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Karl Rumbold and Rosemary Dobson

The Exploration of the Glycerol Metabolic Pathway for ε-poly-L-lysine Production, by Streptomyces albulus. Karl Rumbold and Rosemary Dobson. Introduction. ε-poly-L-lysine (ε-PL ). Glycerol. ε-poly-L-lysine (ε-PL ). Introduction. Introduction. Glycerol ε -poly-L-lysine.

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Karl Rumbold and Rosemary Dobson

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  1. The Exploration of the Glycerol Metabolic Pathway for ε-poly-L-lysine Production, by Streptomycesalbulus Karl Rumbold and Rosemary Dobson

  2. Introduction ε-poly-L-lysine (ε-PL ) Glycerol ε-poly-L-lysine (ε-PL )

  3. Introduction Introduction • Glycerol ε-poly-L-lysine Figure 1: Metabolism of Glycerol (Adapted from Horton et al., 2006 and … Wang et al., 2001)

  4. Aim & Objectives • Aim: • Does the metabolic engineering of the glycerol uptake system lead to improved polylysine production by S.albulus • Objectives: • Grow S. albulus - different glycerol concentrations • Estimate ε-PL produced • Clone glycerol operon into shuttle vectors • Transform S. albulus with glycerol operons from E.coli and S. coelicolor • Grow S. albulus transformants - different glycerol concentrations

  5. Methodology Methodology

  6. Methodology Methodology Cloning sites for restriction enzymes Antibiotic resistance genes Overcome incompatibility Figure 2: pNO33-based shuttle vectors constructed in a study by Hamano et al (2005): pLAE001 and pLAE003 were used for the PEG-mediated protoplast transformation and intergenic conjugation from E. coli to S. albulus (Hamano et al., 2005).

  7. Methodology Methodology

  8. Results Methodology Table 1: ɛ-PL concentrations and biomass accumulation from baffled flask fermentations. Fig 2: The percentage of carbon utilised during 3 day fermentations with S. albulusin minimal media. Glucose was the control carbon source. There is sufficient evidence to suggest that the percentage carbon used is higher from at least one carbon source (Kruskal-Wallis test; H=12.1, d.f.=1, P=11.14).

  9. Results Methodology Fig 3: Gene products and transformations with pLAE001::glpD. A) 1% agarose gel where M: 1Kb Plus molecular marker (Fermentas), 1506bp glpD gene from E. coli (a) and linearised (AscI) 7875bp pLAE001 (b). Confirmation of cloning by restriction digest using AscI and SacI on recombinant plasmid: pLAE001 (c) containing glpD (d); linearised pLAE001::glpD (e); colony PCR of transformed S. albuluswith pLAE001::glpD. B) Rhodococus regeneration media showing black colonies (orange arrow) which are S. albulustransformants, white growth represents overgrowth.

  10. Future tasks • Genome Sequencing • Poly lysine synthase • Meta – transcriptomics

  11. References Methodology • Hamano, Y., Nicchu, I., Shimizu, T. Hoshino, Y., Kawai, T., Nakamori, S., Takagi, H. (2005). Development of Gene Delivery Systems for the ε-Poly-L-Lysine Producer, Streptomyces albulus. Journal of Bioscience and Bioengineering. 99(6):636-641. • Horton,R.H., Moran, L.A., Scrimgeour, K,G., Perry, M.D., Rawn, J.D. (2006). Principles of Biochemistry, 4th edition. Pearson Education Iternational. USA. 526 • Itzhaki, R.F. (1972). Colorimetric method for estimating polylysine and polyarginine. Analytical Biochemistry. 50:569–574. • Wang, J., Gilles, E.D., Lengeler, J.W., Jahreis, K. (2001). Modelling of inducer exclusion and catabolite repression based on a PTS-dependent sucrose and non-PTS-dependent glycerol transport systems in Escherichia coli K-12 and its experimental verification. Journal of Biotechnology. 92:133-158.

  12. Karl.rumbold@wits.ac.za

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