Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2Ps of Pseudomonas stutzeri via site-specific mutation for efficient production of PHA copolymers

ABSTRACT The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2 and GM3 gangliosides, with minor amounts of structures mimicking GD1b and GD2. Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM2 to GM3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

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Cloning and expression of meta-cleavage enzyme (CarB) of carbazole degradation pathway from Pseudomonas stutzeri

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132005000400016 ◽

2005 ◽

Vol 48 (spe) ◽

pp. 127-134 ◽

Cited By ~ 1

Author(s):

Ariane Leites Larentis ◽

Tito Lívio Moitinho Alves ◽

Orlando Bonifácio Martins

Keyword(s):

Active Form ◽

Pseudomonas Stutzeri ◽

Degradation Pathway ◽

Cloning And Expression ◽

Restriction Enzyme Digestion ◽

Site Specific ◽

Recombination System ◽

Pcr Product ◽

Cleavage Enzyme ◽

Site Specific Recombination System

In this work, the 1082bp PCR product corresponding to carBaBb genes that encode the heterotetrameric enzyme 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarB), involved in the Pseudomonas stutzeri ATCC 31258 carbazole degradation pathway, was cloned using the site-specific recombination system. Recombinant clones were confirmed by PCR, restriction enzyme digestion and sequencing. CarB dioxygenase was expressed in high levels and in active form in Escherichia coli BL21-SI using the His-tagged expression vector pDEST TM17 and salt induction for 4h.

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Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000495752 ◽

2018 ◽

Vol 28 (5) ◽

pp. 225-235

Author(s):

Edmar Ramos Oliveira-Filho ◽

Linda P. Guamán ◽

Thatiane Teixeira Mendonça ◽

Paul F. Long ◽

Marilda Keico Taciro ◽

...

Keyword(s):

Carbon Source ◽

Aeromonas Hydrophila ◽

Sole Carbon Source ◽

Ralstonia Eutropha ◽

Pha Synthase ◽

Medium Chain Length ◽

Marker Free ◽

Pha Copolymers ◽

First Time ◽

Induced Mutant

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

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