Mining a South African deep mine metagenome for the discovery of novel biocatalysts

English: The construction and screening of gene libraries prepared from DNA directly isolated from environmental samples is a recent and powerful tool for the discovery of new enzymes of biotechnological interest (Gabor et al., 2004). Standard methods based on the screening of isolated microorganisms are inherently limited to the tiny fraction of cultivable microbial species (<1%); environmental gene banks in principle provide access to the entire sequence space present in nature (Handelsman et al., 1998). Environmental libraries allow the screening of functional classes of genes from thousands of organisms and research in this area will provide an essential backdrop for understanding evolution and biochemical pathways (Rajendran et al., 2008). The metagenomics approach has been shown to be an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms from diverse environments. Before proceeding to the metagenome analysis, we constructed genomic libraries from a South African deep mine isolate Geobacillus thermoleovorans GE-7. The library was screened for lipolytic activity on LB tributyrin (TLB). Active clones were sequenced using 454 technology, and the sequencing results revealed the presence of the lipA and GDSL lipases, of which the latter has not yet been characterized in this organism. This family displays the characteristic G-D-S-L motif instead of the conventional G-X-S-X-G (Xdenotes any amino acid) motif. GDSL lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of ester compounds that are of interest in pharmaceutical, food, biochemical and the biological sector (Akoh et al., 2004; Lämmle et al., 2007). In addition, genes associated with fatty acid degradation, different glycolytic activities, lipolytic activity, spore germination, proper protein folding, antibiotic resistance and the cell w
Afrikaans: Die konstruksie en sifting (‘screening’) van genoombiblioteke wat voorberei is vanaf DNS, geïsoleer direk uit die omgewing is ‘n eietydse en kragtige tegniek vir die ontdekking van nuwe ensieme met biotegnologiese waarde (Gabor et al., 2004). Standaard metodes gebaseer op die sifting van geïsoleerde mikro-organismes is inherent beperk tot die klein breukdeel van kweekbare mikrobiese spesies (<1%). Omgewings genoombiblioteke, daarenteen, lewer meer toegang tot die totale DNS basispaar opeenvolging wat daardie omgewing bied (Handelsman et al. 1998). Omgewings genoombiblioteke bied die navorser die geleentheid om te sif vir funksionele klasse van ensieme vanaf duisende organismes en navorsing in hierdie studieveld sal bydra tot ons huidige insig en begrip van die evolusie van metaboliese we¸ (Short, 1997; Rajendran et al., 2008). Dit is bewys dat die metagenoom aanslag ‘n effektiewe tegniek is vir die isolering van nuwe biokataliste en bruikbare gene van onkweekbare mikro-organismes afkomstig vanaf diverse omgewings. Voordat daar met die metagenoomanalise begin is, het ons ‘n genoombiblioteek berei uit ‘n bakteriese isolaat afkomstig van ’n diep myn: Geobacillus thermoleovorans GE-7. Die biblioteek is gesif vir lipolitiese aktiwiteit m.b.v. LB tributirien plate (TLB). Klone wat aktiwiteit getoon het, se DNS basispaar opeenvolging is bepaal d.m.v. 454 tegnologie en die data het getoon dat die lipA en GDSL lipases teenwoordig was. Die laasgenoemde lipase is nog nie gekaraktiriseer in hierdie organisme nie. Hierdie lipase familie vertoon die kenmerkende G-D-S-L aminosuur motief in plaas van die konvensionele G-X-S-X-G motief (X = dui enige amino suur aan).GDSL lipases is hidrolitiese ensieme met multifunksionele eienskappe soos bv. ‘n wye substraatspesifisiteit en regiospesifisiteit. Hierdie ensieme het ook die potensiaal om die hidrolise en sintese van ester verbindings te bewerkstellig wat van belang is in die farmaseutiese, voedsel, biochemiese en biologiese
National Research Foundation (NRF)
Ernst, Ethel Erickson Trust
Metagenomics Platform