Production of indigo and indirubin using recombinant Escherichia coli harboring

Abstract

본 연구는 일원자탄소대사세균인 Methylophaga aminisulfidivorans MPT에 내포된 flavin-containing monooxygenase 유전자의 upstream에 존재하는 promoter region을 분석하고, flavin-containing monooxygenase 유전자를 포함하는 재조합 대장균을 이용하여 인디고 및 인디루빈을 생산 하고자 하였다. 일반적으로 인디고 및 인디루빈은 화학합성 방법과 식물로부터 추출하는 방법으로 생산되어지고 있다. 이와 같은 생산방식은 생산 중 발생하는 부산물에 의한 환경오염 및 인체 유해성을 가지고 있으며, 생산성의 한계를 가지고 있다. 따라서 본 연구에서는 M. aminisulfidivorans MPT로부터 유래된 flavin-containing monooxygenase를 이용하여 인체 및 환경에 무해하고, 고생산성을 가지는 생물학적 인디고 및 인디루빈 대량생산 공정을 개발하고자 하였다. 또한 M. aminisulfidivorans MPT의 전체 유전자 서열을 분석하였다. M. aminisulfidivorans MPT 유래 flavin-containing monooxygenase 유전자는 1361bp의 염기로 구성되어져 있다. 이 연구에서는 재조합 plasmid pBlue2.0으로부터 약 100bp 간격으로 upsteam region을 제거하여 이들 재조합 plasmid pBlue들을 포함하는 재조합 대장균 E. coli pBlues를 제작하였다. 이 중 upstream region의 길이가 315bp인 pBlue1.7에서 가장 많은 인디고가 생산되었다. pBlue1.7에 포함되어진 fmo 유전자의 전사시작점은 총 6개가 존재하였다. 인디고 생산에 가장 큰 영향을 미치는 전사시작점을 분석한 결과 fmo 유전자의 translation initiation codon으로부터 279번째에 nucleotide T(+1) 였다. fmo 전사시작점으로부터 8bp upstream 위치에 있는 5'-TATCCT-3' 과 14-15 upstream 위치에 있는 5'-TG-3'염기서열이 존재하는 고 있었으며 이들 염기 서열이 fmo의 extended -10 promoter로 작동하였다. 인디고 생산에 적합한 숙주 및 발현시스템을 구축하기 위하여 E. coli, B. subtils 및 C. glutamicum을 숙주를 이용하여 재조합 E. coli pHCE IIB 1.7, E. coli pGEX 4T-11.7, E.coli pBSK1.7 (pBlue1.7), B. subtils pHY300PLK1.7, C. glutamicum pEKE 1.7를 제작하였다. 이들 재조합 미생물로부터 각각 400.2 mg/L, 638.5 mg/L, 672.3 mg/L, 182 mg/L, 377 mg/L의 인디고가 생산되었으며, 이 중 E.coli pBSK 1.7 (pBlue1.7)에서 가장 높은 인디고 생산성을 나타내었다. 인디고의 최적생산을 위하여 반응표면분석법 (response surface methodology, RSM) 중 중심합성계획법 (central composite design, CCD)을 이용하여 배양조건 및 배지의 최적화를 수행하였다. 최적 배양배지의 조성은 tryptophan 2g/L, yeast extract 5g/L, sodium chloride 10g/L이었으며, 최적 배양 조건은 pH 7.0, 30℃, 180rpm이었다. 최적배양조건에서 10L, 100L, 3,000L 배양기를 이용하여 회분식배양을 수행하였다. 그 결과 재조합 E. coli pBlue1.7은 각각 모든 배양기에서 920mg/L의 인디고를 생산하였다. 또한 3,000L 배양기를 이용한 반연속식 배양에서 각각 1차 배양 (911 ± 22 mg/L), 2차 배양 (870 ± 48), 3차 배양 (702 ± 66 mg/L)의 인디고를 생산하였다. 10L 배양기를 이용한 연속식 배양에서는 110시간 동안 23 g의 인디고를 생산하였으며, 생산율은 11.3 mg/L/h이었다. 재조합 E. coli pBlue1.7은 tryptophan 배지에서 주 생산물인 인디고 (920 mg/L) 와 소량의 인디루빈 (5 mg/L)을 생산하였다. 인디루빈의 생산을 증가시키기 위하여 tryptophan 배지에 3 mM cysteien을 첨가한 결과 재조합 E. coli pBlue1.7로부터 95 mg/L의 인디루빈이 생산되었으며 인디고는 5 mg/L이 생산되었다. 배지 내 cysteine 첨가에 따른 E. coli pBlue1.7의 생장률은 약 10배 감소하였으나, FMO의 활성 및 발현양은 영향을 미치지 않았다. 세포내 2-hydroxyindole 및 3-hydroxyindole의 농도를 측정한 결과 cysteine 첨가한 경우 기존에 검출되지 않았던 2-hydroxyindole이 30시간동안 2 μM이 생성되었으며, 3-hydroxyindole은 0.8 μM이 생산되었다. 5L 배양기를 이용한 회분식 최적배양을 통해 223.6 mg/L의 인디루빈을 생산하였다. M. aminisulfidivorans MPT의 전체 유전자 서열의 길이는 3,216,960 bp였으며, 2,984 protein coding genes, 3 rRNA operons, 31 tRNAs 을 포함하는 chromosome 유전자 서열과 190 coding genes, 9 tRNAs 를 포함하는 한개의 plasmid로 구성되어져 있었다. 특히 M. aminisulfidivorans MPT는 메탄올 산화와 관련된 methanol oxidation (mxaFJGIRSACKL), pyrroloquinoline quinone (pqqBCDE) 유전자 클러스터 및 flavin-containing monooxygenase가 존재하였다. 또한 현재까지 알려진 sigma factor와 유사성이 낮은 Sigma 242C FecI-like2C ECF family, RNA polymerase sigma factor RpoD, RNA polymerase sigma-54 factor RpoN, RNA polymerase sigma factor RpoH가 포함되어져 있었다.|Indigo and indirubin are considered to be the oldest natural textile dyes and drugs. They are traditionally extracted from several plant species. Recent studies have shown that indigo and indirubin can be produced via several biological processes through the use of recombinant microorganisms expressing mono- or di-oxygenase enzymes. This study aimed to optimize the production of indigo and indirubin through the use of a bacterial flavin-containing monooxygenase (FMO) gene. A bacterial FMO gene was cloned from Methylophaga aminisulfidivorans MPT, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. The recombinant E. coli produced 160 mg/L of indigo from 2 g/L tryptophan medium. Transcription of the fmo gene was initiated from a thymine residue (+1) 279 bp (TSS1) upstream from the translation initiation codon of the fmo gene. On the basis of the sequenced data from the fmo transcripts, we identified a -35 region (5'-CTGGAA-3') and -10 region (5'-TATCCT-3'). In addition, we also identified the TG motif situated -14, -15 bp upstream from the transcription start site. To increase the production of indigo, upstream length of fmo gene was optimized and the response surface methodology was used. The pBlue 1.7 plasmid was prepared by deleting the sequence upstream of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg/L of indigo in tryptophan medium after 24h of flask culture. Production of indigo was optimized using response surface methodology with a 2n central composite design. The optimal contents of the media that produced the maximum amount of indigo were determined as follows: 2.4 g/L tryptophan, 4.5 g/L yeast extract and 11.4 g/L sodium chloride. In addition, the optimum culture temperature and pH were determined to be 30℃ and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring the pBlue 1.7 plasmid produced 920 mg/L in optimum tryptophan medium after 24 h of cultivation. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of indigo. Indigo productions were performed by 3,000 L repeated batch fermentation and 5 L continuous fermentation. Repeated batch fermentation in a 3,000 L fermenter produced 911 ± 22 mg/L of indigo with 2 g/L tryptophan as a substrate (yield, 46.9%) under the following culture conditions; culture temperature 30℃; pH 7.0; agitation speed 200 rpm; and aeration 3 vvm. We found that sufficient dissolved oxygen (aeration rate and agitation speed) was critical for indigo production. For continuous fermentation in a 5 L fermenter, the volumetric productivity was found to be 11.3 mg/L/h up to 110 h (final accumulated indigo, 23 g) with a constant dilution rate of 0.084/h (constant feeding rate of 0.167 L/h with medium containing 3 g/L tryptophan). Recombinant E. coli pBlue 1.7 can withstand the toxicity of high concentrations of accumulated indigo during batch fermentation. For continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which the plasmid was lost. In a previous study, the recombinant E. coli pBlue 1.7 produced indigo (920 mg/L) and indirubin (≤5 mg/L) in a 5 L fermenter containing tryptophan medium. In this study, we found that indirubin production was greatly increased when 3 mM cysteine was added to the tryptophan medium, although cysteine inhibited the growth of the recombinant E. coli harboring the fmo gene. However, the addition of cysteine did not influence the expression level and activity of FMO in the cell. Cysteine inhibited the synthesis of 3-hydroxyindole from indole but enhanced the synthesis of 2-hydroxyindole, which might increase indirubin production. The optimal culture conditions for indirubin production in tryptophan medium were determined from analysis of the results of the response surface methodology. Under optimal conditions, the recombinant E. coli cells could produce 223.6 mg/L of indirubin from 2 g/L of tryptophan. Thus, indigo and indirubin production systems have potential for use in industrial applications for overcoming the environmental problems associated with synthetic indigo production and for meeting the demand for natural indigo and indirubin in the dye and drug industries. M. aminisulfidivorans MPT is a restricted facultative marine methylotrophic bacterium that grows on methanol, methylated amines, dimethyl sulfide, and dimethyl sulfoxide. Here we present the high-quality draft genome sequence of M. aminisulfidivorans MPT, consisting of a chromosome (3,092,085 bp) and a plasmid (16,875 bp). The M. aminisulfidivorans MPT chromosome was found to harbor 2,984 protein-coding genes, 3 rRNA operons, and 31 tRNAs, whereas the plasmid had 190 coding genes and 9 tRNAs. A gene encoding a novel bacterial FMO, which catalyzes nitrogen-containing compounds or indole by the use of oxygen through an NADPH-dependent pathway, was identified. The information obtained in the current study regarding the M. aminisulfidivorans MPT draft genome sequence should facilitate future studies on the metabolic diversity of the genus Methylophaga.