Mining Xanthomonas and Streptomyces genomes for new pectinase-encoding sequences and their heterologous expression in Escherichia coli.
Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.
Xiao
Zhizhuang
Z
Biotechnology Research Institute, National Research Council Canada, Montreal, QC, Canada.
Boyd
Jason
J
Grosse
Stephan
S
Beauchemin
Manon
M
Coupe
Elizabeth
E
Lau
Peter C K
PC
D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016680:Genome, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016959:Xanthomonas campestris / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase