Metabolic engineering of bacteria

1
Metabolic engineering of bacteria

• Increasing biological production of small
  molecules
• Random screening for overproducing strains
  (genome shuffling)
• Rational engineering of pathways

2
Many biological small molecules are useful

• Antibiotics
• Vitamins
• Amino acids and derivatives (indigo, aspartame)
• secondary metabolites from plants--alkaloids
  (caffeine, theobromine, etc.)
• Etc.
• Synthesis often requires multiple steps and
  enzymes

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Increasing production of antibiotics (and other
small molecules)--traditional methods

• Obtain organism that produces a specific
  compound--Penicillium mold originally made
  micrograms per liter of culture
• Randomly mutagenize the organism and screen for
  increased production, repeat using top producing
  organism
• Outcome grams of penicillum per liter of culture
  (1000-fold increase in production)
• Time consuming and expensive process!

4
An alternative to simple random mutagenesis
genome shuffling
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The shuffling advantage simultaneous
recombination of entire genomes (breeding) with
multiple parents
(new way)
(old way)
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The set-up

• Compare classical strain improvement (CSI) to
  genome shuffling
• Streptomyces sp. produce polyketide antibiotics
• Induce recombination by recursive protoplast
  fusion
• Fuse protoplasts
• Regenerate cell walls, grow as a population (F1)
• Make protoplasts with F1, repeat until F4
• Test with 4 auxotrophy markers (next page)
• Test for increased antibiotic production

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Test of recursive shuffling
Supplements required pro, arg, ura (not
cys) pro, cys, ura (not arg) arg, cys, ura (not
pro) pro, arg, cys (not ura)
4 parental strains
Strain Description
S. coelicolor 268412 proA1 argA1 uraA1
S. coelicolor 268512 proA1 cysD18 uraA1
S. coelicolor 268612 argA1 cysD18 uraA1
S. coelicolor M12412 proA1 argA1 cysD18
Can strains be isolated that can grow in the
absence of pro, arg, ura, and cys (indicating
progeny with all 4 genes wild type)? YES.
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Indicates increased efficiency of recombination
9
Test case increase tylosin production by S.
fradiae?
SF1 was treated with NTG, 11 strains selected
(22000 screened), those 11 strains were shuffled
once (GS1) and then again (GS2)
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Comparing CSI to genome shuffling
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Genome shuffling

• Technique has also been used to generate
  acid-tolerant strains of Lactobacillus (useful
  for production of lactic acid)
• Applicable to eukaryotic microbes?
• Still dont know the mutations that have
  occurred, or what the state of the genome is
  following several fusion events

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Increasing production of a biological compound
rational design

• 1) Increase production of a naturally produced
  commercial compound
• Modify existing genes
• 2) Obtain a new organism that can convert an
  existing compound into a commercial compound
• Introduce new genes
• Modify existing genes

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• Engineering E. coli to produce indigo
• Mutate tryptophan synthase complex to release
  indole
• Introduce napthalene dioxygenase (from
  Pseudomonas putida)

natural source of indigo woad Isatis tinctoria
Pict (painted--with woad)
woad
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Introduce isatin hydrolase (from a soil microbe)
to prevent production of indirubin (color) from
isatin
burgundy
blue
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Potential routes for overproducing biological
compounds

• Remove rate-limiting transcriptional controls
• Remove rate-limiting enzyme allostery controls
• Kinetically enhance rate-limiting enzymes
• Genetically block competing pathways
• Enhance commitment of carbon to the pathway of
  interest
• Enhance transport of compound out of cell

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How to overproduce phenylalanine?

1. Remove feedback inhibition (select strains
   resistant to phenylalanine analogues)
2. Avoid repression (place genes under control of
   non-phe controlled promoters)
3. Remove pathway competition (delete tyr and trp
   specific genes)
4. Overexpress phe-specific genes
5. Increase E4P and PEP synthesis

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Rational metabolic engineering

• Requires at least some knowledge of the
  biochemical pathway required for compound
  synthesis
• Trial and error--try something, see if it works,
  or where new block is (and focus on the new
  block)
• Potentially very labor intensive
• But high degree of control over the organism

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• Non-E.coli Bacterial Cloning
• Homologous recombination

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Cloning in bacteria other than E.coli?

• Utility
• Study bacterial processes and pathways that may
  not be correctly expressed in E. coli, eg.
  pathogenesis, antibiotic production
• properties not available in E.coli, eg. natural
  transformation
• Disadvantages
• Often a poor selection of specialized vectors
• Transformation (by the usual techniques) may be
  difficult

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Necessary components for non-E.coli cloning

• Method for introducing DNA
• Transformation (spontaneous)
• Transformation (chemical, electroporation)
• Conjugation
• Method for replicating DNA
• Plasmid replicon
• Integration into chromosome (homologous
  recombination)
• Cloned gene must be expressed in the non-E. coli
  host (if you want to use the new host as an
  expression vector)

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Natural transformation

• Spontaneous uptake of DNA from the environment
• (Likely to be a major route for horizontal gene
  transfer)
• Fairly common in bacteria-- but this is one thing
  E. coli cannot do!

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Conjugation as a method of transfer

• Promiscuous plasmids--self-transmissible to many
  hosts
• (not a complete substitute for transformation,
  since DNA must often be manipulated in vitro,
  then reintroduced)

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Plasmid Host Range

• Host-range of plasmid replicons is highly
  variable
• E. coli specialized vectors
• have narrow host range
• But their range can be increased by creating
  hybrid plasmids that replicate in E. coli and in
  new host Shuttle Vectors

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Integration by recombination

• If transformed DNA has homology to chromosome (or
  other plasmid), this DNA can be integrated by
  homologous recombination
• Two pieces of DNA with the same sequence RecA
  protein guides a complex that causes strand
  exchange between homologous sequences
• Homologous recombination is rare but spontaneous
  (with a highly predictable frequency 1/1000
  cells will recombine)

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Homologous recombination portrait of a single
cross-over
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Recombination (single crossover)
Transfer plasmid (or linear piece of DNA) into
host in which it cannot replicate Select for
antibiotic marker
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Recombination in genome engineering
(PCR product)
Tet r
recombination
(genome)
gene
flank
flank
(engineered genome)
Cell is Tet r, and red gene is knocked out
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Things that can be easily done with PCR products,
transformation, and recombination.

• Gene deletions (with or without the antibiotic
  resistance gene)
• Addition of tags to chromosomal proteins
• Gene replacement (targeted mutagenesis)

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Recap

• Non-E.coli bacteria can be useful for recombinant
  DNA studies, though not as versatile as E. coli
• Natural transformation is an important feature of
  some species
• Shuttle vectors hybrid plasmids with more than
  one type of replicon to increase host range
• Recombination is an important tool for
  maintaining recombinant DNA and for manipulating
  the genome