Karl Rumbold and Rosemary Dobson
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The Exploration of the Glycerol Metabolic Pathway for ε-poly-L-lysine Production, by Streptomyces albulus Karl Rumbold and Rosemary Dobson
description
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. - PowerPoint PPT Presentation
Transcript of 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
ε-PL
Homopolymer• (discovered by Shima
and Sakai in 1977)
Anti – microbial activity
Aerobic Fermentation
• S. albulus mutant
Degradable + Edible
Introduction
Glycerol
Non-Biodegradable
BiodieselBy-
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
IntroductionIntroduction
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
MethodologyMethodology
Seed Culture• S. albulus CCRC
11814
Shake Flask Fermentation• 30°C, 220rpm• Cell density:660nm
Qualitative Analysis of ε-PL• TLC
Quantitative Analysis• Colormetric method
(Itzahaki, 1972)
Construct Gene Delivery System• Glycerol operon• S. coelicolor + E.
coli
Transform S.albulus• Vectors:
pLAE001 + pLAE003
MethodologyMethodology
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
MethodologyMethodology
Seed Culture• S. albulus CCRC
11814
Shake Flask Fermentation• 30°C, 220rpm• Cell density:660nm
Qualitative Analysis of ε-PL• TLC
Quantitative Analysis• Colormetric method
(Itzahaki, 1972)
Construct Gene Delivery System• Glycerol operon• S. coelicolor + E.
coli
Transform S.albulus• Vectors:
pLAE001 + pLAE003
MethodologyResults
Carbon Source
ɛ-PL Concentration
(mg/L)
Dry Cell Weight (g/L)
Glucose 2.5% 38.91 ± 2.74 3.07 ± 0.69
Glycerol 1% 34.58 ± 7.09 4.48 ± 0.79**
Glycerol 2.5% 31.89 ± 3.33 3.84 ± 1.03**
Glycerol 5% 32.20 ± 0.62* 7.95 ± 5.01**
Glycerol 10% 35.39 ± 5.59 7.08 ± 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).
Glucose 2.5%Glycerol 1%
Glycerol 2.5%Glycerol 5%
Glycerol 10%0
10
20
30
40
50
60
70
80
90
100
Carbon source
% C
arbo
n us
ed
MethodologyResults
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
MethodologyReferences
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):636-641.
2. 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
3. Itzhaki, R.F. (1972). Colorimetric method for estimating polylysine and polyarginine. Analytical Biochemistry. 50:569–574.
4. 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.
