Lectures 10 and 11. Microorganisms as biotechnological tools.

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Lectures 10 and 11. Microorganisms as
biotechnological tools.

• From genes to processes
• Gene resources
• Gene diversity
• Methods of gene discovery
• Expression of genes and production of gene
  products
• Use of organisms and enzymes as catalysts
• Environmental processes
• Fermentation processes
• Biotransformations

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From genes to processes
Gene discovery
Cloning/expression
Production and scale-up
Engineering the catalyst
Application
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Gene discovery

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

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Classical approach to gene discovery 1
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 labelled 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 microbial species diversity range
  from 106 to 107
• International culture collections harbour lt 105
  species
• gt90 (and sometimes gt99) of the microbial
  species present in an environmental sample are
  currently unculturable.

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Accessing the Metagenome refers to 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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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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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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Metagenome screening 3. Random PCR screening
Community DNA extraction
PCR using combination (random/degenerate) primer
pairs
Clone amplicons, select and sequence clones
Align sequences, identify full ORF, design
primers to flanking regions and PCR-amplify
length gene from primary DNA extract
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Metagenome screening 4. Integron screening
Integrons are transposable elements in bacterial
genomes, comprising conserved IS flanking
sequences, an integrase encoding ORF, and a
number of randomly captured ORFs.
IS Integrase ORF1
ORF2 ORF3 IS
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Metagenome screening 4. Integron screening
Community DNA extraction
PCR using degenerate primer pairs to IS flanking
sequences
Clone amplicons, sequence clones and identify ORFs
Design primers to ORF flanking regions and
PCR-amplify length gene from integron clone
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Microorganisms as expression hosts

• Hosts for production of recombinant products
• proteins, enzymes
• products of multi-enzyme pathways
• Host requirements
• Suitable vectors
• Effective transformation
• Ready fermentation and scale-up capacity

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Novel gene to usable gene product

• Use the native organism
• Transfer gene to host organism
• Over-express gene
• Recover gene product (enzyme)

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General strategy
Clone gene
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5
Insert into suitable vector
Transform host cells
Select transformed cell
Culture transformed clone
Extract and purify recombinant product
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Microbial expression hosts E. coli

• ADVANTAGES
• Numerous specific vectors (l phage and pUC
  plasmid derivatives)
• Very high trans-formation efficiencies
• Very well understood fermentations
• Simple cell recovery and lysis

• DISADVANTAGES
• Low expression yields
• No glycosylation of rProtein
• rProtein not exported

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Microbial expression hosts Saccharomyces
cerevisiae

• ADVANTAGES
• Moderate expression yields
• rProtein glycosylated
• rProtein exported
• Very well understood fermentations
• Simple cell recovery and lysis

• DISADVANTAGES
• Low-medium transformation efficiency
• Limited range of vector systems
• rGenes must be stably incorporated

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Microbial expression hosts Pichia pastoris and
Kluveromyces sp.

• ADVANTAGES
• Moderate expression yields
• rProtein glycosylated
• rProtein exported
• Simple cell recovery

• DISADVANTAGES
• Low-medium transformation efficiencies
• Difficult fermentations
• Limited range of vector systems
• rGenes must be stably incorporated
• Unusual codon usages

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Microbial expression hosts Trichoderma reesii

• ADVANTAGES
• High expression yields
• rProtein glycosylated
• rProtein exported
• Simple cell recovery

• DISADVANTAGES
• Low-medium transformation efficiencies
• Difficult fermentations
• Limited range of vector systems
• rGenes must be stably incorporated
• Unusual codon usages

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Microbial expression hosts Bacillus

• ADVANTAGES
• High transformation efficiencies
• Very high expression yields (gt10 g/L)
• Simple fermentations
• rProtein exported
• Simple cell recovery

• DISADVANTAGES
• Limited range of vector systems
• Optimised vector systems are proprietary
• No glycosylation

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A typical E. coli plasmid vector

• Blue/white screening
• SP6 or T7 RNA polymerase promoters
• Amp resistance selection

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Expression development

• Multicopy plasmids
• Multiple selective (resistance) markers
  (Ampicillin, Tetracycline, Kanamycin,
  Thiostreptin)
• Different promoters (ITPG, Trp,
  temperature-inducible, aTet)
• Multiple promoters (shuttle vectors for different
  hosts)
• Multiple ori sequences
• N-terminal tags (e.g., polyHis) with cleavage site

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Steps to using novel gene products

• Scale up fermentation of recombinant organism
• Recover and purify expression product
  (downstream processing)
• Manipulate expression product for specific
  application (immobilisation, cross-linking etc)

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Applications of microorganisms and microbial
products in biotechnology

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Microbial fermentations

• First generation biotechnological processes
• Winemaking
• Methane generation
• Amino acid production
• Antibiotic production

