Engineering Delftia acidovorans DSM39 to produce polyhydroxyalkanoates from slaughterhouse waste.

The inexpensive agricultural fatty by-products could be usefully converted to polyhydroxyalkanoates (PHAs) by properly selected and/or developed microbes. Delftia acidovorans DSM39 is a well-known producer of PHAs with high molar fractions of 4-hydroxybutyrate (4HB), but unable to grow on fatty substrates. The aim of this study was to construct a recombinant strain of D. acidovorans DSM39 using fats-containing waste such as udder, lard and tallow, to produce PHAs. The lipC and lipH genes of Pseudomonas stutzeri BT3, proficient lipolytic isolate, were successfully co-expressed into D. acidovorans DSM39 and the resulting recombinant strain displayed high extracellular enzymatic activity on corn oil. The PHAs production from corn oil achieved high levels (26% of cell dry weight, with about 7% of 4HB). Surprisingly, the recombinant strain produced greater values directly from slaughterhouse residues such as udder and lard (43 and 39%, respectively, with almost 7% of 4HB). Moreover, this work proved the ability of the recombinant D. acidovorans strain to produce PHAs with significant percentage of 4HB, without the supplementation of any precursor in the liquid broth. This research paves the way to the efficient one-step conversion of fatty residues into PHAs having valuable properties exploitable in several medical and industrial applications.

Polyhydroxyalkanoate biosynthesis by Hydrogenophaga pseudoflava DSM1034 from structurally unrelated carbon sources.

In the present paper we report the exclusive microbial preparation of polyhydroxyalkanoates (PHA) containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) as comonomers through the use of unexpensive carbon sources such as whey from dairy industry. Polymers were produced by growing H. pseudoflava DSM 1034 in minimal medium supplemented with sucrose, lactose or whey without any co-substrate added. The chemical and physical properties of the polymers were fully characterized by GPC, DSC, TGA analyses and the composition by GC and (1)H NMR examinations to especially confirm the content of different monomeric units. The presence of 4HB units into PHA samples is particularly aimed in thermoplastic applications where greater flexibility is required and conventional rigid PHAs tend to fail. Usually the insertion of 4HB into chain backbone consisting of 3-hydroxyalkanoates requires expensive carbon sources mostly of petrochemical origin. According to our study the production of P(3HB-co-3HV-co-4HB) terpolymer can be obtained directly by the use of lactose or waste raw materials such as cheese whey as carbon sources. Although the amount of 4HB in the produced terpolymers was usually low and not exceeding 10% of the total molar composition, a PHA containing 18.4% of 4HB units was produced in 1 step fermentation process from this structurally unrelated carbon sources. The crystallinity of the terpolymer is basically to be markedly affected with respect to that of conventional PHAs, thus obtaining a comparatively less rigid material and easier to be processed.

Polyhydroxyalkanoates production by engineered Cupriavidus necator from waste material containing lactose.

Cupriavidus necator DSM 545 is a well-known polyhydroxyalkanoates (PHAs) producer, but unable to grow on lactose. The aim of this study was to construct a recombinant strain of C. necator that can use lactose-containing waste material such as cheese whey, to produce PHAs. One of the intracellular PHA depolymerases (phaZ1) of C. necator was chosen to insert the lacZ, lacI and lacO genes of Escherichia coli. This would have the effect to allow polymer production on lactose and, at the same time, to remove part of the PHA intracellular degradation system. Disruption of phaZ1 was achieved by gene replacement after isolating a fragment of this gene and interrupting it with a cartridge containing the lac genes and a synthetic promoter. Growth and polymer production studies of the genetically modified (GM) strain mRePT in lactose, whey permeate and hydrolyzed whey permeate as carbon sources, were performed. Lower PHA degradation and higher yields were obtained compared to the wild-type strain. Inactivation of the putative depolymerase gene phaZ3 on mRePT recombinant strain was also reported.

Effect of poly-3-hydroxybutyrate synthase mutation on the metabolism of Ensifer (formerly Sinorhizobium) meliloti.

In order to investigate the effect of poly-3-hydroxybutyrate synthase mutation (phbC) on the synthesis of exopolysaccharides (EPS) and glycogen, on the symbiotic properties and on the survival under specific conditions of Ensifer meliloti (formerly Sinorhizobium), a new stable phbC mutant of Ensifer meliloti 41 was isolated and characterized.Under poly-3-hydroxybutyrate accumulation conditions, the phbC -minus mutant (strain 41003) accumulates more glycogen and less exopolysaccharides as compared to the wild-type strain, and grows poorly in pyruvate as carbon source. The inactivation of aniA, encoding for a global carbon flux regulator, restores in E. meliloti 41003 the ability to grow on pyruvate, indicating a new role for this gene. Survival studies of E. meliloti 41 and 41003 under carbon free medium in both liquid and soil microcosms showed prolonged survival of E. meliloti 41 under these adverse conditions as compared to the mutant strain unable to accumulate the polyester. On the other hand, the accumulation of P(3HB) gave no significant advantage in survival under oxygen-limiting conditions. In both strains, E. meliloti 41 and 41003, nodule-inducing ability on alfalfa plants and acetylene reduction activity did not significantly differ from each other, although the mutant strain was less competitive in terms of root colonization.

Resuscitation of viable but not culturable Sinorhizobium meliloti 41 pRP4-luc: effects of oxygen and host plant.

A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained, S. meliloti 41/pRP4-luc, and its parental strain served as a model system for viable but not culturable (VBNC) resuscitation experiments in both in vitro and soil samples. Incubation under oxygen (02) concentrations varying from 1% to atmospheric levels did not result in resuscitation. A demonstration of recovery was attained through exposure to the appropriate concentrations of antibiotics, bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation ratio was 1 recovered VBNC cell in every 10(5) 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC+) bacteria. Although isolated VBNC rhizobia were unable to nodulate Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated bacteria maintained their symbiotic properties. Soil experiments showed that the lack of O2 leads to onset of VBNC status as in liquid microcosm, but the number of recoverable and culturable cells decreased more drastically in soil.