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Cloning of a Serratia marcescens Gene Encoding Chitinase
R L Fuchs1, S A McPherson, D J Drahos
1Biological Sciences Department, Corporate Research Laboratories, Monsanto Company, St. Louis, Missouri 63198.
Applied and Environmental Microbiology
|March 1, 1986
Summary
Serratia marcescens produces five chitinases, with a 57-kilodalton protein being the most abundant. Researchers identified a DNA fragment encoding this chitinase, enabling its expression in Escherichia coli and Pseudomonas fluorescens.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Serratia marcescens is known for its chitinase production.
- Chitinases are enzymes that degrade chitin, a major component of fungal cell walls and arthropod exoskeletons.
- Understanding the genetic basis of chitinase production can lead to biotechnological applications.
Purpose of the Study:
- To isolate and characterize the genes responsible for chitinase production in Serratia marcescens.
- To express these genes in a heterologous host for further study.
- To compare the expression efficiency of Serratia marcescens promoters in different bacterial hosts.
Main Methods:
- Construction of a cosmid library of Serratia marcescens DNA in pLAFR1.
- Screening of the library in Escherichia coli for chitin degradation.
- Isolation and characterization of positive clones using restriction endonuclease digestion.
- Expression of chitinase genes in Escherichia coli and Pseudomonas fluorescens.
Main Results:
- Five unique chitinolytic proteins were identified with masses of 21, 36, 48, 52, and 57 kilodaltons.
- A common 9.5-kilobase EcoR1 fragment containing the gene for the 57-kilodalton chitinase was identified in four independent clones.
- Chitinase expression in E. coli and P. fluorescens was driven by an S. marcescens promoter.
- Higher chitinase levels were observed in E. coli compared to P. fluorescens, suggesting differential promoter recognition.
Conclusions:
- A specific DNA fragment encoding the abundant 57-kilodalton chitinase from Serratia marcescens was isolated.
- This fragment can be expressed in heterologous hosts like E. coli and P. fluorescens.
- Escherichia coli appears to be a more efficient host for expressing Serratia marcescens chitinase due to potentially better recognition of the native promoter.