Quantitation of Tolyporphins, Diverse Tetrapyrrole Secondary Metabolites with Chlorophyll‐Like Absorption, from a Filamentous Cyanobacterium–Microbial Community

• Published in: Phytochemical analysis 2018-03, Vol.29 (2), p.205-216
• Main Authors: Zhang, Yunlong, Zhang, Ran, Hughes, Rebecca‐Ayme, Dai, Jingqiu, Gurr, Joshua R., Williams, Philip G., Miller, Eric S., Lindsey, Jonathan S.
• Format: Article
• Language: English
• Subjects: absorption spectroscopy with multicomponent analysis; Chlorophyll - chemistry; Chlorophyll A; Chromatography, High Pressure Liquid - methods; Cyanobacteria - growth & development; Cyanobacteria - metabolism; growth curve; HPLC; mass spectrometry; nitrogen deprivation; Porphyrins - analysis; Porphyrins - chemistry; Reference Standards; Reproducibility of Results; Spectrum Analysis - methods; Tetrapyrroles - analysis
• ISSN: 0958-0344; 1099-1565
• DOI: 10.1002/pca.2735

Introduction Tolyporphins are unusual tetrapyrrole macrocycles produced by a non‐axenic filamentous cyanobacterium (HT‐58‐2). Tolyporphins A–J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a. Identification and accurate quantitation of the various tolyporphins in these chlorophyll‐rich samples presents challenges. Objective To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a. Methodology Chromatographic fractionation of large‐scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small‐scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a, and by inference the amounts of tolyporphins A–M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a. The reported absorption spectral data for the various tolyporphins required re‐evaluation for quantitative purposes. Results and Discussion The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a. Under soluble‐nitrogen deprivation after 35–50 days, tolyporphin A represents 1/3–1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8‐fold that of chlorophyll a. Conclusions The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd. Tolyporphins A–M, produced by a filamentous cyanobacterium (HT‐58‐2), absorb in the same spectral region as chlorophyll a. Fractionation by chromatography of large‐scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small‐scale (2–50 mL) cultures, HPLC separation, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a. Absorption spectroscopy with multicomponent analysis of lipophilic extracts afforded the ratio (up to 1.8‐fold) of all tolyporphins to chlorophyll a.