Bioprocess, biomolecular and genetic engineering as related to human health
Bioengineering Protein Sweeteners
Among Dr. Bang's research efforts is a project that focuses on over-production of a small protein sweetener, brazzein, in recombinant microorganisms, which will be used for human consumption to alleviate health problems associated with sweet carbohydrates.
Lew P. Christopher:
Biomass Bioprocessing to Pharmacologically Active Products
Dr. Christopher is Director of the Center for Bioprocessing Research & Development. The BME component of his research is focused on isolation and/or fermentation of plant biomass and its building block monomers and oligomers to obtain high-value products with biological activities, useful in preparation of improved pharmacological products. Examples of current and recent projects are provided below:
- Production of Optically Pure Lactic Acid by Homolactic Fermentative Bacteria
Lactic acid is a building block chemical with multiple product applicability that has been recently identified by DOE as one of the “top-ten” biorefinery production platform chemicals. It is widely used as an anti-microbial agent, and its optically pure form is a starting material in synthesis of chiral compounds for drug formulations and therapy. Recently, we have isolated a bacterial strain with high potential for lactic acid production. This strain was identified as Enterococcus faecalis and partially characterized. Subsequently, its full genome was sequenced and the isolate E. faecalis CBRD01 was deposited with ATCC. Our current objective is to lower the optimal pH for growth of this organism (pH optimum of 7.0 for the wild strain) closer to the pKa of lactic acid of 3.8 using “directed evolution” while retaining the ability of the strain to produce high levels of lactic acid. We use strain improvement through genome shuffling to improve the acid tolerance of E. faecalis CBRD01. Other strategies under investigation include error-prone whole genome amplification, and cloning of the two LDH genes from E. faecalis into a suitable yeast host.
- Lignin Biodegradation to Low Molecular Weight Aromatics
Low molecular weight phenolics and aromatics have been recently used in drug formulations and products to combat, however, their biological properties have not been fully explored. This project aims at biodegradation of lignin, the most-abundant naturally occurring polymer on earth, employing selected lignolytic microorganisms (fungi and bacteria) and laccase-mediator systems to obtain, characterize and evaluate bioactive aromatics for potential biomedical applications.
- Isolation of Natural Compounds of Pharmacological Importance
There is an increasing need for the discovery and development of pharmacologically active products. This can be accomplished in two ways, either by synthesizing new compounds or discovering new naturally occurring compounds in nature. The first has a high-cost for development and is associated for potential rapid drug resistance. The latter is a low-cost alternative with the greatest difficulty in finding these natural bioactive compounds in nature. When integrating new technology into medicinal therapy, materials from a biological origin are generally more desirable over their synthetic counterparts due to the fact that these materials are usually readily available in large quantities at a low cost. Recent projects in this field have concentrated on isolation and characterization of extracts from corn cobs, lichens, and milkweed seed oil that have shown high anti-oxidant, anti-microbial, and anti-tumor activities.
Linda C. Deveaux:
Molecular Mechanisms of Stress Survival
Research in the DeVeaux lab involves using microbial model organisms to study the molecular and genetic mechanisms underlying innate and acquired tolerances to environmental stresses.
Radiation resistance: We are investigating the gene regulatory responses to various extreme stresses, including high doses of ionizing radiation. By delineating the genetic determinants for maintaining genomic integrity under extreme conditions, we can ultimately develop ways to manipulate overall survival. We have created mutants of one model Archaeon, Halobacterium salinarum, which are more resistant to ionizing radiation than any other organism reported. The single common change in seven independently isolated mutants is constitutive expression of one operon encoding a putative Replication Protein A (RPA) homolog. Eukaryotic Replication Protein A is known to function in DNA replication and repair by binding to single-stranded DNA, and directing the appropriate enzymes to the site of action. In bacteria this protein is SSB, or Single-Stranded DNA Binding protein. There are five RPA homologs annotated in the genome of H. salinarum, suggesting specialization of function for repair of DNA damage. Genes for two of the five proteins are up-regulated in response to both UV and ionizing radiation. We are using genetic techniques to delineate the roles of each homolog, and biochemical methods to delineate the protein interactions necessary for the specialized function of each homolog. The SSB protein of Deinococcus radiodurans, a highly resistant bacterium, is also regulated in response to DNA damage. We have recently demonstrated that high levels of this protein are necessary for the extreme radiation resistance of D. radiodurans, but not for normal growth. We are currently investigating the role of this SSB, as well as a second, unique pentameric SSB, in the extreme resistance of this organism.
Ultimately, we hope to discover ways to manipulate the expression of these highly conserved and ubiquitous proteins. Such methods may be extended to human cells, allowing control of cellular radiation sensitivity.
Richard R. Sinden:
DNA Structure and Function; therapeutic approaches to DNA structure associated genomic instability leading to disease
- DNA structure and supercoiling
The Sinden lab studies DNA, alternative conformation of DNA, and DNA supercoiling. Alternative conformations of DNA include cruciforms, left-handed Z-DNA, intramolecular triplex DNA, unwound DNA structures, and G-quadruplex structures. In 1995 we discovered a new alternative DNA structure called slipped-strand DNA. We have shown that many of these structures exist in the chromosomes of living bacterial cells. We work to understand the biological roles and consequences of these alternative DNA conformations in mammalian cells.
- Molecular mechanisms of spontaneous and genotoxicant-induced mutation
A second area of research interest involves understanding the molecular mechanisms of mutagenesis. An exciting correlation exists between DNA sequences that form alternative structures and mutations that cause cancer and human genetic disease. That is – mutations do not really occur randomly, rather they are often templated by the DNA sequence itself. In other words, certain DNA sequences (DNA repeats) are their own worst enemy. These DNA sequences are prone to, or better perhaps, programmed for, self-directed mutation. We work to understand these molecular mechanisms of spontaneous mutagenesis that involve alternative DNA conformations. In addition, we have shown that many types of mutations occur preferentially on either the leading or lagging strand during replication. Currently, we are investigating the genetic instability of DNA sequences that form four-stranded, G-quadruplex structures. These sequences are associated with genetic instabilities associated with cancer.
- DNA repeat and genomic instabilities associated with human genetic neurodegenerative disease
A third area of interest involves understanding the molecular basis of certain human genetic diseases, including Huntington disease, myotonic dystrophies, fragile X syndrome, spinocerebellar ataxias, and Friedreich ataxia. This area of research integrates the above two focus areas: DNA structure and spontaneous mutagenesis. Currently, more than 40 human genetic neurodegenerative diseases are caused by the massive expansion of (CTG)n•(CAG)n, (CGG)n•(CCG)n, (GAA)n•(TTC)n, (CCTG)n•(CAGG)n, or (ATTCT)n•(AGAAT)n DNA repeats. All these DNA repeats form one or more alternative DNA conformations, including hairpins, slipped strand DNA, parallel DNA, triplex DNA, and unwound structures, which are likely involved in their genomic instability (i.e., expansion or deletion mutations). We have developed genetic assays for studying the deletion of DNA repeats in a model bacterial system. A goal of our laboratory is to understand the molecular basis for the expansion (and deletion) mutations and to find a therapeutic approach for reducing repeat length.
We are currently investigating the role of tryptanthrins and coralyne derivatives as chemicals that may stabilize intramolecular triplex DNA and lead to increased rates of deletion of the Friedreich Ataxia (GAA)n•(TTC)n repeats. With such an approach, one may be able to prevent or delay onset of repeat expansion diseases.