Melanin biopolymer synthesis using a new melanogenic strain of Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing 4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi

Abstract

Background: Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report the high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD).

Results: Melanin synthesis of F. kingsejongi strain was confirmed via melanin synthesis inhibition test, melanin solubility test, genome analysis, and structural analysis of purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6 × His-tagged and native forms of HPPD. The specific activity of F. kingsejongi HPPD was 1.2 ± 0.03 μmol homogentisate/min/mg-protein. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h.

Conclusions: Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large-scale bacterial melanin production. Furthermore, F. kingsejongi strain could serve as a model to elucidate the regulation of melanin biosynthesis pathway and its networks with other cellular pathways, and to understand the cellular responses of melanin-producing bacteria to environmental changes, including nutrient starvation and other stresses.

Keywords: 4-Hydroxyphenylpyruvate dioxygenase; Flavobacterium kingsejongi; Melanin.

Fig. 1

Biosynthesis of three classes of microbial melanin (eumelanin, pheomelanin, and pyomelanin) from the precursor l-tyrosine

Fig. 2

Structural analysis of the melanin purified from the F. kingsejongi culture broth. A UV–vis spectra analysis of F. kingsejongi melanin and commercial eumelanin (as a control). B FT-IR spectra of F. kingsejongi melanin and commercial eumelanin (as a control). C ¹H NMR spectra of F. kingsejongi melanin. D ¹H NMR spectra of commercial melanin (as a control)

Fig. 3

Heterologous expression of the eight putative proteins from F. kingsejongi involved in melanin synthesis in E. coli; organization of genes involving homogentisate pathway of F. kingsejongi. A E. coli cultures expressing eight F. kingsejongi candidate genes after 2 days of growth at 30 °C on LB agar plates supplemented with 1 g/L tyrosine. Plate sections are 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). B Time-course monitoring of culture broth of E. coli expressing the putative HPPD. C Organization of genes encoding homogentisate pathway enzymes in F. kingsejongi

Fig. 4

In vitro activity of purified 6 × His-tagged HPPD and native HPPD in crude protein extract. A HPPD activity was investigated using 4-HPP as a substrate for purified 6 × His-tagged HPPD. Red and blue arrows indicate peaks corresponding to homogentisate (product) and 4-HPP (substrate), respectively. B Tyrosinase activity was investigated using tyrosine as a substrate for purified 6 × His-tagged HPPD. C HPPD activity was investigated using 4-HPP as a substrate for native HPPD in a crude protein extract. Red and blue arrows indicate peaks corresponding to homogentisate (product) and 4-HPP (substrate), respectively. D Tyrosinase activity was investigated using tyrosine as a substrate for native HPPD in a crude protein extract

Fig. 5

Multialignment of amino acid sequences of five Flavobacterium HPPD and homology modelling of the five Flavobacterium HPPDs and R. pickettii HPPD. A Amino acid sequences of F. kingsejongi HPPD and the four phylogenically close Flavobacterium HPPDs were aligned. The grey background represents 100% homology identities of amino acids between the five HPPDs, the red background represents 80% homology identities, and the brown background represents 50% homology identities. Amino acid sequences braced in red brackets represent the active site for 4-HPP binding. The yellow background indicates the binding residues in the active site for HPPD-4-HPP complex. B 3D molecular docking models for the HPPD-4-HPP complexes in the active site region of the HPPDs of F. kingsejongi (1), F. microcysteis (2), F. endophyticum (3), F. noncentrifugens (4), Flavobacterium sp. BFFFF1 (5), and R. pickettii (6) are presented. A molecular atom stick model of the substrate 4-HPP is presented. Hydrophobic, hydrogen bonding, and ionic interactions are indicated as dashed grey, blue, and yellow lines, respectively. Cavity sizes of each active site of the protein–ligand models are presented

Fig. 6

Batch fermentation of melanin-producing F. kingsejongi in a 5-L bioreactor. A The kinetics of cell growth and the rates of glucose and tyrosine consumption were monitored by measuring optical density at 600 nm (OD₆₀₀) and quantifying the concentrations of glucose and tyrosine present in the culture broth. OD₆₀₀ is represented by filled circles (●), residual glucose concentration by green triangles (▲), and residual tyrosine concentration by red rectangles (■). B Supernatant was collected from the culture at set time intervals and visually inspected. C The kinetics of F. kingsejongi melanin production were monitored by quantifying the melanin present in the culture broth during batch fermentation. D RT-PCR (left) and qRT-PCR (right) analysis of the hpd gene encoding HPPD in F. kingsejongi cells collected at 5, 19, and 50 h of fermentation culture. The tuf gene was used as a reference

Fig. 7

Batch bioreactor fermentation of melanin-producing recombinant E. coli expressing the F. kingsejongi hpd gene encoding HPPD. A The kinetics of cell growth and the rates of glucose and tyrosine consumption were monitored by measuring optical density at 600 nm (OD₆₀₀) and quantifying the concentrations of glucose and tyrosine present in the culture broth. OD₆₀₀ is represented by filled circles (●), residual glucose concentration by green triangles (▲), and residual tyrosine concentration by red rectangles (■). B Supernatant was collected from the culture at set time intervals and visually inspected. C The kinetics of melanin production by recombinant E. coli were monitored by quantifying the melanin present in the culture broth during batch fermentation