Textile Dye Decolorizing Synechococcus PCC7942 Engineered With CotA Laccase

Abstract

Cyanobacteria are prokaryotic phototrophs capable of achieving high cellular densities with minimal inputs. These prokaryotic organisms can grow using sunlight as energy source and carbon dioxide as carbon source what makes them promising candidates as microbial cell factories for the production of numerous compounds such as chemicals, fuels, or biocatalysts. In this study, we have successfully designed and constructed using synthetic biology approach two recombinant strains of Synechococcus elongatus PCC7942 for heterologous expression of the industrially relevant Bacillus subtilis CotA laccase. One of the strains (PCC7942-NSI-CotA) was constructed through integration of the laccase gene into neutral site I of the cyanobacterial genome whilst the other (PCC7942-NSII-CotA) targeted neutral site II of the genome. Of the two strains the one with CotA laccase integrated in neutral site II (PCC7942-NSII-CotA) was superior in terms of growth rate and enzymatic activity toward typical laccase substrates: ABTS [2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] and syringaldazine. That may suggest that two of the traditionally used neutral sites of S. elongatus PCC7942 are not equally suitable for the expression of certain transgenes. The PCC7942-NSII-CotA produced protein was capable of decolourising three classes of dyes namely: anthraquinonic-, azo-, and indigoid-type over 7 days of incubation making the strain a potentially useful microbial cell factory for the production of broad-spectrum biodegradation agent. Interestingly, presence of additional synthetic redox mediator ABTS had no effect on the degradation of these dyes.

Keywords: DNA assembly; cyanobacterium; dye decolorization; laccase; microbial cell factory.

Figure 1

Analysis of successful transformants of PCC7942-NSI-CotA and PCC7942-NSII-CotA strains and the wild type PCC7942-WT strain. (A) PCR products confirming genomic arrangement of the transgenic strains and controls labeled in convention Strain ID: Forward primer ID_Reverse primer ID. Strain IDs and primer IDs can be found in Table 1, M, molecular weight marker. Actual PCR product sizes and sizes of markers are shown on the figure at the bottom and on the left, respectively. (B) Design features of the plasmid pCV0063 and the corresponding strain PCC7942-NSI-CotA. (C) design features of the plasmid pCV0062 and the corresponding strain PCC7942-NSI-CotA. Binding sites for primers from Table 1 are shown sections (B,C) of the figure.

Figure 2

Cultivation of transgenic strains PCC7942-NSI-CotA and PCC7942-NSII-CotA in BG 11 growth medium: (A) growth curve of PCC7942-NSII-CotA (squares ■), PCC7942-NSI-CotA (triangles ▴) and PCC7942-WT (circles •); (B) laccase activity curve in cell-free culture medium of PCC7942-NSII-CotA (squares ■), PCC7942-NSI-CotA strain (triangles ▴), PCC7942-WT (circles •).

Figure 3

Expression analysis of the PCC7942-NSII-CotA and wild type PCC7942-WT: (A) RT-PCR products confirming expression of the laccase in strain PCC7942-NSII-CotA. Strains and controls labeled in convention Strain ID: Forward primer ID_Reverse primer ID. Strain IDs and primer IDs can be found in Table 1. Size of the CotA laccase gene is 1539 bp, and C-phycocyanin beta subunit gene, that was used as positive control, 519 bp. M, molecular weight marker. (B) SDS-PAGE of the CotA expressing S. elongatus PCC7942-NSII-CotA. Total cell extract of PCC7942-NSII-CotA and PCC7942-WT were fractionated by SDS/PAGE and stained with EZBlue™ Gel Staining Reagent. Lanes: PCC7942-WT: wild-type S. elongatus PCC7942; PCC7942-NSII-CotA: NSII-targeted, CotA-expressing transformant of S. elongatus PCC7942, M, molecular weight marker, Arrow indicates CotA band confirmed by peptide sequencing.

Figure 4

Decolorization of tested dyes (0.1 g/l) by cell-free culture medium containing CotA laccase produced by the recombinant cyanobacterium PCC7942-NSII-CotA (light gray bars) and by the cell-free culture medium of the control strain PCC7942-WT (dark gray bars) during 7 days of incubation. Following decolorization conditions were used: (A) No ABTS mediator in 0.05M phosphate-citric acid buffer (pH 5.5), (B) 10 mM (final) ABTS mediator in 0.05M phosphate-citric acid buffer (pH 5.5); (C) No ABTS mediator in 0.05M phosphate-citric acid buffer (pH 7.5); (D) 10 mM ABTS mediator in in 0.05M phosphate-citric acid buffer (pH 7.5). Abbreviations: Reactive Blue 19 (RB19); Reactive Blue 4 (RB4); Reactive Black 5 (RB5), Acid Red 18 (AR18), Acid Red 27 (AR27), Acid Yellow 18 (AY18), Reactive Red 11 (RR11) Reactive Orange 5 (RO5), and Indigo carmine (IC).

Figure 5

UV–Vis spectra of Indigo carmine (0.1 g/l) biodegraded by PCC7942-NSII-CotA produced recombinant laccase at times: 0, 3, 7 days. T = 30°C, pH = 7.5 Arrows indicate characteristic decreases in absorbance throughout the decolorization experiment.