Transcript Metabolic engineering of bacteria

Metabolic engineering of bacteria
• Increasing biological production of small
molecules
• Random screening for overproducing strains
(genome shuffling)
• Rational engineering of pathways
Many biological small molecules are useful
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Antibiotics
Vitamins
Amino acids and derivatives (indigo, aspartame)
“secondary metabolites” from plants--alkaloids
(caffeine, theobromine, etc.)
• Etc.
• Synthesis often requires multiple steps and
enzymes
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• Obtain organism that produces a specific compound-Penicillium mold originally made micrograms per liter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!
An alternative to simple random
mutagenesis: genome shuffling
The shuffling advantage: simultaneous recombination
of entire genomes (breeding) with multiple parents
(old way)
(new way)
The setup
• 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
Test of recursive shuffling
4 parental strains
Strain
Supplements required:
Description
S. coelicolor
268412
S. coelicolor
268512
S. coelicolor
268612
argA1 cysD18 uraA1
S. coelicolor M12412
proA1 argA1 cysD18
proA1 argA1 uraA1
proA1 cysD18 uraA1
pro, arg, ura (not cys)
pro, cys, ura (not arg)
arg, cys, ura (not pro)
pro, arg, cys (not ura)
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….
Indicates increased
efficiency of recombination
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)
Comparing CSI to genome shuffling
Genome shuffling
• Technique has also been used to generate acidtolerant strains of Lactobacillus (useful for
production of lactic acid)
• Applicable to eukaryotic microbes?
• Still don’t know the mutations that have occurred,
or what the state of the genome is following
several fusion events
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
Engineering E. coli to produce indigo
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Mutate tryptophan synthase complex
to release indole
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Introduce napthalene dioxygenase
(from Pseudomonas putida)
natural source of indigo:
woad [Isatis tinctoria]
woad
Pict (“painted”--with woad)
Introduce isatin hydrolase (from
a soil microbe) to prevent
production of indirubin (color)
from isatin
blue
burgundy
Potential routes for overproducing
biological compounds
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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
How to overproduce phenylalanine?
1) Remove feedback inhibition
(select strains resistant to
phenylalanine analogues)
2) Avoid repression (place
genes under control of nonphe controlled promoters)
3) Remove pathway
competition (delete tyr and
trp specific genes)
4) Overexpress phe-specific
genes
5) Increase E4P and PEP
synthesis
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
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Homologous recombination
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
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)
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!
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)
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”
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)
Homologous recombination: portrait of a single cross-over
Recombination (single crossover)
Transfer plasmid (or linear piece of DNA) into host in
which it cannot replicate
Select for antibiotic marker
Recombination in genome engineering:
(PCR product)
Tet r
recombination
(genome)
flank
gene
flank
(engineered
genome)
Cell is Tet r, and red gene is knocked out
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)
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