2. Excess Production of Primary Metabolites
• Understanding of the biochemistry and genetics of microorganism
• Led to excrete excess primary metabolites
• Primarily eliminate feedback inhibition
• Auxotrophic mutants – no longer produce the desired end product
– due to a block in one of the steps in the pathway
– elimination of end product inhibition or repression is achieved
• Growth occur by adding required end product in low amount
• Feedback inhibition is avoided
• Desired intermediate product is excrete
• Both branched and unbranched pathway can be manipulated
3. • Select the antimetabolites resistant mutants
• Enzyme structure is changed
• Corresponding enzyme lacks the allosteric control site
• Constitutive enzyme produced and overproduction
• Mutants – block in an allosterically regulatble enzyme
• Suppressor mutation – lead to restoration of enzyme activity
• Non allosterrically controllable enzyme
4. Regulation and overproduction of secondary metabolites
• Structural genes – code for enzyme involved in secondary metabolite biosynthesis
• Regulatory genes – control secondary metabolite synthesis
• Resistance genes – keep antibiotic producing strains immune to their own produccts
• Permeability genes – control the uptake and excretion of substances
• Regulatory genes – control primary metabolism, indirectly affect to their own products
5. Affect Of Regulatory Mechanisms On The Products Of
Secondary Metabolism
• Induction
• End product regulation
• Phosphate regulation
• Autoregulation
6. Induction
• In batch fermentation – carbon and nitrogen sources are primarly metabolitzed
• Trophophase and idiophase are formed
• Secondary metabolites – called idiolites
• In trophophase – repress enzyme involved in secondary metabolism
• Composition of culture medium – arrange significant fraction to slowly metabolize
substrate
• Organism – grow under suboptimal condition
• Growth and secondary metabolite formation occur in parallel
7. End product Regulation
• Antibiotic – inhibit their own biosynthesis
• Feedback regulation mechanism – with chloramphemical and penicillin
• Repress the first enzyme of biosynthetic pathway
• Concentration of end product inhibit corresponds to the production level
• Isolation of less sensitive strain by antibiotic – inhibit end product
– produce higher yields
8. Catabolite Regulation
• General regulatory mechanism
• Key enzyme – repress, inhibit, inactivate when a commonly used substrate is added
• Catabolite repression – by the substrate of ccarbon and nitrogen sources
• Carbon source – inhibit biosynthesis of different secondary metabolites by rapidly
fermentation
• Carbon catabolite regulation mechanism – depend on the organism and metabolite
• Nitrogen source – inhibit in antibiotic fermntaion
– regulation mechanism have not been completely understood
9. Phosphate Regulation
• Culture medium – require inorganic phosphate (Pi)/ 0.3-300 mM for growth
• Much lower P concentration – inhibit secondary metabolite production
• 1 mMPi – unimpeded secondary metabolite production
• 10 mMPi – complete inhibition
• Pi – control the metabolic pathway not only the first stage but also the biosynthesis of
secondary metabolites
• Several mechanism have been studied.
10. • First type – P stimulate primary metabolism
• Consumption of mechanism – for the metabolic change from trophase to idiophase
• By adding Pi at the end of trophophase – delay idiophase
• By adding Pi in the idiophase – growth resume and antibiotic synthetic stops
• Second type – P inhibit or repress phosphatase enzymes in secondary metabolite
biosynthesis
• Third type – excess P shift carbohydrate metabolism from PPP to EMP
NADPH – limiting factor in the synthesis of biosynthesis
P restricts the induction of secondary metabolite production
11. Autoregulation
• Type of self-regulation in some actinomycetes
• Eg. Streptomyce species which produce antibiotics
• Factor A – affect the development of streptomycin resistance and spore formation
– induce the enzyme streptomycin phosphotransferase
– streptomycin resistance property
• Streptomycin formation – shift in the metabolism of the carbohydrate source
• Factor A deficient mutants – high in the activity of glucose-6-phosphate dehydrogenase
• High yielding strain – not demonstrate this enzyme
12. • Factor A – add into mutants
– decrease this enzyme activity
• Assume – absence of this enzyme block pentose phosphate cycle
– Glucose is channeled to streptomycin formation pathway
• Factor A – consider as analogus hormone
