Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB-4 strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB-4 strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB-4 (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs.

Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli.

BACKGROUND: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli. MATERIALS AND METHODS: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized. RESULTS: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours. CONCLUSION: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Optimization of the Expression of Genes Encoding Poly (3-hydroxyalkanoate) Synthase from Pseudomonas aeruginosa PTCC 1310 in Escherichia coli.

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates (PHAs). To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa (P.aeruginosa) PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli (E.coli) BL21 (DE3) cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37 °C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes.

Análisis de las polihidroxialcanoato sintasas (PhaC1 y PhaC2) en una cepa de Pseudomonas fluorescens IBUN S1602, aislada en suelos colombianos / Analysis of polyhydroxyalkanoate synthases (PhaC1 and PhaC2) in a strain of Pseudomonas fluorescens IBUN S1602 isolated from Colombian soil

La cepa Pseudomonas fluorescens IBUN S1602 conforma el grupo de aislamientos provenientes de suelos colombianos de caña de azúcar, que acumula polihidrioxialcanoato (PHA), fue seleccionada como promisoria para escalamiento comercial por tener afinidad por sustratos alternativos y económicos como el glicerol, aceites usados, suero de leche, entre otros. Dada la importancia de la enzima sintasa en la síntesis de los PHAs, en el presente trabajo se realizó el análisis molecular de los genes phaC1 y phaC2 que codifican las enzimas sintasas tipo II (PhaC1 y PhaC2). Para la obtención de los amplímeros requeridos en la secuenciación, se utilizó la técnica de PCR bajo condiciones estandarizadas para iniciadores diseñados reportados en las bases de datos. Se identificaron dos fragmentos de 1680 pb y 1683 pb correspondientes a phaC1 y phaC2. El análisis comparativo de las secuencias proteicas resultantes de estos genes demuestra que la sintasa IBUN S1602 contiene la región α/β hidrolasa y 8 residuos de aminoácidos conservados, que son características de las sintasas examinadas a nivel mundial. Se analizó la estructura enzimática a nivel primario y se predijo la secundaria. Se concluyó que las sintasas de la cepa Pseudomonas fluorescens IBUN S1602 presentan alta homología con las sintasas tipo II que se reportan para Pseudomonas. Los resultados obtenidos contribuyen al entendimiento básico de la biosíntesis de PHA, la cual permitirá, en un futuro, el aumento de la calidad de PHA debida a la modulación del nivel de sintasa que se exprese en un organismo recombinante, con el fin de variar el peso molecular del biopolímero, propiedad esencial en el estudio de aplicaciones industriales.

The strain Pseudomonas fluorescens IBUN S1602 forms the group of isolates from colombian sugarcane soil´s, which accumulates polyhydroxyalkanoate biopolymer (PHA) and was selected as promising for commercial scale by having affinity for economic and alternative substrates such as glycerol, oils, whey, among others. Given the importance of the synthase enzyme in the synthesis of PHAs, was realized the molecular analysis of genes phaC1 and phaC2 which encode type II synthases (PhaC1 y PhaC2). To obtain the amplimers required in the sequencing, was used the PCR technique under standardized conditions for primers designed based on the updated review in databases. Were identified two fragments of 1680 bp and 1683 bp for phaC1 and phaC2. Comparative analysis of the resulting protein sequences of these genes shows that the IBUN S1602 synthases containing the region α/β hydrolase and 8 conserved amino acid residues that are characteristic of synthases examined worldwide. Enzyme structure was analyzed at the primary level and was predicted the secondary. It is concluded that synthase strain Pseudomonas fluorescens IBUN S1602 has high homology with type II synthases that are reported for Pseudomonas. The results contribute to basic understanding of the biosynthesis of PHA, and will allow in the future, increasing the quality of PHA due to modulation of the level of synthase is expressed in a recombinant organism, in order to vary the weight molecular biopolymer, an essential property in the study of industrial applications.