Ch 12. Synthesis of Commercial Products by Recombinant Microorganisms
Restriction Endonucleases Vitamins and Amino Acids Dyes Antibiotics Biopolymers
Restriction Endonucleases
Vitamins and Amino Acids
Dyes Antibiotics Biopolymers
Production of Restriction Endonucleases
Figs. 12.1, 12.2, 12.3
Widely used for recombinant DNA technology
Several hundred are known from a variety of different bacterial species (Components of the restriction-modification system) Aerobes, anaerobes, phototrophs, thermophiles Each has different growth requirements
Several hundred are known from a variety of different bacterial species (Components of the restriction-modification system)
Aerobes, anaerobes, phototrophs, thermophiles
Each has different growth requirements
Commercial production of many enzymes by one organism (recombinant E. coli) allows fermentation conditions to be standardized
Growth media composition and pH Temperature and aeration Fermentor design
Growth media composition and pH
Temperature and aeration
Fermentor design
Clone both genes of RM system (restriction enzyme and methylase)
Methylase protects E. coli host's DNA from cleavage by the restriction enzyme Methylase gene should be expressed before the restriction enzyme
Methylase protects E. coli host's DNA from cleavage by the restriction enzyme
Methylase gene should be expressed before the restriction enzyme
The restriction enzyme should have a leader peptide so that it will be transported from the cytoplasm to the periplasm
This separates the enzyme from the host's chromosome to further protect it from being cleaved
Cloning and Commercial Production of Restriction Enzymes
Target genes encode both th methylase and the restriction enzyme
I. Create genomic DNA library
1. Digest genomic DNA of organism that produces the desired restriction enzyme 2. Insert into cloning vector 3. Transform E. coli strain that doesn't produce its own restriction enzyme
1. Digest genomic DNA of organism that produces the desired restriction enzyme
2. Insert into cloning vector
3. Transform E. coli strain that doesn't produce its own restriction enzyme
II. Screen library
4. Infect libary w/ bacteriophage l 5. Isolate colonies -Cells w/ cloned restriction enzyme gene are resistant to lysis by l -Screen for expression of modification enzyme
4. Infect libary w/ bacteriophage l
5. Isolate colonies
-Cells w/ cloned restriction enzyme gene are resistant to lysis by l -Screen for expression of modification enzyme
-Cells w/ cloned restriction enzyme gene are resistant to lysis by l
-Screen for expression of modification enzyme
III. Transfer genes to expression vector for large-scale commercial production
Commercial Production of Indigo from Tryptophan by Recombinant E. coli
See Figs. 12.8 aand 12.9
Large market for indigo as a dye for blue jeans
Chemical synthesis produces toxic wastes
nahA genes of Pseudomonas encodes naphthalene dioxygenase
Cloning of nahA and expression in E. coli results in production of indigo
1. E. coli tryptophanase converts trp to indole 2. Naphthalene dioxygenase transforms indole to an unstable intermediate (indole and naphthalene have similar chemical structures) 3. Intermediate dehydrates, producing indoxyl 4. Indoxyl oxidizes, producing indigo
1. E. coli tryptophanase converts trp to indole
2. Naphthalene dioxygenase transforms indole to an unstable intermediate
(indole and naphthalene have similar chemical structures)
3. Intermediate dehydrates, producing indoxyl
4. Indoxyl oxidizes, producing indigo
Produces fewer toxic wastes than chemical synthesis
Not yet economically competitive with chemical process, however
Production of Amino Acids
800,000 tons produced/year worth $5 billion
See Table 12.1 Sources 1. Hydrolysis of proteins 2. Microbial fermentation Mainly by nonrecombinant Gram positive bacteria Ex. Corynebacterium glutamicum Brevibacterium spp.
See Table 12.1
Sources
1. Hydrolysis of proteins
2. Microbial fermentation
Mainly by nonrecombinant Gram positive bacteria Ex. Corynebacterium glutamicum Brevibacterium spp.
Mainly by nonrecombinant Gram positive bacteria
Ex. Corynebacterium glutamicum Brevibacterium spp.
Ex.
Corynebacterium glutamicum
Brevibacterium spp.
