Production of poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) with flexible 3-hydroxyhexanoate content in Aeromonas hydrophila CGMCC 0911

Abstract

Aeromonas hydrophila CGMCC 0911 isolated from lake water was found to be able to synthesize a polyhydroxyalkanoate (PHA) copolymer (PHBHHx) consisting of 3-hydroxybutyrate (HB) and 4-6 mol% 3-hydroxyhexanoate (HHx). The wild-type bacterium accumulated 49% PHBHHx containing 6 mol% HHx in terms of cell dry weight (CDW) when grown on lauric acid for 48 h. When A. hydrophila CGMCC 0911 expressed the Acyl-CoA dehydrogenase gene ( yafH) of Escherichia coli, the recombinant strain could accumulate 47% PHBHHx, while the HHx content reached 17.4 mol%. The presence of changing glucose concentration in the culture changed the HHx content both in wild type and recombinant A. hydrophila CGMCC 0911. When 5 g l(-1) glucose was added to a culture containing 5 g l(-1) lauric acid as co-substrate, 45% PHBHHx/CDW consisting of 8.8 mol% HHx was produced by wild-type A. hydrophila CGMCC 0911 compared with only 5% in the absence of glucose. When the recombinant A. hydrophila CGMCC 0911 was grown on a mixed substrate containing lauric acid and 8-10 g l(-1) glucose, the HHx content could be further increased to 35.6 mol%. When the glucose concentration exceeded 10 g l(-1), cell growth, PHA content and mole percentages of HHx in PHBHHx were significantly reduced.

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

“…A few organisms produce PHAs with a broader range of monomer chain lengths (C 4 to C 12 ) (4,10,21,23). The purple and phototrophic bacterium Rhodospirillum rubrum is known to produce PHA composed of both short-and medium-chain-length monomers, and it can produce up to 50% (dry weight) PHA (4,19,45).…”

Inactivation of type I polyhydroxyalkanoate synthase in Aeromonas hydrophila resulted in discovery of another potential PHA synthase

“…hydrophila possessing PhaC utilizes lauric acid to produce only PHBHHx through the beta-oxidation pathway. The wild type cannot produce PHA and grows poorly when high concentrations of glucose were used as carbon source [17]. However, after PhaC was inactivated, the resultant mutant produced mcl PHA and also grew well on lauric acid or high concentrations of glucose, showing that the mutant still had the ability to express active PHA synthase.…”

“…A. hydrophila CGMCC 0911 possessed type I PHA synthase (PhaC) and produced only PHBHHx from lauric acid. Growth of the strain was inhibited when octanoate was used as carbon source, while grew on glucose or gluconate only traces of PHA were detected in the cells [17]. In this paper we report, for the Wrst time, that inactivation of PhaC resulted in synthesis of mcl PHA in A. hydrophila.…”

“…The resistant clones were conWrmed for double crossover events by PCR method. Primers P1 (5Ј-TTGGAT CCTGGAGACCGATGATGAATATG-3Ј), P2 (5Ј-A ATAAGCTTGGCCTTGGCCGTGCTCTTC-3Ј) and P3 (5Ј-ATGAATTCTTAAGGCAGCTTGACCACG G-3Ј) were designed according to the organization of type I PHA synthesis genes in A. hydrophila [17]. PCR products, ampliWed from genomic DNA of resistant clones and the wild type, were sequenced and analyzed (data not shown).…”

“…hydrophila and P. putida strains were cultivated in 500 ml conical Xasks containing 100 ml mineral salt medium [17] supplemented with 10 g l ¡1 lauric acid, 20 g l ¡1 glucose , 20 g l ¡1 gluconate or 10 g l ¡1 octanoate and shaken at 30°C and 200 rpm. Kanamycin (50 g ml -1 ) was added to medium when needed.…”