Departamento de Biología Molecular, Facultades de Veterinaria y de Biología, Universidad de León, 24071 León, España.
Microb Biotechnol. 2008 Mar;1(2):170-6. doi: 10.1111/j.1751-7915.2007.00016.x.
The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium-chain-length poly-hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC-phaC1) or only phaC2 (P. putida U Δpha pMC-phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC-phaC1 strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5-phenylvaleric, 6-phenylhexanoic, 7-phenylheptanoic, 8-phenyloctanoic or 9-phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6-phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC-phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3-hydroxy-5-phenylvaleryl-CoA. The possible influence of the putative three-dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed.
在活体中研究了参与中链长度聚羟基烷酸（mcl PHAs）生物合成的两种聚合酶（PhaC1 和 PhaC2）的底物特异性。对于这类实验，使用了两种从整个 pha 基因座缺失的遗传工程突变体（P. putida U Δpha）衍生的重组菌株。这些细菌仅表达 phaC1（P. putida U Δpha pMC-phaC1）或仅表达 phaC2（P. putida U Δpha pMC-phaC2），根据添加到培养液中的前体积累不同的 PHAs。因此，P. putida U Δpha pMC-phaC1 菌株能够合成几种脂肪族和芳香族 PHAs，当使用己酸、庚酸、辛酸、癸酸、5-苯基缬草酸、6-苯基己酸、7-苯基庚酸、8-苯基辛酸或 9-苯基壬酸作为前体时；当使用癸酸（脂肪族 PHAs）或 6-苯基己酸（芳香族 PHAs）作为前体时，聚合物的积累最高。然而，尽管它合成类似的脂肪族 PHAs（当己酸是前体时积累最高），但另一种重组菌株（P. putida U Δpha pMC-phaC2）仅在聚合单体为 3-羟基-5-苯基缬酰辅酶 A 时积累芳香族 PHAs。讨论了假定的三维结构对 PhaC1 和 PhaC2 不同催化行为的可能影响。
