BIO 414 Applied and Industrial Micro

metabolism

the chemical processes of a living organism responsible for converting substrates to products

catabolic pathways

enzymatic breakdown of organics enables controlled extraction of useful energy (ATP) plus carbon skeletons liberating energy for cell growth, maintenance, movement and biosynthesis (heterotrophic)

anabolic pathways

energy in to facilitate the build up of complex molecules (autotrophic)

amphibolic

pathways that have both anabolic and catabolic functions

antipleuritic replenishment

a metabolic reaction that replenishes intermediates of other pathways

convergent pathways

multiple pathways exist to move a common substrate to product and can be differentially regulated

3 major glycolytic pathways

Embden Meyerhof-Parnas Pathway (EMP)

Pentose Phosphate Pathway (PPP)

Entner-Doudoroff Pathway (ED)

primary metabolites

log phase (trophophase) growth, essential life processes

secondary metabolites

products of pathways active during later stationary phase and not essential for growth or survival

what are examples of primary metabolites

ethanol and CO2 from fermentation

what is an example of a secondary metabolite

antibiotics

anabolic primary metabolites industrial importance

amino acids, enzymes, citric acid, nucleic acids

what stage are secondary metabolites synthesized

idiophase

microbial secondary metabolites

penicillin and streptomycin

role and types of secondary metabolites valuable to micro

antibiotics, pigments, toxins, pheromones, enzyme inhibitors, receptor agonists, growth promoters

how can we direct metabolism

control fermentor conditions or limit nutrients

bioprospecting

investigation of living things to see how they can be commercially useful to humans

stages of bioprospecting

collection, preparation, screening, and commercialization

qualities of ideal industrial microorganisms

1. efficient product production (high yield)

2. safe, non pathogenic and resists contamination

3. utilizes diverse, low cost available carbon sources

4. limited need for growth factors

5. biosynthetic pathways well characterized

6. genetically stable

7. amenability to genetic manipulation

8. full genome sequence is available

9. easily harvested

10. ready lysis

11. few coproducts to minimize purification issues and cost

12. wastes produced do not require special processing

optimal medium

must be available as needed, economical, and easy to deliver

sources of industrial microbes

culture collection, isolation of organisms from nature (soil, water, air, leaves)

isolation strategies: shotgun screening

nonspecific approach screens free living organisms from different environments

isolation strategies: targeted approach

specifically samples environments where desired organisms are most likely

enrichment culture

selected media and fermentor conditions preferentially encourage growth of particular target type of organism over others

why are continuous cultures convenient for enriching organisms

constant flow of nutrients, conditions such as pH and temp can be gradually adjusted and agar platings can determine when organism has developed

martinus Beijerinck

founders of virology

conceived of the enrichment culture

discovered nitrogen fixation

Lourens Baas Becking’s idea

selective enrichment

what might metabolites be screened for

antimicrobial activity, cell growth promotion effects, enzyme inhibition, morphological changes, animal or chemical tests

methods for strain improvement

select a production strain from broad natural population, customize growth conditions and media, introduce new genetic properties

how can we perform highly targeted direct genome manipulation

insertion of foreign DNA, but it is costly

physical methods of mutant generation

ionizing radiation and UV radiation

chemical methods of mutant generation

mutate resisting DNA, dividing DNA, and induce frameshift mutations

three major classes of chemical mutagens

1. act on DNA of resting or nondividing organisms

2. DNA analogues which can be incorporated during active DNA replication

3. those that cause frame shift mutations

what type of chemicals act on resting DNA

nitrous acid, NTG, alkylating agents

base analogues

resemble nucleotides and are incorporated into dividing DNA in place of natural base causing a transition mutation (point mutation)

frameshift mutagens

insert between adjacent base pairs pushing nucleotides far enough apart that an extra nucleotide may be added to the growing chain during replication

what are the benefits and disadvantages of UV as a mutagen

benefits: simple and effective and widely used

disadvantages: absorbed by plastic and some glass, not effective on opaque or colored organisms

working stock disadvantages

likelihood of contamination, loss of genetic and phenotypic characteristics, high time and labor costs, loss of productivity

how can we prevent mutations and preserve useful strains

by slowing and stopping metabolism

methods for preservation of microorganisms

reduction in temperature, dehydration, limitation of nutrients available to the organism (induce spores)

what do cryoprotectants prevent

ice crystal formation, membrane lysis and death

lyphophilization

freeze drying by first freezing the material and reducing pressure causing frozen water in the material to sublimate

advantages of lyophilization

removal of water at low temp

thermolabile materials can be dried

compatible with aseptic operations

more precise fill weight control

sterility can be maintained

reconstruction is easy