Biomedical subjects

Soazig C Delamarre

Publications and source records attributed to Soazig C Delamarre.

3 recordsLinked to original sources

PubMed2005

Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha.

Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (P(phb)), acetoin (P(acoE), P(acoD), and P(acoX)) or pyruvate (P(pdhE)) metabolism were constructed and tested. The promoters were representative either of the enterobacterial sigma70 (P(phb), P(acoE), and P(pdhE))- or sigma54 (P(acoD) and P(acoX))-dependent promoters. To obtain polymers consisting of C4-C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 (Ps)) tandemly linked to the W. eutropha genes encoding beta-ketothiolase (phbA (We)) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB (We)) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the P(acoD) and P(acoX) promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters-independent of the addition of acetoin-resulted in the production of PHA polymers with high 3HB fractions (90-100 mol%) and with high 3HO contents (70-86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the P(acoE) promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.

Acetoin↗

PubMed2005

Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2.

A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha ( Ps-1) and pha ( Ps-2)) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha ( Ps-2) locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1( Ps-2)-phaC1 ( Ps-2)-phaZ ( Ps-2)-phaC2 ( Ps-2)-phaD ( Ps-2). The pha ( Ps-1) locus that contains phaC1 ( Ps-1)-phaZ ( Ps-1) appears to have arisen by a duplication event that placed it downstream of a gene (ORF1( Ps-1)), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 ( Ps-1) and phaC1 ( Ps-2) were investigated by heterologous expression in Wautersia eutropha PHB(-)4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1( Ps-1) to confer PHA synthesis, however, appeared reduced compared to that of PhaC1( Ps-2), since cells harboring PhaC1( Ps-1) accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1( Ps-2). Primary sequence analysis revealed that PhaC1( Ps-1) had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1( Ps-2)). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 ( Ps-1) produces a protein product in the native strain, it is likely that PhaC1( Ps-1) may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.

Acyltransferases↗

PubMed2004

Engineering of chimeric class II polyhydroxyalkanoate synthases.

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 (phaC1Po) 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 phaC1Po genes that produced more PHA than the native phaC1Po. 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 (Mw) 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 phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, 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.

Acyltransferases↗