Biosynthesis Of Indigoids By Comamonas Sp.MQ And Its Augmented Systems

Indole and its derivatives are typical N-heteroaromatic compounds existing in coke plant effluents. The microbial transformation of indoles will produce valuable indigoid pigments such as indigo and indirubin, which can bring both economic and environmental benefits. To date, numerous researches have been conducted using pure cultures (wild strains or recombinant Escherichia coli) to produce indigoids. However, the reported wild strains are limited to a few genera, and most of the strains belong to Pseudomonas. Furthermore, few attempts have been made to explore the possibility of microbial community for indigo production. Herein, the production of indigoids from indoles was investigated by wild strain, recombinant E. coli, and the activated sludge systems, respectively. The main contents are as follows:Due to limited genera were reported for indigo production, it was necessary to screen novel indigo producing strains. Two activated sludge systems induced by different aromatics were operated in parallel:naphthalene+indole (G1) and phenol+indole (G2). The results proved the both systems were capable of producing indigo from indole, and G1possessed a better capability of indigo production, from which a new bacterial strain MQ was isolated. Based on16S rRNA gene sequence analysis, strain MQ was identified as Comamonas sp., a novel genus for indigo production. The characteristics of indigo production by growing and resting cells of strain MQ were investigated in detail. The optimal conditions for the growing cells were:indole50mg/L, naphthalene200mg/L. Under the conditions,32.2mg/L indigo was produced. As for the resting cells,53.1mg/L indigo was produced under the following optimal conditions: temperature25℃, rotational speed150r/min, biomass2.16(OD660), indole201mg/L and pH6.9. According to thin-layer chromatography and liquid chromatography-mass spectrometry (LC-MS) analysis, the blue product was confirmed to be indigo. A possible pathway for indigo production by strain MQ was proposed. Indole was firstly oxidized by naphthalene dioxygenase to cis-indole-2,3-dihydrodiol, which was subsequently dehydrated to form indoxyl. Two molecules of indoxyl were condensed spontaneously to form indigo.The complete5.2kbp naphthalene dioxygenase (NDO) genes were successfully amplified from strain MQ by Thermal Asymmetric Interlaced-PCR and homologous amplification. Based on the sequence analysis, the NDO genes belonged to nag gene cluster in an order of nagAa-nagG-nagH-nagAb-nagAc-nagAd. Then, the genes were transferred into in E. coli BL21(DE3)(designated as ND_IND) and successfully expressed based on the analysis of SDS-PAGE and crude enzyme activity. The recombinant strain ND_IND was able to produce various indigoid pigments from most indole derivatives such as indole, methylindoles, nitroindoles, chloroindoles, etc. According to the analysis of UV-vis spectra and LC-MS, the major products derived from indoles should be indigo with different substituent groups.The recombinant strain ND_IND was then used to produce indigoids from indole and tryptophan. When using indole as the substrate, about205.1mg/L indigo was produced under the optimal conditions:biomass2.50(OD600), indole300mg/L, glucose30g/L and pH7.0. The effects of metal ions and typical compounds in coking wastewater on indigo production were investigated, which indicated that most of the metal ions, as well as quinoline, could inhibit indigo production. When using tryptophan as the substrate, the preferable induction conditions for NDO expression were30℃with0.50mmol/L IPTG after the cells were grown to OD6001.20. The effects of tryptophan medium composition on indigoids production were optimized by response surface methodology, and150.2mg/L indigo and9.4mg/L indirubin were produced, respectively. Meanwhile, the indirubin production was further improved by adding2-oxindole and isatin to the media. About58.0mg/L indirubin was obtained by the addition of500mg/L2-oxindole after1-h induction, which was approximately increased by6.2-fold.The activated sludge system (B1) and other two systems inoculated with wild strain MQ (B2) and recombinant strain ND_IND (B3) were constructed to investigate the indigo production for the first time. The results indicated that the two augmented groups (B2and B3) produced higher yields of indigo compared with the non-augmented group (B1). The microbial communities were analyzed by Illumina high-throughput sequencing, and the results revealed that the inoculated strain MQ in B2were dominant in the early days, while the recombinant strain ND_IND could not survive in the activated sludge system. QPCR analysis showed that the nagAc gene from strain MQ was strongly correlated with indigo yields at early stage, suggesting the inoculated strains could play an important role in indigo production. According to the detrended correspondence analysis and the nonparametric tests, the three communities varied significantly during the operation. Comamonas, Alcaligenes, Aquamicrobium and Pseudomonas were the major genera in the three groups (>1%on average). Comamonas and Pseudomonas, as commonly reported indigo-producing strains, showed no positive relationship with indigo yields based on Pearson correlation test. The other two genera Alcaligenes and Aquamicrobium were positively correlated with indigo yields, which were rarely reported for indigo production previously and could be the novel indigo producing strains.