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Winemaking
Red grapes (Cabernet sauvignon, Merlot, Pinot
noir, Pinotage)
Crushing
Grape must (skins plus juice)
S. cerevisiae primary fermentation
Removal of grape skins
Fermentation product (wine) 12-14 EtOH, high

tannin, high malic acid
Maloloactic (Lactobacillus sp. secondary
fermentation Maturation in oak casks
Mature wine
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Factors in primary fermentation

• Can use either natural yeast populations or
  cultured yeasts
• Natural yeast fermentations are not solely S.
  cerevisiae
• Conversion of fructose to EtOH
• Extraction of pigments
• Degradation of complex CHO
• Production of glycerol
• Generation of minor flavour substances
• Can generate chemical faults (H2S)

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Methane generation

• CH4 is generated by methanogenic Archaea
• Methanococcus, Methanosarcina, Methanospirillum,
  Methanobacterium
• Utilise simple substrates (CO2, CH3OH, HCOOH,
  C2H5COOH)
• Process is strictly anaerobic
• CO2 H2 ? CH4 2H2O
• Methanogenesis processes always operate as
  microbial co-cultures

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A typical methanogenesis fluidized-bed sludge
bioreactor
Removal, scrubbing and use of methane for
power generation.
Disposal or recycle of degraded organic fraction
Co-culture of heterotrophic, Fermentative and
acetogenic bacteria and methanogenic archaea
Domestic, agricultural or industrial waste sludge
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Processes of methane biosynthesis
Organic material (cellulose, proteins, starch,
lipids)
Heterotrophs polymer degradation (Bacillus,
Clostridium)
Alcohols, fatty acids
Acetogenic, H2-producers
Acetate
CO2, H2
Methanogens
CH4, CO2
CH4
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Amino acid production

• Amino acids are important industrial products
• Food additives
• Pharmaceutical intermediates
• Biochemical research
• L-Glu, L-Val, DL-Ala, L-Gln, L-Pro are all
  produced industrially via microbial fermentation

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Industrial production of amino acids

• Yields of up to 30g/L can be generation by
• Strain selection (auxotrophs, regulatory
  mutants)
• Mutation (? substrate uptake, ? product
  excretion, ?synthesis, ?degradation/side
  reactions)
• Fermentation and media development
• Corynebacterium (E,K,L), Brevibacterium
  (A,L,P,K), Bacillus (A), Arthrobacter (E),
  Escherichia (D), Pseudomonas (D), Microbacterium
  (E,P,V)

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Antibiotic Production

• There are approx. 8000 known antibiotics
• More than 100 are produced commercially by
  microbial fermentation
• 70 of these are produced by Streptomyces spp.
• Strain selection and development has resulted in
  very high product yields (e.g., 1100 g/L
  Penicillin)

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Common antibiotics and their sources

• Bacitracin
• Cephalosporin(s)
• Chloramphenicol
• Cycloheximide
• Hygromycin
• Penicillin
• Streptomycin
• Tetracycline(s)
• Vancomycin

• Bacillus subtilis
• Cephalosporium sp.
• S. venezuelae
• S. griseus
• S. hygromyces
• P. chrysogenum
• S. griseus
• S. aurofaciens
• S. orientalis

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Industrial evolution of penicillin production
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Industrial penicillin fermentation
1000
100
Lactose
g/L of lactose, ammonia and cell biomass
g/L of penicillin
Penicillin
50
500
Cell biomass
Ammonia
0
0
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Biotransformations

• The use of cells or enzymes as catalytic tools
  for the conversion of chemical compounds
• Example 1 Nitrile conversions
• Example 2 Chiral resolution

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1. Synthesis of acrylamide and nicotinic acid
Acrylonitrile
Acrylamide
Rhodococcus rhodochrous cells
3-Cyanopyridine
Nicotinamide
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Commercial uses of nitrile hydratase
biotransformation productsNitrile hydratase
converts cyanide to amide by water
addition widely distributed intracellular
enzyme specific for aliphatic (linear, cyclic
and heterocyclic substrates)

• Nicotinic acid
• Animal feed supplement
• Health food supplement

• Acrylamide
• Absorbent polymers
• Flocculants
• Construction

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Industrial development of NHase

• Third generation R. rhodochrous whole cell
  biocatalyst
• Generation 1. Native organism
• Generation 2. NHase gene cloned and re-expressed
  at high levels in parent organism
• Generation 3. Gene manipulated to increase
  thermostability, reduce substrate and product
  inhibition

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Fluidized bed continuous bioreactor
Product out, to DSP
P
Immobilised or cross-linked R. rhodochrous cells
Concentration
S
Column path-length
Substrate feed (0.5 1M )
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Chiral resolution of racemic amino acids
Aspergillus L-specific aminoacylase
acyl-DL-amino acid
L-amino acid acyl-D-amino acid
Chemical or enzymic racemisation
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Industrial production of chirally pure amino acids

• Racemic amino acids are synthesised chemically
• Chiral resolution using chemical methods is a
  multistep process
• Some enzymes have very high chiral specificity
  (e.g., L-specificity)
• 100 yield is obtained by racemising the
  unhydrolysed fraction