Журнал управления прибрежной зоной

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Novel Molecular Methods for Discovery and Engineering of Biocatalysts from Uncultured Marine Microorganisms

Agustinus Robert Uria, Yusro Nuri Fawzya, and Ekowati Chasanah

Metagenomics is a powerful cultivation-independent approach, which can be applied to gain access to the
biocatalysts from uncultured marine microorganisms. Discovery of marine biocatalysts by this approach, in
general, involves four main steps. First, a metagenomic library containing a pool of biocatalyst-encoding
genes is constructed from a marine environment, which can be done by various methods, including cloning of
enzymatically-digested DNA, uncut DNA, and PCR-amplified products. Second, the metagenomic library is
screened for the genes of interest by employing the activity assay of expression product, in situ hybridization,
or Polymerase Chain Reaction (PCR). Third, the obtained target genes, both functional and phylogenetic genes,
are sequenced and analysed by using bioinformatic tools in order to gain information on the functional and
structural properties as well as the microbial sources of the encoded biocatalysts. Finally, the target genes are
expressed in suitable microbial hosts, thereby producing the corresponding recombinant biocatalysts. All
existing methods in engineering of marine biocatalysts for the performance improvement can be classified into
two main strategies: (i) rational design and (ii) directed evolution. Rational design, which may include the use
of resctriction enzyme(s) and splicing by overlap extension (SOE), requires information on the biocatalyst`s
structural and functional properties to alter specific amino acid(s). Whereas directed evolution, including
error-prone PCR technique and gene shuffling, needs no such information