October 1, 2011 (Vol. 31, No. 17)
Arnold L. Demain, Ph.D.
Jose L. Adrio, Ph.D.
Combination of Fermentation, Genetics, and Biotechnology Produced a Formidable Force
While fermentation has been important since prehistoric times, it really took off at the beginning of the 20th century when microorganisms were used to make organic acids.
By the 1940s, antibiotic production had become the new application and this moved microbes to the forefront of industrial biology. From the 1940s to the 1970s, the discovery of new antibiotics continued to contribute to the importance of microbial fermentations. Also at this time, production of primary metabolites such as amino acids, organic acids, and vitamins pushed the fermentation field into the spotlight.
Production was dependent on making mutants that produced higher titers of products, but this was difficult, time-consuming, and slow. Thanks to the discovery of recombinant DNA and the establishment of the biotechnology industry in California in 1971–1973, entirely new ways of improving microorganisms became available and it revolutionized the fermentation industry.
Microbes are one of the greatest sources of metabolic and enzymatic diversity. Emerging recombinant DNA and genomic techniques have led to efficient new expression systems, modification of biosynthetic pathways leading to new metabolites by metabolic engineering, and enhancement of catalytic properties of enzymes by directed evolution.
Complete sequencing of industrially important microbial genomes is taking place very rapidly and there are already dozens of genomes sequenced. Functional genomics and proteomics are indeed major tools used in the search for new molecules and development of higher-producing strains.
Advantages of Microbes
Natural products are superior to synthetic compounds for making pharmaceuticals. Microbes are better than plants or animals for manufacturing commercial levels of such compounds.
Microbes have a number of advantages, including rapid uptake of nutrients supporting high rates of metabolism and biosynthesis; ability to carry out a wide variety of reactions; facility to adapt to a large array of different environments; ease of genetic manipulation, both in vivo and in vitro, to increase production, to modify structures and activities, and to make entirely new products; simple screening procedures; and a wide diversity.
Microbial products are diverse, ranging from large molecules such as proteins, nucleic acids, carbohydrate polymers, or even cells, to small molecules that are usually divided into primary metabolites, i.e., those essential for vegetative growth, and secondary metabolites, which are not essential for growth. The primary metabolites include amino acids, organic acids, alcohols, and vitamins.
Production of a particular primary metabolite by deregulated organisms may inevitably be limited by the inherent capacity of the particular organism to make the appropriate biosynthetic enzymes. Recent approaches utilize modern genetic engineering techniques to correct such deficiencies and develop strains overproducing primary metabolites.
There are two ways to accomplish this: increase the number of copies of structural genes coding for these enzymes and the frequency of transcription.
Novel genetic technologies are important for the development of overproducers. Genome-based strain reconstruction leads to the development of a superior strain which contains mutations crucial to hyperproduction but not other unknown mutations, which accumulate by brute-force mutagenesis and screening.
Also important are genome-sequencing projects involving hundreds of genomes, the availability of sequences corresponding to model organisms, new DNA microarray and proteomic tools, and new techniques for mutagenesis and recombination DNA technology.
The improvement of product formation or cellular properties via modification of specific biochemical reactions or the introduction of new properties with the use of recombinant DNA technology is known as metabolic engineering. Analytical methods are combined to quantify fluxes and to control them with molecular biological techniques in order to implement suggested genetic modifications.
The overall flux through a metabolic pathway depends on several steps, not just a single rate-limiting reaction. Amino acid production is one of the fields with many examples of this approach.
Reverse (inverse) metabolic engineering is another technique that involves choosing a strain that has a favorable cellular phenotype, evaluating and determinating the genetic and/or environmental factors that confer that phenotype, and finally, transferring that phenotype to a second strain via direct modifications of the identified genetic and/or environmental factors.
Molecular breeding techniques are based on mimicking natural recombination by in vitro homologous recombination. DNA shuffling not only recombines DNA fragments but also introduces point mutations at a very low controlled rate. Unlike site-directed mutagenesis, this method of pooling and recombining parts of similar genes from different species or strains has yielded remarkable improvements in a short amount of time.
Whole-genome shuffling is another technique that combines the advantage of multiparental crossing allowed by DNA shuffling with the recombination of entire genomes through recursive protoplast fusion.
Systems biology is an integrated, systemic approach to the analysis and optimization of cellular processes by introducing a variety of perturbations and then measuring the system response. Altered phenotypes are created by molecular biological techniques or by altering environments. Further characterization of the phenotype leading to maximal product formation is analyzed and quantified through the use of genome-wide high-throughput omics data and genome-scale computational analysis.
