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

ε-PL Homopolymer (discovered by Shima and Sakai in 1977) Anti – microbial activity Aerobic Fermentation S. albulus mutant Degradable + Edible Introduction Glycerol Non- Biodegradable Biodiesel By- product Many Applications Carbon Source ε-poly-L-lysine (ε-PL )Glycerol ε-PL Homopolymer Anti- microbial activity Aerobic Fermentation Degradable + Edible ε-poly-L-lysine (ε-PL )

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

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 Aim & Objectives

Methodology

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). Cloning sites for restriction enzymes Overcome incompatibility Antibiotic resistance genes

Methodology

Results Carbon Source ɛ-PL Concentration (mg/L) Dry Cell Weight (g/L) Glucose 2.5%38.91 ± ± 0.69 Glycerol 1%34.58 ± ± 0.79** Glycerol 2.5%31.89 ± ± 1.03** Glycerol 5%32.20 ± 0.62*7.95 ± 5.01** Glycerol 10%35.39 ± ± 2.55* 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. albulus in 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).

Methodology Results 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. albulus with pLAE001::glpD. B) Rhodococus regeneration media showing black colonies (orange arrow) which are S. albulus transformants, white growth represents overgrowth.

Genome Sequencing Poly lysine synthase Meta – transcriptomics Future tasks

Methodology References 1.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): Horton,R.H., Moran, L.A., Scrimgeour, K,G., Perry, M.D., Rawn, J.D. (2006). Principles of Biochemistry, 4 th edition. Pearson Education Iternational. USA Itzhaki, R.F. (1972). Colorimetric method for estimating polylysine and polyarginine. Analytical Biochemistry. 50:569– 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: