Silvana Povolo

Poly-β-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41

Summary: Genes encoding β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti.

Abstract. During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-β-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and β-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.

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.

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 (O2) 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 105 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.

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.

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.

Production of Polyhydroxyalkanoates from Fatty Wastes

The production of polyesters from triglyceride containing substrates was investigated. A first filter step based on lipase activity was followed and those bacteria potentially able to degrade oils or animal fats were further tested for their polymer accumulation properties, selected and kept for further studies. In a second step, bacteria were directly grown on animal fats and/or vegetable oils, and polyhydroxyalkanoates (PHAs) accumulation was verified under appropriate incubation conditions. Each substrate, whether of animal or vegetable derivation, supported the growth of a number of the newly isolated strains and among those, some strains were also found to produce reasonably high amounts of PHA. The repeat-unit composition of the polyesters was determined by gas chromatography (GC) analysis of the ß-hydroxyalkanoate methyl esters from the hydrolyzed polymer and some class of co-polymers were also detected. These properties, coupled with the ability of some of the selected isolates to grow and produce lipases on a minimal medium, could be considered as promising in view of possible industrial applications. The overall results indicate that PHAs could be produced from waste containing considerable amounts of fat, oil and grease (FOG), that generally need to be treated for their disposal.

Poly(hydroxyalkanoate) Production by Cupriavidus necator from Fatty Waste Can Be Enhanced by phaZ1 Inactivation

Although PHAs are regarded as an effective substitute for conventional plastics for a number of medical and agricultural applications1 and food packaging2, their full-scale manufacturing is hampered by high production costs3. Factors affecting the cost of PHAs include raw materials availability, suitable process design, and downstream processing4,5. Since almost 50 % of the total production costs can be attributed to the carbon source for microbial growth and polymer production, the selection of renewable, cheap carbon feedstock, specially generated from industrial or agricultural by-products, can provide a way to reduce the price6,7,8. To that end, different industrial by-products, such as whey9,10, molasses11, starch, and waste oils and glycerol, have been investigated as start materials for PHA production12,13,14,15. In this perspective, fatty residues from slaughterhouse represent a promising raw material. For Europe, the amount of animal lipids, also causing expensive disposal problems, is estimated a half a million tons per year16. However, PHA production from waste oils or fats requires microorganisms that should be both excellent PHA producers as well as equipped with enzymatic activities allowing hydrolysation of triglycerides. Cupriavidus necator (formerly Ralstonia eutropha) is one of the best known bacteria among PHA-producing microorganisms15,17. The production of different kinds of PHAs by C. necator using several inexpensive feedstock, including glycerol, has been recently reported18,19,20,21,22, indicating that some strains of this bacterial species could be used for the conversion of fatty residues into PHA. As an alternative, the use of costly commercial enzymes or genetic modification of microorganisms exhibiting high PHA product yields would be required23. In the case of C. necator, its lipase activities could most likely be improved by physiological/technological studies in terms of biomass production and PHA content, but the help of commercial enzymes may still be necessary. A possible strategy to help increase the final PHA yield in a bacterial strain already possessing both high polymer production ability and acceptable triglyceride hydrolytic activity, could originate from the relevant findings regarding C. necator PHA metabolism, physiology, Poly(hydroxyalkanoate) Production by Cupriavidus necator from Fatty Waste Can Be Enhanced by phaZ1 Inactivation

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.

Biosynthesis of Polyhydroxyalkanoates and their Regulation in Rhizobia

During free living reproductive growth Rhizobium spp. is capable to accumulate poly-s-hydroxybutyrate (PHB) and to synthesize intracellular glycogen. These bacteria can also produce and excrete exopolysaccharides and s-1,2-glucan. Rhizobia provide an excellent model to investigate the connection between cellular metabolism and polyester accumulation. We have shown that under oxygen-limiting conditions, free-living cells of Sinorhizobium meliloti 41 can use intracellular glycogen to generate ATP, while maintaining their PHB content. PHB synthesis serves as an alternative pathway for storage/regeneration of reducing equivalents. We have described genes involved in PHB biosynthesis in S. meliloti encoding for s-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA-synthase (phaC) together with an open reading frame, referred to as aniA. Under oxygen-limiting conditions (such as conditions in the bacteroid state) aniA is actively expressed, and a mutation in this gene generates an overproduction of extracellular polymeric substances (EPS). This finding suggests that the production of EPS could be directly or indirectly regulated by aniA. Therefore, in S. meliloti, aniA is likely to be involved in carbon/energy flux regulation that, in turn, is dependent upon oxygen availability. By hybridization studies we revealed, in various soil bacteria the presence of genes with sequence similarity to aniA of S. meliloti 41. These results will be important to gain a deeper insight into aniA function in the control of PHB (more generally PHA) and EPS biosynthesis.