Increasing Tryptophan Production
Fig. 12.10
Traditional industrial bacterium for production of amino acids by fermentation Rate-limiting step of Trp biosynthesis is catalyzed by antharnilate synthetase trpE encodes anthranilate synthetase Gene dosage of trpE was increased in C. glutamicum
Traditional industrial bacterium for production of amino acids by fermentation
Rate-limiting step of Trp biosynthesis is catalyzed by antharnilate synthetase
trpE encodes anthranilate synthetase
Gene dosage of trpE was increased in C. glutamicum
1. C. glutamicum DNA library was created using C. glutamicum-E. coli shuttle vector
2. Library was screend by complementation of a C. glutamicum mutant that needed anthranilic acid for growth
o Suggests that vector contains trpE
3. Vector transferred to C. glutamicum for expression
Eosinophilia Myalgia Syndrome
1989 Eosinophilia myalgia epidemic in U.S.
1500 cases --28 deaths high blood eosinophil counts muscle pain
1500 cases --28 deaths
high blood eosinophil counts
muscle pain
Associated with ingestion of large amounts of trp as dietary supplement
Trp manufacturer traced to Showa Denko KK of Japan Changed fermentation process in 1988 1. Wild type => genetically engineered strain -overproduced phosphoribosylpyrophosphate -a trp precursor, increased trp biosynthesis
Trp manufacturer traced to Showa Denko KK of Japan
Changed fermentation process in 1988 1. Wild type => genetically engineered strain -overproduced phosphoribosylpyrophosphate -a trp precursor, increased trp biosynthesis
Changed fermentation process in 1988
1. Wild type => genetically engineered strain
-overproduced phosphoribosylpyrophosphate -a trp precursor, increased trp biosynthesis
-overproduced phosphoribosylpyrophosphate
-a trp precursor, increased trp biosynthesis
2. Product purification (downstream processing) -reduced amount of activated charcoal
2. Product purification (downstream processing)
-reduced amount of activated charcoal
Several byproducts were produced which weren't removed by purification and contaminated product Ex. 1,1'-ethylidene-bis[L-tryptophan] which is converted to 1,2,3,4-tetrahydro-b-carboline 3-carboxylic acid by the acidic pH of the stomach May have triggered an autoimmune reaction
Several byproducts were produced which weren't removed by purification and contaminated product
Ex. 1,1'-ethylidene-bis[L-tryptophan] which is converted to 1,2,3,4-tetrahydro-b-carboline 3-carboxylic acid by the acidic pH of the stomach May have triggered an autoimmune reaction
Ex. 1,1'-ethylidene-bis[L-tryptophan] which is converted to 1,2,3,4-tetrahydro-b-carboline 3-carboxylic acid by the acidic pH of the stomach
May have triggered an autoimmune reaction
Antibiotics
Only 1-2% are useful and commercialized High costs of development and clinical testing Many have toxic side-effects
Only 1-2% are useful and commercialized
High costs of development and clinical testing
Many have toxic side-effects
To treat infections by resistant strains None are known for some pathogens
To treat infections by resistant strains
None are known for some pathogens
1. Modify existing antibiotics
Reduce side effects Broaden spectrum of activity Increase stability
Reduce side effects
Broaden spectrum of activity
Increase stability
2. Engineer producer strains with increased yields
3. Lower production costs
Cloning Genes Encoding Enzymes of Antibiotic Biosynthetic Pathways
-Biosynthesis may involve 10-30 different enzyme-catalyzed reactions with as many genes
-May be close to each other on chromosome, or
-May be scattered around the chromosome
One approach
1. Create DNA library using wild-type antibiotic-producing organism
2. Transform mutants of the same organism that have defects in antibiotic biosynthetic pathway
3. Screen colonies for complementation of the mutations by cloned genes that reestablish the ability to synthesize the antibiotic
4. Screen for additional genes of the pathway
Use DNA isolated from the initial positive clones and to synthesize labeled DNA probes -can use random primer method (Ch. 4) or -sequence cloned DNA and design complementray probes that will hybridize to 5' and 3' ends of the cloned DNA Rescreen library for colonies that hybridize to labeled probes Positive colonies may contain cloned genes encoding enzymes that catalyze additional steps of biosynthetic pathway if the genes are located nearby (upstream or downstream) on the chromosome (May require several rounds of probe design and screening to get all genes for a complete pathway)