Secondary metabolites are a group that includes antibiotics, pesticides, pigments, toxins, pheromones, enzyme inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, antitumor agents, immunosuppresants, cholesterol-lowering agents, plant protectants and animal and plant growth factors—these metabolites have tremendous economic importance. This remarkable group of compounds is produced by certain restricted taxonomic groups of organisms and usually formed as mixtures of closely related members of a chemical family.
The antibiotics include β-lactams (penicillins, cephalosporins, cephamycins, clavulanic acid, and carbapenems), aminoglycosides, macrolides (erythromycin, oleandomycin, pikromycin, tylosin and amphotericin B), and polyenes.
Metabolic engineering has been used to replace the normal promoter to increase antibiotic production many-fold. High-level expression of positive regulatory genes has led to major increases in antibiotic production.
The production of antibiotics in heterologous hosts via combinatorial biosynthesis is becoming popular in antibiotic production and discovery. Over 200 new polyketides have been made by combinatorial biosynthesis, which also has been used to construct macrolides with new sugar moieties.
An important new strategy to improve the discovery of new antibiotics is genome mining, which has come about due to advances in microbial genomics. Mining of whole-genome sequences and genome scanning allows the rapid identification of more than 450 clusters of genes (in antibiotic-producing cultures) that encode the biosynthesis of new bioactive products. Genome mining also allows one to predict structure based on gene sequences.
Research in this area includes mining of whole-genome sequences, genome scanning, and heterologous expression. The discovery of novel chemistry has also been a result. Genomics could provide a huge group of new targets against which natural products can be screened.
Biopharmaceutical products, a key part of the pharmaceutical industry, are biotechnology’s major contribution. These products can be categorized into four major groups: protein therapeutics with enzymatic activity (e.g., insulin), protein vaccines, protein therapeutics with special targeting activity (e.g., monoclonal antibodies) and protein diagnostics (e.g., biomarkers).
Biologics accounted for over $80 billion in sales in 2008. Six of these therapeutic proteins were among the best-selling drugs in the U.S. in that year. Monoclonal antibodies and Fc-fusion proteins made up 43% of this market value. The market reached over $90 billion in 2010.
Genetic engineering allows the production of massive quantities of desired proteins. Protein quality, functionality, production speed, and yield are the most important factors to consider when choosing the right expression system for recombinant protein production.
The major organisms used for protein production are mammalian cells (mainly CHO cells) and E. coli. CHO cells constitute the preferred system for producing monoclonal antibodies and some other recombinant proteins. By 2006, production of therapeutic proteins by mammalian systems reached $20 billion.
A relatively new system is PER.C6, which has been reported to produce 27 g/L of a monoclonal antibody.
Some recombinant proteins are also made by yeasts (Saccharomyces cerevisiae, Pichia pastoris), molds, insect cells, plant cells, transgenic plants, and transgenic animals. Some fungi such as Chrysosporium lucknowense can make native protein at a level of 100 g/L. It has been genetically converted into a nonfilamentous, less viscous, low protease-producing strain that is capable of producing high yields of heterologous proteins.
Directed evolution of proteins includes DNA shuffling, whole-genome shuffling, heteroduplex, random chimeragenesis of transient templates, assembly of designed oligonucleotides, mutagenic and unidirectional reassembly, exon shuffling, Y-ligation–based block shuffling, nonhomologous recombination, and the combining of rational design with directed evolution. Application of such technologies have led to protein titers of 5–10 g/L. Indeed, claims have been made that P. pastoris can make 20–30 g/L of recombinant proteins.
The properties of many enzymes modified by genetic approaches including DNA shuffling, result from changes in substrate specificity, increased or modified enantioselectivity, resistance to feedback inhibition, kinetic parameters (Vmax, Km or Ki), pH optimum, thermostability, stability in organic solvents, and increased biological activity.
The best is yet to come from the combination of industrial microbiology and recombinant DNA technology. Many of the new techniques are carried out by small companies and academic groups that could play a major role in rescuing us from the antibiotic crisis that we are now experiencing.
Arnold L. Demain, Ph.D. ([email protected]), conducts research at the Charles A. Dana Research Institute for Scientists Emeriti, Drew University. José L. Adrio, Ph.D., is head of the Neuron BioIndustrial Divison, Neuron BPh.