Taylor, Paul Phillip (1986) The cloning and expression of amylolytic genes in "Escherichia coli" and their role in starch utilization. PhD thesis, University of Warwick.

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Abstract

The structural genes for the K.pneumoniae maltohexaose (G6)-producing amylase, the K.pneumoniae pullulanase and the B.licheniformis α-amylase were cloned and expressed in E.coli. Plasmids carrying the G6-producing amylase, conferred starch utilization on E.coli, but this growth was much slower than growth on glucose. On SDS-PAGE gels, the putative G6-producing amylase had an Mᵣ 67,000. Strains carrying the pullulanase plasmid (pPT14) were able to utilize pullulan as their sole carbon source. On SDS-PAGE the cloned pullulanase comigrates with purified pullulanase (Mᵣ 120,000+). Certain insertions and deletions in the cloned pullulanase fragment reduce growth rate on pullulan. The phenotypes expressed by malB strains and mutants which reduce plasmid copy number suggested that at least one other gene product was required in addition to the pullulanase gene, for effective utilization of pullulan by E.coli. A possible candidate for one of these extra gene products was a protein (Mᵣ 94,000) which was expressed in maxicells. Possible mechanisms of pullulan utilization are discussed.

Strains carrying the B.licheniformis α-amylase plasmids (pPT80, pPT81, pPT8Ti and pPX2) could all grow on starch. A copy number of approximately 50 was required for efficient growth (i.e. as good as growth on glucose). When expressed in E.coli the α-amylase is found almost entirely in the periplasmic space. The evidence suggests that the α-amylase is poorly expressed in E.coli and only relatively small amounts of enzyme are required for starch utilization. A model for starch utilization in E.coli is discussed. The whole of the 3.466 kb insert carrying the B.licheniformis α-amylase gene was sequenced. The insert had three open reading frames, only one of which formed a complete gene. This open reading frame corresponded to the B.licheniformis α-amylase gene, coded for a protein of Mᵣ 58,492 and had a 29 amino-acid putative signal sequence. The protein was very homologous to both the B.amyloliquefaciens and B.stearothermophilus α-amylases. The B.licheniformis α-amylase was put under tac promoter control on a high copy number plasmid. Induction with IPTG caused lethality. Uninduced α-amylase levels were sufficient to allow E.coli to grow on starch. The mRNA start points for both the wildtype gene and the tac construct were determined. The effect of removing the major part of the α-amylase signal sequence was studied. This was achieved by a combination of site-directed and linker mutagenesis of the α-amylase DNA. This construct was poorly expressed in E.coli and did not exhibit a lethal phenotype.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany Q Science > QP Physiology Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Escherichia coli -- Genetics, Starch -- Synthesis, Amylases
Official Date:	August 1986
Dates:	Date Event August 1986 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Edwards, Mark
Extent:	xxix, 232 leaves : illustrations
Language:	eng

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