Robinson, Susan Clare;
(1998)
Enhanced production of a recombinant, thermostable [alpha]-amylase in streptomyces lividans: Effects of plasmid construction and culture condition.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Members of the genus Streptomyces are well known for their ability to produce a wide range of antibiotics. In addition to this valuable attribute, many of these bacteria also secrete a variety of proteins. This has led to their being considered as alternative host organisms for the production of heterologous gene products. This work examines the potential of Streptomyces lividans as a host for the secretion of recombinant proteins using the production of a thermostable a-amylase as a model system. Two new streptomycete expression vectors were constructed. Incorporation of the sti region from pIJ101, which carries a site for second strand initiation of plasmid replication, was found to increase levels of a-amylase production by over 1000-fold. This was likely due to improvements in plasmid copy number and form. The study confirmed that use of promoter arrays to drive recombinant gene expression can be an effective way to achieve high level protein production. Over one and a half times more a-amylase was produced when Pmel was placed in tandem with Pamy in front of the a- amylase gene. As expected, medium composition also played an important role in determining the behaviour of the system. The most favourable condition for high level enzyme production was found to be minimal medium lacking rapidly metabolisable sugars. Glucose was found to repress production of the a-amylase in the S. lividans system, likely through the action of the Reg1 protein. A statistical experimental approach allowed the identification of other influential medium variables, with succinate, calcium and phosphate levels proving key. Unexpectedly, above optimal growth temperatures were found to significantly boost levels of a-amylase production from the S. lividans host. Possible reasons for this phenomenon are explored and discussed. In summary, this work highlights the strong potential of the Streptomyces system. It demonstrates that S. lividans can provide a viable, and competitive, alternative to E. coli as a host for the production of heterologous proteins.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Enhanced production of a recombinant, thermostable [alpha]-amylase in streptomyces lividans: Effects of plasmid construction and culture condition |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | Pure sciences; Applied sciences; Streptomyces |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10097762 |
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