Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering

1. Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering Applied and Environmental Microbiology, Aug 2006, 72(8): 5283-5288

2. Commercial importance of Streptomyces Biosynthesis of Antibiotics (Streptomycin, Erythromycin, Tetracycline, Chloramphenicol, etc.) Antifungals (Amphotericin B) Alkaloids (Physostigmine) Anti-cancer compounds (Migrastatin) Used as host for heterologous gene expression (Curr Opin Biotechnol 2 (5): 674-81)

3. Morphology Highly heteromorphous in contrast to unicellular organisms In submerged cultures, hyphae are present as Mycelium: dispersed hyphal filaments Pellet: Spherical agglomerate of hyphal elements Rheology � science dealing with deformation and flow of matterRheology � science dealing with deformation and flow of matter

4. Fermentation broth rheology Filamentous growth related problems Highly viscous broth and slow growth rate Effect on gas-liquid mass transfer rate Higher power inputs required for frequent agitation and oxygen transfer Complex and expensive downstream processing

5. Fermentation broth rheology Pellet growth related problems Autolysis at the centre of large pellets due to nutrient limitation � effect on cellular metabolism & product synthesis Density of pellets is imp- compact n fluffy pellets Filamentous growth of Aspergillus niger is preferred for pectic enzyme production, whereas the pelleted form is preferred for citric acid productionDensity of pellets is imp- compact n fluffy pellets Filamentous growth of Aspergillus niger is preferred for pectic enzyme production, whereas the pelleted form is preferred for citric acid production

6. Factors affecting Rheology Inoculum size, type and age Genetic factors Biosynthesis or addition of polymers, surfactants and chelators Shear forces Temperature and pressure Medium viscosity

7. ssgA Actinomycete-specific protein family member Involved in control of peptidoglycan maintainance Stimulates the formation of septa Enhanced protein levels of ssgA result in mycelial fragmentation

8. To analyze the effects of the enhanced expression of ssgA on the morphology, growth, and productivity of industrial streptomycetes A construct overproducing ssgA was introduced into model system S. coelicolor A3(2) S. lividans (the preferred host for industrial enzyme production) S. limosus (a producer of amylases), S. rimosus (a producer of oxytetracycline) S. roseosporus (a producer of daptomycin) S. venezuelae (a producer of chloramphenicol)

9. Results : Change in morphology Fragmented growth of strains that normally grow as pellets (S. coelicolor, S.limosus, and S. roseosporus) No effect on morphology of originally fragmented strains (S. rimosus and S. venezuelae)

10. S.roseosporus growth rate

11. Effect of ssgA overexpression in S.coelicolor (minimal) GSA2: ssgA overproducer M145: Parent Glu-dehydrogenase diaphorase coupled assayGlu-dehydrogenase diaphorase coupled assay

12. Effect of ssgA overexpression in S.coelicolor (minimal) GSA2 shows smaller mycelial structures and protruding hyphae Glucose utilization M145 � 70h GSA2 � 35h Specific growth rate M145 � 0.14 h-1 GSA2 � 0.20 h-1 (43% increase)

13. Effect of ssgA overexpression in S.coelicolor (TSBS) Biomass conc. 2.5g/L GSA2: 3.5 h M145: 7 h Specific growth rate M145: 0.33 h-1 GSA2: 0.55 h-1 (67%) Stationary phase reached M145: 12 h GSA2: 7h Production of antibiotic GSA2: 5300 units (max) M145: 500 units (max)

14. Effect of ssgA overexpression in S.lividans Tyrosinase production assay 1326: 0.55 after 35 h GSAL1: 0.94 after 20 h Tyrosinase productivity (units/time): 2.5X of 1326 GSAL1 produced more melanin than 1326 Specific growth rate 1326: 0.20 h-1 GSAL1: 0.29 h-1 (45%)

15. Discussion Improved growth rates in modified strains with reduced lag phase and smaller avg mycelium size S.coelicolor fermentation: Cultures reach same total biomass but modified strain fermentation time is reduced by half S.lividans fermentation: Strong improvement in enzyme productivity Approx. 10 fold increase in antibiotic production

16. Conclusion Over-expression of ssgA � efficient means for directed strain improvement Fragmented growth makes growth in micro-titer plates feasible � important for HT screening Reduced energy requirements