glu Asp Val Met 195 200 205 Gly Phe Val Asn Phe Leu Ser Phe Asp Asp Lys Gln Ile Asn Thr Glu 210 215 220 Tyr Ser Ala Leu Met Ser Lys Val Met Ser Asn Gly Asn Gly Arg Ile 225 230 235 240 Lys Phe Pro Ile Asn Glu Pro Ala Glu Gly Lys Lys Lys Ser Gln Ile 245 250 255 Glu Glu Tyr Leu Glu Phe Tyr Gly Gly Pro Gly Ile Gln His Val Ala 260 265 270 Ile Ala Thr Asp Asp Ile Ile Lys Thr Val Ser Glu Leu Lys Ser Arg 275 280 285 Gly Val Glu Phe Leu Ser Ala Pro Pro Arg Ser Tyr Tyr Glu Ala Ile 290 295 300 Pro Ser Arg Leu Gly Ala His Met Asp Met Met Lys Glu Asp Leu Asn 305 310 315 320 Glu Ile Glu Lys Leu Ala Ile Met Val Asp Ala Asp Glu Glu Gly Tyr 325 330 335 Leu Leu Gln Ile Phe Thr Lys Pro Val Gln Asp Arg Pro Thr Leu Phe 340 345 350 Phe Glu Ile Ile Gln Arg Met Gly Ala Arg Gly Phe Gly Ala Gly Asn 355 360 365 Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Glu Gln Glu Lys Arg Gly 370 375 380 Thr Leu 385 <210> 2 <211> 1161 <212> DNA <213> artificial sequence <220> <223> 4-hydroxyphenylpyruvate dioxygenase from Flavobacterium kingsejongi <400> 2 atgtcaaaag aagtaaaatc agtagaatac ggattagaga aaatatttga aggcgcacag 60 gatttcctgc cgcttttagg aacagattat gtagagtttt atgttggtaa tgccaaacag 120 gcagcgcatt tttataaaac agcattcggc taccagtcac ttgcttatgc aggtttggaa 180 acgggtgtac gtgacagggc ttcctatgta ttgaaacagg ataaaatacg tttggtactg 240 actacgccat taaatgagga ctctccaatc aacgagcacc ttaaaaaaca tggtgatggg 300 gtaaaagtag cagcgctttg ggttgaagat gccagaagtg ctttcgaaga gaccactaaa 360 agaggagcaa ggccttttat ggagccaacg gttgaaaaag atgaatttgg agaagtagta 420 cgttccggaa tctacaccta tgggggaaacg gtacatattt ttgtagaaag aaaaaattac 480 aatggtgtgt ttcttccggg ctacaaagaa tggaagtctg attataaccc cgctccaaca 540 ggattaaaat acatcgacca catggtgggg aatgtaggct ggggtgaaat gaatacttgg 600 gtaaaatggt atgaagatgt gatggggttc gtgaatttcc tttcttttga tgacaaacaa 660 atcaatacgg agtattctgc tttgatgagt aaggtaatgt ccaatgggaa tggaagaatc 720 aagtttccaa tcaatgaacc ggcagaagga aagaaaaaat cacagattga ggaatatttg 780 gagttttatg gcggccctgg tatacagcac gttgctatag cgacagatga tattatcaaa 840 acggtttctg aattgaaatc ccgtggagta gaattccttt ctgcaccacc gagatcgtat 900 tatgaagcaa ttccatcccg tttaggagct catatggata tgatgaaaga agacctgaac 960 gaaattgaaa aactggcgat tatggtagat gccgatgaag aaggatacct gcttcagatt 1020 tttaccaaac cagttcagga caggccaacc cttttctttg aaattatcca gagaatgggc 1080 gccagaggtt ttggagccgg aaactttaaa gctttatttg agtctattga acgcgaacaa 1140 gaaaaaagag gaacgttata a 1161 <210> 3 <211> 45 <212> DNA <213> artificial sequence <220> <223> Tryptophan 2,3-dioxygenase Forward Primer <400> 3 gctctagagc aggaggatta caaaatgtac catcaggtta atgaa 45 <210> 4 <211> 40 <212> DNA <213> artificial sequence <220> <223> Tryptophan 2,3-dioxygenase Reverse Primer <400> 4 aaggaaaaaa gcggccgctt aaacgttttt ttcttctccc 40 <210> 5 <211> 47 <212> DNA <213> artificial sequence <220> <223> 4-Hydroxyphenylpyruvate dioxygenase Forward Primer <400> 5 gctctagagc aggaggatta caaaatgtca aaagaagtaa aatcagt 47 <210> 6 <211> 41 <212> DNA <213> artificial sequence <220> <223> 4-Hydroxyphenylpyruvate dioxygenase Reverse Primer <400> 6 aaggaaaaaa gcggccgctt ataacgttcc tcttttttct t 41 <210> 7 <211> 46 <212> DNA <213> artificial sequence <220> <223> Homogentisate 1,2-dioxygenase Forward Primer <400> 7 gctctagagc aggaggatta caaaatgctg aaaggttttg aactaa 46 <210> 8 <211> 39 <212> DNA <213> artificial sequence <220> <223> Homogentisate 1,2-dioxygenase Reverse Primer <400> 8 aaggaaaaaa agcggccgct tactctaccc aggacttat 39 <210> 9 <211> 44 <212> DNA <213> artificial sequence <220> <223> 3-Hydroxyanthranilate 3,4-dioxygenase Forward Primer <400> 9 gctctagagc aggaggatta caaaatggca atagcaaaac cgtt 44 <210> 10 <211> 39 <212> DNA <213> artificial sequence <220> <223> 3-Hydroxyanthranilate 3,4-dioxygenase Reverse Primer <400> 10 aaggaaaaaa gcggccgctt atctttttag ggcataccg 39 <210> 11 <211> 46 <212> DNA <213> artificial sequence <220> <223> Phytanoyl-CoA dioxygenase Forward Primer <400> 11 gctctagagc aggaggatta caaaatggat cactataaag aacaag 46 <210> 12 <211> 35 <212> DNA <213> artificial sequence <220> <223> Phytanoyl-CoA dioxygenase Reverse Primer <400> 12 aaggaaaaaa gcggccgctt agacaacctg tcccc 35 <210> 13 <211> 46 <212> DNA <213> artificial sequence <220> <223> Phytanoyl-CoA dioxygenase, hypothetical protein Forward Primer <400> 13 gctctagagc aggaggatta caaaatggat cactataaag aacaag 46 <210> 14 <211> 37 <212> DNA <213> artificial sequence <220> <223> Phytanoyl-CoA dioxygenase, hypothetical protein Reverse Primer <400> 14 aaggaaaaaa gcggccgctc agcgttgaaa aacctcc 37 <210> 15 <211> 45 <212> DNA <213> artificial sequence <220> <223> Putative dioxygenase Forward Primer <400> 15 gctctagagc aggaggatta caaactacac gttatgaaaa ggaaa 45 <210> 16 <211> 41 <212> DNA <213> artificial sequence <220> <223> Putative dioxygenase Reverse Primer <400> 16 aaggaaaaaa gcggccgctt atccattcac tttaatagtg t 41 <210> 17 <211> 41 <212> DNA <213> artificial sequence <220> <223> Hypothetical protein Forward Primer <400> 17 gctctagagc aggaggatta caaaatggca acactgcagc c 41 <210> 18 <211> 45 <212> DNA <213> artificial sequence <220> <223> Hypothetical protein reverse primer <400> 18 gctctagagc aggaggatta caaaatgtac catcaggtta atgaa 45

Claims (12)

4-hydroxyphenylpyruvate dioxygenase (HPPD) derived from Flavobacterium kingsejongi represented by the amino acid sequence of SEQ ID NO: 1.
A gene encoding the 4-hydroxyphenylpyruvate dioxygenase (HPPD) of claim 1.
The gene according to claim 2, wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2.
A recombinant vector comprising the gene of claim 2.
A recombinant microorganism in which the gene of claim 2 or the recombinant vector of claim 4 is introduced into a host cell.
The recombinant microorganism according to claim 5, wherein the host cell is E. coli.
The recombinant microorganism according to claim 5, wherein the recombinant microorganism produces melanin.
The recombinant microorganism according to claim 7, wherein the melanin is pyomelanin.
Culturing the recombinant microorganism of claim 5 to biosynthesize melanin; and recovering the biosynthesized melanin.
[Claim 10] The method of claim 9, wherein the melanin is pyomelanin.
Biosynthesis of melanin by culturing Flavobacterium kingsejongi; and recovering the biosynthesized melanin.
The method of claim 11, wherein the melanin is pyomelanin.

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