Foong Choon Pin, Lau Nyok-Sean, Deguchi Shigeru, Toyofuku Takashi, Taylor Todd D, Sudesh Kumar, Matsui Minami
Synthetic Genomics Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Kanagawa, 230-0045, Japan.
Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800, Gelugor, Penang, Malaysia.
BMC Microbiol. 2014 Dec 24;14:318. doi: 10.1186/s12866-014-0318-z.
Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host.
A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant).
A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.
日本海域具有独特的纬度和深度范围。本研究首次对从日本海沟和南海海槽采集的不同深度（24米至5373米）的远洋海水DNA样本中的I类和II类聚羟基脂肪酸酯合成酶（PhaC）的多样性进行了研究。PhaC是微生物中的关键酶，它决定了聚合形成聚羟基脂肪酸酯（PHA）的单体单元类型，从而影响这种热塑性聚合物的物理化学性质。通过基因组步移确定了完整的推定PhaC序列，并在异源宿主中评估了新发现的PhaC的活性。
从全基因组扩增的海水DNA中总共扩增出76个推定的phaC PCR片段。其中55个克隆含有保守的PhaC结构域，并根据其序列相似性分为20个遗传组。基于与已知PHA生产者不同的系统发育谱系，11个遗传组具有未公开的PhaC活性。通过IAN-PCR确定了三个完整的DNA编码序列，并且一种PhaC能够在重组食酸铜杆菌PHB-4（PHB阴性突变体）中产生聚（3-羟基丁酸酯）。
在本研究中发现了一种与海杆菌属具有密切同源性的新的功能性PhaC。对从未培养细菌中分离出的所有phaC基因进行的系统发育分类表明，海水和其他环境资源中蕴藏着大量具有尚未研究活性的PhaC。通过基因组步移对这些基于计算机预测的PhaC进行功能评估，为这些酶的聚合能力提供了新的见解。
