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Cloning, characterization, and expression in Streptomyces lividans 66 of an extracellular lipase-encoding gene from
C Pérez1, K Juárez, E García-Castells
1Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F.
Abstract:
A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.
Insights
Researchers cloned an extracellular lipase gene from Streptomyces sp. M11 into S. lividans. The gene encodes a 28-kDa protein with a signal peptide, suggesting potential for industrial applications.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Extracellular lipases are valuable industrial enzymes.
- Streptomyces species are known producers of diverse bioactive compounds, including enzymes.
- Understanding lipase gene expression is crucial for biotechnological applications.
Purpose of the Study:
- To clone and characterize a gene encoding an extracellular lipase from Streptomyces sp. M11.
- To analyze the genetic features and expression of the lipase gene in a heterologous host.
- To identify regulatory elements for lipase production.
Main Methods:
- Gene cloning using the pIJ486 vector in Streptomyces lividans 66.
- Protein expression analysis and molecular weight determination (28-kDa).
- N-terminal amino acid sequencing and nucleotide sequencing of the lipase gene (lip).
- Signal peptide identification and motif analysis (Gly-His-Ser-Met-Gly).
- S1 mapping to identify the lipase gene promoter.
Main Results:
- A 6-kb DNA fragment containing the lipase gene was successfully cloned and expressed.
- The cloned gene product is a 28-kDa extracellular lipase with a 48 amino acid signal peptide.
- A lipase active-site motif was identified within the nucleotide sequence.
- High-level expression may require additional genes, as subcloning the structural gene alone reduced activity.
- The lipase gene promoter shares similarities with other Streptomyces vegetative promoters.
Conclusions:
- The extracellular lipase gene from Streptomyces sp. M11 has been successfully cloned and its product characterized.
- The identified signal peptide and active-site motif are consistent with functional lipase activity.
- Further genetic elements may be necessary for optimizing lipase production in S. lividans.
- The promoter characteristics suggest regulation typical of Streptomyces vegetative growth phases.
