Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

Clemente, Thomas E.; Shah, Devang; Tran, Minhtien; Stark, David; Padgette, Stephen R.; Dennis, Douglas; Brückener, K; Steinbüchel, Alexander; Mitsky, Timothy A.

doi:10.1007/s002530051636

Cited by 30 publications

(25 citation statements)

References 20 publications

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“…NC_004722.1 [20]. This result is consistent with many studies in different bacteria such as; Rhodospirillum rubrum [26], Bacillus megaterium [18], Bacillus cereus SPV [25] and Bacillus thuringiensis R1 [4].…”

Section: Discussionsupporting

confidence: 79%

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Rabey¹

2017

JIG

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Poly 3-hydroxybutyrate (PHB) is the most known degradable biopolymers, produced by some genera of bacteria under unfavorable growth conditions. Isolation, cloning, and expression of PHB genes from PHB producing Bacillus cereus was achieved. A highest Poly 3-hydroxybutyrate producer bacterium was selected and identified as Bacillus cereus MM7, based on morphological and physiological characters in addition to 16S rRNA gene sequences. The four genes of Bacillus cereus MM7 that involved in Poly 3-hydroxybutyrate production were successfully amplified using specific primers which were designed depending on the sequence alignment of the known PHA biosynthetic genes from Bacillus cereus, cloned in E. coli DH5α and completely sequenced. Moreover, the expression of the four genes responsible for PHA biosynthetic in E. coli XL1-Blue and E coli MJ 109 was assayed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the current study Bacillus cereus was able to synthesize PHB. PHB genes were isolated, cloned and expressed in E. coli DH5α. The PHB encoding proteins were analyzed in SDS-PAGE and the PHB corresponding protein bands were analyzed using mass spectrometry to nominate their amino acid sequence. Keywords: Pre cultureThe pre culture was prepared in 250 ml Erlenmeyer flasks containing 50 ml of nutrient broth [16]. Each flask was inoculated with a loop of the tested cultures and incubated in a 130 rpm speed rotary shaker at 30˚C for 24h.

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Section: Discussionsupporting

confidence: 79%

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Rabey¹

2017

JIG

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“…Previous studies identified and characterized two PHA polymerases (PhaCs) (5,12) in R. rubrum. Using these two sequences as the query, homology searches of the R. rubrum genome of strain ATCC 11170 revealed three genes encoding PHA polymerases, Rru_A0275, Rru_A2413, and Rru_A1816, which we designated phaC1, phaC2, and phaC3, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…This metabolically versatile bacterium can grow under aerobic or anaerobic conditions or in the presence or absence of light, and in the latter condition it can use a variety of different carbon substrates (39,43). Previous studies and genome sequencing have revealed that R. rubrum can express three PHA polymerases (5,12). Because of its metabolic flexibility, R. rubrum offers the potential for converting many different carbon sources to PHA, and therefore there are considerable industrial interests in exploring this flexibility (6,35).…”

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confidence: 99%

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Jin

Nikolau

2012

J Bacteriol

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This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C 5 and C 6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.

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“…Most strains of the genus Methylobacterium grow slowly, and many of them do not grow at all on nutrient agar (4). Strains from the genus Rhodobacter and Rhodospirillum are very difficult to grow and must be cultivated on special medium under anaerobic conditions (2,17). However, none of the recombinant R. eutropha transformants expressing the chimeric constructs carrying (in-frame) phaC fragments originating from those bacteria was able to produce PHAs.…”

Section: Discussionmentioning

confidence: 99%

Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

Niamsiri

Delamarre

Kim

et al. 2004

Appl Environ Microbiol

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PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1 Po ) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB ؊ 4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1 Po genes that produced more PHA than the native phaC1 Po . Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (M w ) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1 Po , and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1 Po , developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

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Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

Cited by 30 publications

References 20 publications

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

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