Use DNA isolated from the initial positive clones and to synthesize labeled DNA probes
-can use random primer method (Ch. 4) or -sequence cloned DNA and design complementray probes that will hybridize to 5' and 3' ends of the cloned DNA
-can use random primer method (Ch. 4) or
-sequence cloned DNA and design complementray probes that will hybridize to 5' and 3' ends of the cloned DNA
Rescreen library for colonies that hybridize to labeled probes
Positive colonies may contain cloned genes encoding enzymes that catalyze additional steps of biosynthetic pathway if the genes are located nearby (upstream or downstream) on the chromosome
(May require several rounds of probe design and screening to get all genes for a complete pathway)
To construct a recombinant strain with increased antibiotic production:
Assemble all genes on one vector and optimize expression to increase amount of antibiotic produced, or Overexpress cloned genes for the enzymes that encode rate-limiting or slow steps of the pathway
Assemble all genes on one vector and optimize expression to increase amount of antibiotic produced, or
Overexpress cloned genes for the enzymes that encode rate-limiting or slow steps of the pathway
Engineering Antibiotics with Altered Chemical Structures May give antibiotic new or improved properties
Genes encoding enzymes for biosynthetic pathways of two related antibiotics can be combined in one organism
Intermediate products of one pathway may be transformed by enzymes of the introduced pathway, resulting in altered antibiotic stuctures
Exs. (See Fig. 12.17)
All biosynthetic genes were cloned and inserted into plasmids pIJ2315 and pIJ2303
Transformed with pIJ2315 -Produced Mederrhodine A, new antibiotic
Transformed with pIJ2315
-Produced Mederrhodine A, new antibiotic
Transformed with pIJ2303 Produced Dihydrogranatirhodine, new antibiotic
Transformed with pIJ2303
Produced Dihydrogranatirhodine, new antibiotic
Production of Industrial Polymers
Ex. Xanthum gum
High molecular weight biopolymer (Fig. 12.24) Synthesized by Xanthomonas campestris Most important bacterial polymer used by industry High viscosity, stable to heat and pH extremes
High molecular weight biopolymer (Fig. 12.24)
Synthesized by Xanthomonas campestris
Most important bacterial polymer used by industry
High viscosity, stable to heat and pH extremes
Many uses:
Thickener of processed foods, paints and well drilling mud
Xanthan Gum from Whey Waste
Whey: Waste product of cheese manufacturing Disposal is a problem and costly Contains high conc. of lactose, ~3.5-4%
Whey: Waste product of cheese manufacturing
Disposal is a problem and costly Contains high conc. of lactose, ~3.5-4%
Disposal is a problem and costly
Contains high conc. of lactose, ~3.5-4%
Xanthomonas campestris grows on glucose but not lactose 1. lacZY genes cloned from E. coli (Fig. 12.25) lacZ encodes b-galactosidase lactose --> glucose + galactose lacY encodes lactose permease transports lactose into cell
Xanthomonas campestris grows on glucose but not lactose
1. lacZY genes cloned from E. coli (Fig. 12.25)
lacZ encodes b-galactosidase lactose --> glucose + galactose lacY encodes lactose permease transports lactose into cell
lacZ encodes b-galactosidase
lactose --> glucose + galactose
lacY encodes lactose permease
transports lactose into cell
2. Inserted into broad host range plasmid with a constitutive promoter from X. campestris bacteriophage Introduced into X. campestris Grows on lactose present in whey and produces xanthan gum (Table 12.5) Former waste product converted to marketable product
2. Inserted into broad host range plasmid with a constitutive promoter from X. campestris bacteriophage
Introduced into X. campestris
Grows on lactose present in whey and produces xanthan gum (Table 12.5) Former waste product converted to marketable product
Main Page
Comments or questions regarding this page or this class: haddock@micro.siu.edu
Comments or questions regarding this department: microbiology@micro.siu.edu
SIUC / College of Science / Microbiology / Microbiology 421 http://www.micro.siu.edu/micr421/index.html
Last updated: December 2, 2004 /jdh
Comments and questions related to web server: webmaster@science.siu.edu