Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

Abstract

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Biodiversity and Habitats of Polar Region Polyhydroxyalkanoic Acid-Producing Bacteria: Bioprospection by Popular Screening Methods

“…isolates contained genes of a usual PHA synthase (provisionally named class V) besides the class I synthase. Class III and IV synthase genes were not amplified by the primers used and currently, there are no wide-range primer sets to detect them [ 29 ]. Microscopy observation of the Nile Red stained cells also required cautious examination.…”

Switching from petro-plastics to microbial polyhydroxyalkanoates (PHA): the biotechnological escape route of choice out of the plastic predicament?

“…PHA bioproduction can be carried out either using established wild type strains, new mesophilic (60,61) or extremophilic (62, 63) microbial isolates, or, to an increasing extend, by using genetically modified strains. Such genetic modifications ("genome editing") of PHA-accumulating microbes have originally been carried out to understand the principal biochemical, enzymatic, genetic, and metabolic features of PHA biosynthesis and intracellular degradation of these biopolyesters.…”

Advances in Polyhydroxyalkanoate (PHA) Production

“…Different substrate feeding strategies using volatile fatty acids (VFAs) were investigated, showing a dynamic trend in biomass formation and monomeric PHA composition dependent on the substrate feed. Metabolic flux analysis was used to gain deeper insights into the goings-on in the MMC during the cultivation; revealing the correlation of reducing equivalents’ generation to the intracellular carbon flux, thus to the PHA composition on the monomeric level, which can be considered as a significant outcome of this study [ 24 ].…”

“…Second, new insights into metabolic processes during intracellular PHA mobilization [ 22 ], metabolic flux analysis of PHA production by mixed microbial cultures (MMCs) [ 23 ], and novel molecular diagnostic techniques to trace new PHA production strains from diverse environments in a convenient manner [ 24 ] are presented.…”