Production of 3-hydroxy-n-phenylalkanoic acids by a genetically engineered strain of Pseudomonas putida

Abstract

Overexpression of the gene encoding the poly-3-hydroxy-n-phenylalkanoate (PHPhA) depolymerase (phaZ) in Pseudomonas putida U avoids the accumulation of these polymers as storage granules. In this recombinant strain, the 3-OH-acyl-CoA derivatives released from the different aliphatic or aromatic poly-3-hydroxyalkanoates (PHAs) are catabolized through the beta-oxidation pathway and transformed into general metabolites (acetyl-CoA, succinyl-CoA, phenylacetyl-CoA) or into non-metabolizable end-products (cinnamoyl-CoA). Taking into account the biochemical, pharmaceutical and industrial interest of some PHA catabolites (i.e., 3-OH-PhAs), we designed a genetically engineered strain of P. putida U (P. putida U DeltafadBA-phaZ) that efficiently bioconverts (more than 80%) different n-phenylalkanoic acids into their 3-hydroxyderivatives and excretes these compounds into the culture broth.

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442

“…2). Remarkably, the phaZ gene showed a 96% identity with the homologous gene of P. putida GPo1 and U strains (26,29,30). To confirm the functionality of the phaZ gene of P. putida KT2442, we tested its capacity to complement the mutant strain P. putida U (PhaZ Ϫ ) by constructing the plasmid pPAZ2, a phaZ shuttle expression vector derived from pBBR1MCS5, able to replicate in Pseudomonas strains.…”

“…Identification of the Products Released from PHA after Enzymatic Hydrolysis-For the identification of the PhaZ hydrolysis products, four reaction mixtures were subjected to enzymatic hydrolysis in parallel with 250 g of P(HO-co-HX)w at different reaction times (1,12,30, and 360 min). The first three mixtures were developed in the presence of 1.8 g of PhaZ as a standard turbidimetric assay.…”

“…This pha cluster is composed of two synthase-coding genes (phaC1 and phaC2), a putative regulatory gene (phaD), two coding phasin genes having structural and regulatory functions (phaF and phaI) (24,26), and the phaZ gene located between the synthase-coding genes whose product is similar to members of the family V of bacterial lipolytic enzymes containing a potential lipase box (27). Interestingly, the first report describing a process of mcl-PHA mobilization demonstrated the existence of self-hydrolysis of the granules in a strain of Pseudomonas oleovorans (15,28), whereas an mcl-PHA intracellular depolymerase activity was ascribed to the phaZ gene firstly in Pseudomonas putida GPo1 (formerly known as P. oleovorans GPo1) (26) and later in P. putida U only by using mutagenesis/complementation experimental approaches (29,30). Nevertheless, a biochemical demonstration that phaZ encodes a depolymerase enzyme remained to be presented.…”

Two New Alkaloids from the Roots of <i>Baphicacanthus cusia</i>

“…On the basis of the comparison of their NMR spectroscopic data with those reported in the literature, the 28 known compounds were identified to be 2-benzoxazolinone (3), 6) tryptanthrin (4), 7) 2-hydroxy-1,4-benzoxazin-3-one (5), 8) 3-(2′-hydroxyphenyl)-4(3H)-quinazolinone (6), 9) 1H-indole-3-carbaldehyde (7), 10) benzouracil (8), 11) 2H-1,4-benzoxazin-3-one (9), 12) 3-carboxyindole (10), 13) acanthaminoside (11), 14) 4(3H)-quinazolinone (12), 15) deoxyvasicinone (13), 16) acanthaminoside isomer (14), 14) lupeol (15), 17) betulin (16), 18) betulinic acid (17), 19) ursolic acid (18), 20) lup-20(29)-en-3β,30-diol (19), 21) maslinic acid (20), 22) guaiacylglycerol-β-ferulic acid ether (21), 23) (2S,3R,4S)-lyoniresinol-3α-O-β-D-glucopyranoside (22), 24) (2R,3S,4R)-lyoniresinol-3α-O-β-D-glucopyranoside (23), 24) (+)-5,5′-dimethoxy-9-O-β-D-glucopyranosyl secoisolariciresinol (24), 25) tyrosol (25), 26) β-hydroxy-benzenepentanoic acid (26), 27) acteoside (27), 28) acteoside isomer (28), 28) loliolide (29), 29) hispiduloside (30). 30) Among these, compounds 3-14 belong to alkaloids (Fig.…”

Non-Edible Vernonia galamensis Oil and Mixed Bacterial Cultures for the Production of Polyhydroxyalkanoates

“…However, the performance of A. latus may be susceptible to extreme temperature, pH, carbon to nitrogen ratio in the feed, concentration of substrates, trace elements, ionic strength, agitation intensity and dissolved oxygen level [29,30]. Likewise, wild type Pseudomonas oleovorans, is the best characterized mcl-PHA producer; however, it does not utilize substrates such as fructose or glucose, but grows on substrates such as fatty acids or carbon sources which can undergo fatty acid de novo synthesis (n-alkanoic acids, n-alkanals and n-alkanes) [14,[31][32][33][34]. Pseudomonas spp.…”