De Novo Assembly of the Polyhydroxybutyrate (PHB) Producer Azohydromonas lata Strain H1 Genome and Genomic Analysis of PHB Production Machinery

Abstract

Polyhydroxybutyrate (PHB) is a biodegradable natural polymer produced by different prokaryotes as a valuable carbon and energy storage compound. Its biosynthesis pathway requires the sole expression of the phaCAB operon, although auxiliary genes play a role in controlling polymer accumulation, degradation, granule formation and stabilization. Due to its biodegradability, PHB is currently regarded as a promising alternative to synthetic plastics for industrial/biotechnological applications. Azohydromonas lata strain H1 has been reported to accumulate PHB by using simple, inexpensive carbon sources. Here, we present the first de novo genome assembly of the A. lata strain H1. The genome assembly is over 7.7 Mb in size, including a circular megaplasmid of approximately 456 Kbp. In addition to the phaCAB operon, single genes ascribable to PhaC and PhaA functions and auxiliary genes were also detected. A comparative genomic analysis of the available genomes of the genus Azohydromonas revealed the presence of phaCAB and auxiliary genes in all Azohydromonas species investigated, suggesting that the PHB production is a common feature of the genus. Based on sequence identity, we also suggest A. australica as the closest species to which the phaCAB operon of the strain H1, reported in 1998, is similar.

Keywords: PHA; bacteria biopolymers; biodegradable; bioeconomy; bioplastic; polyhydroxyalkanoates.

Figure 1

Circular map of the candidate plasmid generated with the web-based implementation of Proksee. Orange: CDS univocally annotated by PGAP; gray: CDS without a predicted function; blue: predicted transposases; green: TA system-inferred proteins.

Figure 2

Linear representation of the transposes localized on the megaplasmid. The red line corresponds to the plasmid reported in a linearized fashion while the blue bars spanning above are the projections of the predicted transposases on the plasmid. The potential cluster of transposases is visible in the last quarter (from 310 kbp to 410 kbp) of the plasmid from the IS21 family transposase (left side) to the ISL3 family transposase (right side).

Figure 3

Heatmap of the ANI values. The heatmap is calculated by OrthoAniU between each pair of Azohydromonas isolates assemblies, including self-comparison (main diagonal). Darker shades of the color indicate higher ANI values. Accession numbers are listed in Table S1.

Figure 4

Heatmap of the dDDH values. The heatmap has been computed using the Genome-to-Genome Distance Calculator (GGDC-3.0) with recommended settings between each pair of Azohydromonas isolates assemblies. Self-comparison is on the main diagonal and a darker color suggests higher dDDH values. Accession numbers are listed in Table S1.

Figure 5

Heatmap of blastp sequence similarity analysis among collection of representative PhaCs classes (I–V). Darker shades of the color mark higher identity values. Self-comparison occupies the main diagonal.

Figure 6

Portion of the MSA of candidate PhaC proteins computed by the online version of Clustal Omega and visualized with JalViewer. Colored columns are the 8 conserved residues. Residues of the catalytic triad are highlighted with red stars on the top of the corresponding column.