Cloning and characterization of poly(3‐hydroxybutyrate) biosynthesis genes from <i>Pseudomonas</i> sp. USM 4‐55

BackgroundCyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN−) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required.ResultsIn this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha−, deleted in putative mcl-PHA synthases; Spha−, deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC–MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha− and Spha−, respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers.ConclusionsThese results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain benefits of this type of approaches.

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“…61-3 [ 23 ], Pseudomonas sp. USM 4-55 [ 42 ], Azotobacter sp. FA8 [ 43 ] and Azotobacter vinelandii [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity

et al. 2015

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“…strain 61-3 (Matsusaki et al, 1998), and Pseudomonas sp. USM 4-55 (Tan et al, 2010), the phbA, phbB and phbC genes are clustered in the same locus and are organized in a single transcriptional unit. In all these cases, upstream of the phbBAC operon and in the opposite direction, the gene called phbR is located.…”

Section: Phbrmentioning

confidence: 99%

The Modification of Regulatory Circuits Involved in the Control of Polyhydroxyalkanoates Metabolism to Improve Their Production

Velázquez-Sánchez

Espı́n

Peña

et al. 2020

Front. Bioeng. Biotechnol.

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Poly-(3-hydroxyalkanoates) (PHAs) are bacterial carbon and energy storage compounds. These polymers are synthesized under conditions of nutritional imbalance, where a nutrient is growth-limiting while there is still enough carbon source in the medium. On the other side, the accumulated polymer is mobilized under conditions of nutrient accessibility or by limitation of the carbon source. Thus, it is well known that the accumulation of PHAs is affected by the availability of nutritional resources and this knowledge has been used to establish culture conditions favoring high productivities. In addition to this effect of the metabolic status on PHAs accumulation, several genetic regulatory networks have been shown to drive PHAs metabolism, so the expression of the PHAs genes is under the influence of global or specific regulators. These regulators are thought to coordinate PHAs synthesis and mobilization with the rest of bacterial physiology. While the metabolic and biochemical knowledge related to the biosynthesis of these polymers has led to the development of processes in bioreactors for high-level production and also to the establishment of strategies for metabolic engineering for the synthesis of modified biopolymers, the use of knowledge related to the regulatory circuits controlling PHAs metabolism for strain improvement is scarce. A better understanding of the genetic control systems involved could serve as the foundation for new strategies for strain modification in order to increase PHAs production or to adjust the chemical structure of these biopolymers. In this review, the regulatory systems involved in the control of PHAs metabolism are examined, with emphasis on those acting at the level of expression of the enzymes involved and their potential modification for strain improvement, both for higher titers, or manipulation of polymer properties. The case of the PHAs producer Azotobacter vinelandii is taken as an example of the complexity and variety of systems controlling the accumulation of these interesting polymers in response to diverse situations, many of which could be engineered to improve PHAs production.

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“…LDC-5, 26 and Pseudomonas sp. USM 4-55 53 can accumulate two types of PHA, SCL-PHA e.g. P(3HB) and MCL-PHA.…”

Section: Nitrogen Sourcementioning

confidence: 99%

“…USM 4-55 was clustered in the phbBAC operon which may be a unique property of the Pseudomonads that can produce both SCL-PHA and MCL-PHA whereas in the earliest operon model investigated, C. necator, its operon was arranged as phbCAB. 53 However, the genetic organization in P. mendocina PSU will be further studied. To corroborate that this strain can produce two types of PHA granules, GC-MS and 1 H NMR were used to determine the monomer compositions of the produced PHA.…”

Section: Nitrogen Sourcementioning

confidence: 99%

Efficient production of polyhydroxyalkanoates (PHAs) fromPseudomonas mendocinaPSU using a biodiesel liquid waste (BLW) as the sole carbon source

Chanasit

Hodgson

Sudesh

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.

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Cloning and characterization of poly(3‐hydroxybutyrate) biosynthesis genes from Pseudomonas sp. USM 4‐55

Cited by 7 publications

References 33 publications

Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity

Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity

The Modification of Regulatory Circuits Involved in the Control of Polyhydroxyalkanoates Metabolism to Improve Their Production

Efficient production of polyhydroxyalkanoates (PHAs) fromPseudomonas mendocinaPSU using a biodiesel liquid waste (BLW) as the sole carbon source

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