R-3-hydroxyhexanoate

For large-scale recombinant production of bacterial polymers, non-polymer producing bacteria were exposed to biosynthesis pathways. Polymers such as PHA, CGP (cyanophycin granule peptide), HA (hyaluronic acid), and PGA [poly-y-glutamate] were produced by these methods [89, 85-96]. For example, recombinant E.coli [89] was fermented for the lai e-scale production of PHA [89]. In addition the PHB biosynthesis genes of Ralstonia eutropa were harbored in E.coli to produce poljmers such as PHA composed of (R)-S-hydroxybutyrate and (R)-3-hydroxyvalerate and/or (R)-3-hydroxyhexanoate which showed preferable properties for use in industrial applications [97-99, 85-96]. 

The second PHA synthesis pathway (pathway II) is related to fatty acid uptake by microorganisms. After fatty acid P-oxidation, acyl-CoA enters the PHA monomer synthesis process. Enzymes including 3-ketoacyl-CoA reductase, epimerase, (I )-enoyI-CoA hydratase/enoyl-CoA hydratase I, acyl-CoA oxidase (putative), and enoyl-CoA hydratase I (putative) were found to be involved in supplying the PHA precursor 3-hydroxyacyl-CoA for PHA synthesis. Pseudomonas putida, Pesudomonas aeruginosa, and A. hydrophila are able to use pathway n to synthesize medium-chain-length (mcl) PHA or copolymers of (/ )-3-hydroxybutyrate (R3HB) and (R)-3-hydroxyhexanoate (PHBHHx). 

Taking into account the advantages and the limitations of both kind of polymers, different approaches based on biochentical, genetic and bioprocess innovation have attempted to obtain hybrid scl-mcl copolymers scl C -CJC copolymers, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate]/poly[(/ )-3-hydroxybu-tyrate-co-(R)-3-hydroxyhexanoate Fig. 2 to generate new PHAs with different or improved physiochemical properties and broader biotechnological applications (Lee et al. 2000b Lu et al. 2004 Ramsay et al. 1990 Hanggi 1995 Lenz and Marchessault 2005 Wei et al. 2009). 

C) containing as monomers (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate, (/ )-3-hydroxy-7-oxooctanoate and (R)-3-hydroxy-5-oxooctanoate (in polymer A) and (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate, 8-acetoxy-(R)-3-hydroxyoc-tanoate, 6-acetoxy-(R)-3-hydroxyhexanoate and 4-acetoxy-(R)-3-hydroxybutyrate (in polymer B) were accumulated (Jung et al. 2000). 

Most of the mcl-PHA production strains - with the exception of Pseudomonas putida GPol - accumulate alkanoic mcl-PHA also from unrelated carbon substrates through the fatty acid de novo synthesis pathway. Consequently, glucose may result in polymers containing (R)-3-hydroxy fatty acids with even carbon numbers, e.g., (R)-3-hydroxydecanoate, (R)-3-hydroxyoctanoate, (R)-3-hydroxyhexanoate,... 

P. putida KTOY06 was genetically engineered by the deletion of the genes of 3-ketoacyl-coenzyme A (CoA) thiolase (fadA) and 3-hydroxyacyl-CoA dehydrogenase (fadB). The P-oxidation pathway was weakened and therefore the carbon source tetradecanoic acid was converted to a mcl-PHA containing 31 9 mol% (R)-3-hydroxytetradecanoic acid as the main component, whereas the (R)-3-hydroxyhexanoic acid content remained almost constant at 3 mol%. The mechanical properties were influenced by the content of (R)-3-hydroxydodecanoic or (R)-3-hydroxytetradecanoic acid, respectively, as can be derived from Table 1. 

P(3HB) poly[(R)-3-hydroxybutyrate], UHMW-P(3HB) ultra-high-molecular-weight poly[(/J)-3-hydroxybutyrate], P(3HB-co-3HV) poly[(l )-3-hydroxybutyrate-co-(/f)-3-hydroxyvalerate], P(4HB) poly[(R)-4-hydroxybutyrate], poly(4-hydroxybutyrate) P(3HB-co-3HH) poly[(/J)-3-hy-droxybutyrate-co-(R)-3-hydroxyhexanoate]... 

