Lecture 23/24: Microbial Biotechnology

Biotechnology

Use of biological processes or organisms for the production of goods of services

Microbes using in biotechnology applications

Modified versions of naturally occurring organisms

Sources of microbes

• Scientists build and maintain culture collections

• depending on the organism, they are usually stored in a freeze-dried or frozen state and must be revived after shipment to the end user

• Help promote confirmation of findings through repeatable independent research

Bioprospecting

Searching for useful new microbes to cultivate and add to collections

Fermentation

Controlled and regulated aerobic/anaerobic culture of microbes to produce desired substances

Bioreactors

Maximize cell density and product yield

Fed-batch reactors

Concentrated nutrient (feed) is added in a controlled manner until the maximum concentration of cells is reached, and then the biomass is harvested

Chemostats

Some amount of biomass is continually removed (effluent) as the same amount of nutrient solution is added (feed). The addition of nutrient solution to the chemostat can continue indefinitely.

Primary metabolite

A product a metabolic processes required for growth of the microbe (e.g. alcohol)

Secondary metabolite

Not required for microbial growth, often produced during stationary phase (e.g. antibiotics)

Random mutagenesis

By chemical/radiation exposure is followed by screening for desired mutations

• drawbacks include little info obtained about mutations and the possibility of negative mutations

• Screening can also be difficult, labor-intensive, and costly

Mutagenesis: Site-directed

Specific mutations at specific sites within a DNA molecule - allows researchers to alter microbes in a much more rational way

Mutagenesis: Oligonucleotide-mediated site directed

• The cloning of the DNA has to be mutated into a vector that produces single-stranded DNA molecules

  • These vectors are modified from bacteriophage, such as M13, that have single-stranded genomes 

• A complementary oligonucleotide containing the desired sequence change is allowed to anneal, and then DNA synthesis is carried out by DNA polymerase 

• Once the complementary strand is completed, double-stranded plasmids carrying the desired mutation can be recovered after transformation of E.coli

Mutagenesis: PCR site-directed

• Researchers design complementary primers with the desired mutation and conduct PCR

• The product is subjected to digestion with the Dpnl restriction enzyme, which cleaves its recognition site (GATC) only when it is methylated during propagation within an E.coli cell

  • It will only cleave the original template DNA that has been replicated in the E.coli cell 

  • This will enrich for the desired mutated product 

• The products are introduced into a host cell by transformation, and the presence of the correct mutation can easily be confirmed by DNA sequence analysis 

Mutagenesis: Directed enzyme evolution

• Use progressive rounds of random mutation to “direct” and select for desired traits 

  • Starting with a gene encoding the enzyme of interest, mutagenesis is carried out to generate a library of variant genes

  • These libraries can then be screened for improved gene function, with the best-performing genes being subjected to mutagenesis again, and the screening process repeated 

  • The process is similar to that described slide 7 (primary and secondary metabolite), but here it is limited to a single gene rather than the entire genome 

Error-prone PCR

Amplify the gene you’re inserting in a sloppy way, leading to random mistakes - see what effect the mistakes have on the expressed product

DNA shuffling

Take bits you’ve already mutated, shear them up, and stick them back together randomly - see what effect the chimeras have on the expressed product

CRISPR-Cas Genome Editing

• Includes Clusters Regularly Interspaced Short Palindromic Repeats (CRISPR) and a CRISPR - associated (Cas) enzyme 

• When bacteria is exposed to a pathogen, it inserts a segment of the pathogen’s DNA into its repeat segment. After, it transcribed this into CRISPR DNA (crDNA) that then circulates within the cell. When the pathogen is encountered, the crRNA binds and targets the pathogen for destruction 

Expression vector

• Used to mass-produce recombinant proteins

• Must contain an E.coli promoter, operator, and terminator

• Eukaryal genes must be reformatted to remove introns

• A Shine-Dalgarno sequence must be added to the sequence to promote eventual translation

• Start, stop codons must be added

Red Biotechnology

• Used in the pharmaceutical/medical applications

• Major uses of microbes

  1. Producers of secondary metabolites with therapeutic properties

  2. Hosts for the production of recombinant human proteins

Secondary metabolites

Are therapeutics. Antibiotics like penicillin or statins, inhibitors of cholesterol synthesis

White Biotechnology

Industrial applications. Basic principle in this field is to use microbial conversion of low-cost biomass to products with a higher value and industrial use. Examples include biofuel production, bioplastics, etc

Biorefinery concept

Converting biomass, living or recently living biological substance, into a number of products, including chemicals, energy and materials

Biofuels: Ethanol

• Yeast ferment sugars to to produce ethanol during anaerobic fermentation of sugars

• Even industrial yeast strains can only tolerate a maximum of 15% ethanol

• Ethanol can be used in internal combustion engines with little modification - a reasonably alternative to gasoline

Waste biomass

• Agriculture and forestry operations could be used as feedstock

• Mostly composed of lignin/cellulose (difficult to breakdown)

Biofuels: Butanol and acetone

Butanol can also be used in internal combustion engines and has properties more similar to gasoline than ethanol

Green Biotechnology

Agriculture application. Modern agriculture involves the large amount of pesticide/herbicide and synthetic fertilizers. Genes can also be inserted to make plants look better

Agrobacterium - Nature’s genetic engineer

• A. tumefaciens causes crown gall tumors on plants

  • It does so by carrying a tumor-producing plasmid

  • Part of the plasmid is transferred into plant cells 

  • This makes it a good delivery system for gene insertion into plants (transgenic plant production) 

  • It represents a cross-kingdom transfer of DNA (bacteria to plants)

Agrobacterium - mediated plant transformation

• The opine and phytohormone genes within the T-DNA are placed by the gene to be introduced into the plant, along with suitable markers for selection and screening, on a plasmid that can be manipulated in E.coli

• To introduce the gene into a plant, the plasmid is transferred to Agrobacterium that contains no T-DNA, but does contain a modified pTi with vir genes that facilitate the transfer of the genes within the T-DNA of the introduced plasmid

Protoplast formation

Removal of the cell wall prior to DNA introduction

Biolistics

Metal fragments with DNA coating fired into plant cells

Herbicide resistance

• Broad-spectrum herbicide Roundup poisons plants but not mammals 

• Genetically engineering Roundup-resistant plants means farmers can use it year-round 

  • A plasmid containing a gene for a resistant form of the enzyme EPSP was introduced into plants using biolistics by Monsanto scientists