Industrial Biotechnology

1
Industrial Biotechnology

• Lectures 6. Biocatalyst/Gene discovery
• Classical enrichment/selection
• Plate selection
• Liquid enrichments
• Cloning genes from single organisms
• Metagenomic approaches
• Libraries
• PCR strategies
• Metagenome sequencing

2
Module Theme

• From genes to products
• The identification of novel genes
• Expression of genes and production of gene
  products (proteins, enzymes)
• Fermentation processes
• Down-stream processing
• Applications of enzymes as industrial catalysts
• Theory and practice of biotransformation
  processes
• Industrial applications

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Scope of Industrial Biotechnology

• First generation processes
• Fermentation based processes using native
  organisms
• Wine- and beer-making
• Baking
• Second generation processes
• Use of native or engineered enzymes/genes to
  enhance first generation processes
• Textile industry
• Starch industry
• Third generation processes
• Direct use of recombinant and engineered systems
• Plant, insect and animal cell culture

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Gene/Biocatalyst discovery

• Microorganisms are a genetic resource
• Genes
• Gene products (proteins, enzymes)
• Multigene pathways and cassettes
• Control sequences
• Primary and Secondary metabolites (pigments,
  antibiotics)

5
The path to an industrial organism/biocatalyst
Environmental sample
Selected organism
Production from native organism
Cloning, expression and recombinant production
6
Solid media isolation1. Growth on selective media

• Prepare solid media containing target compound as
  sole C and/or N source (example, thermophilic
  NHase producers)
• Spread soil/water etc on plate and incubate under
  selected conditions
• Pick colonies, re-streak on selective media.
• Characterise isolates
• Advantages
• Restricts range of organisms which grow
• May identify multiple positives
• Disadvantages
• Limitations of culturing methods apply
• Favours fast-growing isolates

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Solid media isolation1. Growth on selective
substrate

• Prepare liquid media containing target substrate
  in presence of nutrient background (example,
  X-Gal for b-galactosidase producers)
• Spread soil/water etc on plate and incubate under
  selected conditions
• Pick colonies which produce zone of activity,
  re-streak on selective media.
• Characterise isolates
• Advantages
• Positives east to detect
• May identify multiple positives
• Disadvantages
• Unlimited growth (numerous negatives)
• Limitations of culturing methods apply
• Favours fast-growing isolates

8
Liquid media isolationBatch or Continuous

• Prepare liquid media containing target compound
  as sole C and/or N source (example, thermophilic
  NHase producers)
• Inoculate soil/water etc into media and
  incubate/recycle under selected conditions
• Streak onto selective media.
• Characterise isolates
• Advantages
• Restricts range of organisms which grow
• Highly selective for positive organism
• Many organisms at very low density will grow in
  liquid media but not on solid media
• Disadvantages
• Limitations of culturing methods apply
• Only single organism isolated
• Favours fast-growing strains

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Classical approach to cloning novel genes
Isolate pure cultures from target source
Identify target enzymes
Isolate genomic DNA, restrict and prepare
Shotgun library
Purify enzyme and obtain N-terminal sequence
Prepare labeled DNA probe from N-terminal
sequence and screen library by colony
hybridisation
Identify positive clone(s), isolate gene, clone,
sequence and express in host
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Classical approach to gene discovery 2
Isolate pure cultures from target source
Isolate genomic DNA, restrict and prepare
Shotgun library
Screen library for positive clone(s) by activity,
complementation, Western blotting etc.
Isolate gene,clone, sequence and express in host
Identify target enzymes
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Limitations of classical gene discovery methods

• A very small proportion of the total microbial
  genome diversity is isolated
• Estimates of global microbial species diversity
  range from 106 to 107
• International culture collections harbour lt 105
  species
• gt99 of the microbial species present in most
  environmental samples are currently
  unculturable.

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Accessing the Metagenome the full
complement of genomes available in any environment

• New genetic screening methods avoid the
  limitations of culturing
• Isolation of community DNA represents all
  organisms present in a sample
• Manipulation of community DNA extracts for
  identification of target genes

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Metagenome screening 1. Expression library
screening
Community DNA extraction
Preparation of multigenomic library
Expression screening
Analysis of cloned gene and gene product
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Metagenome screening 2. Gene-specific PCR
screening
Community DNA extraction
PCR using degenerate primers
Clone amplicons, select and sequence clones
Label amplicon and screen multigenomic
library For full-length gene by colony
hybridisation
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Limitations in metagenomic screening methods

• Depends on efficient DNA extraction technology
• Screening methods must be very sensitive if low
  frequency genes/genomes are to be detected
• Screening technology will limit range of genes
  accessed
• Technology currently only effective for
  prokaryotic genes (i.e., genes with no introns)

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Metagenomic sequencing

• Isolate total community DNA
• Shotgun clone into sequencing vectors
• Sequence!!!!!!!!!!!!!
• Prepare contigs
• Identify gaps and chromosome walk to complete
• Identify ORFs and annotate
• Scale of task Assume 100 genomes of approx. 5Mbp
  5 x 108 bp
• At least 10x redundancy is necessary to obtain a
  complete genome 5 x 109 bp (equivalent to a
  human genome).