Figure 16.6 Polyhydroxyalkanoates (PHA) that have been produced in sufficient quantity for application research, including poly-(R)-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3-hy-droxyoctanoate (PHO) (up part left to right). Random copolymers of (R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate (PHBV), (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate (PHBHHx), and (R)-3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) (bottom part left to right).

The Solid-State Structure, Thermal and Crystalline Properties of Bacterial Copolyesters of (R)-3-Hydroxybutyric Acid with (R)-3-Hydroxyhexanoic Acid... 

Figure 3.71 AFM phase images at 6-minute intervals of lamellar growth of poly(R-3-hy-droxybutryrate-co-R-3-hydroxyhexanoate) at 85°C. A uniform right-handed twist is evident. From Xn et al. [103] with permission from the American Chemical Society.

As shown in Figure 7 ethyl 3-hydroxyhexanoate, isolated from purple passion fruit possessed the (R)-configuration, comparable to the hydroxyacid ester obtained by the reduction with baker s yeast. In contrary to that methyl 3-hydroxyhexanoate, which was isolated from aroma extracts of pineapple, consisted of the (S)-enantiomer (91 %). ... 

Figure VIII shows the enantiomeric composition of various hydroxy- and acetoxyacid esters and of if -hexa-and -octalactone isolated from pineapple. Methyl 3-hydroxyhexanoate and methyl 3-acetoxyhexanoate are mainly of the (S)-configuration corresponding to intermediates of B-oxidation. The optical purity of the 5-acetoxy esters is lower than of the 3-acetoxy derivatives. The lactones were mainly of the (R)-configuration. Figure IX presents a possible pathway to explain the formation of these compounds. Methyl (S)-(+)-3-hydroxyhexanoate and methyl (S)-3-acetoxyhexanoate may be derived from (S)-3-hydroxyhexanoyl-CoA by transacylation with methanol and acetyl-CoA, respectively. The biosynthesis of 5-hydroxyacids is still unknown, but they may be formed by elongation of 3-hydroxyacids with malonyl-ACP. This hypothesis could explain their varying enantiomeric composition relative to the 3-hydroxyacids. However, hydration of unsaturated acids and/or the reduction of 5-oxoacids may be involved.

Figure VIII. Capillary GC-separation of the (R)-(+)-PEIC-derivatives of chiral (main) constituents isolated from pineapple. 3-Hydroxyhexanoate and 3-aeetoxyhexanoate were separated as the (R)-(+)-MTPA-derivatives.

PHB-co-PHV [54] is obtained from Azotobacter chroococcum [58, 63). The biodegradation is slower for the copolymers than poly-3-hydroxybutyrate. 3-hydroxy-n-phenylalkanoic acids and 3-hydroxyaliphatic acids are obtained from Pseudomonas putida [59]. Poly (3-hydroxyoctanoic acid) and poly (6-hydroxyhexanoic acid) and poly (3-hydroxyoctanoic acid) [64], Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes are isolated from the plasma membranes of bacteria [65,66]. Polyhydroxyoctanoate is produced by feeding octanoic acid to Pseudomonas oleovorans [67]. 

Qu, X. H. Wu, Q. Liang, J. Zou, B. and Chen, G. Q. Effect of 3-hydroxyhexanoate content in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on in vitro growth and differentiation of smooth muscle cells. Biomater. 2006 May, 27(15), 2944-2950. Sangsanoh, R et al. In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds. Biomacromole. 2007, 8(5), 1587-1594. 

Abe H, Doi Y, Aoki H, Akehata T, Hori Y, Yamaguchi A (1995) Physical properties and enzymatic degradability of copolymers of (R)-3-hydroxybutyric and 6-hydroxyhexanoic acids. Macromolecules 28 7630-763... 

Zhao W, Chen GQ (2007) Production and Characterization of terpolyester poly(3-hydroxybu-tyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB. Process Biochem 42 1342-1347 Zheng LZ, Li Z, Tian HL, Li M, Chen GQ (2005) Molecular cloning and functional analysis of (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transacylase from Pseudomonas mendocina LZ. FEMS Microbiol Lett 252 299-307... 

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