The Altman laboratory has two main research interests.
Metabolic engineering involves the microbial production of biochemical products through the modification of metabolic pathways. To accomplish this key metabolic enzymes are cloned and overproduced and/or metabolic enzymes that produce off-pathway products are eliminated using gene knockouts. We have investigated the production of a number of important biochemicals using metabolic engineering strategies.
Because of the importance of industrial enzymes and therapeutic proteins, a longstanding interest of our laboratory has been developing approaches to increase the overproduction of recombinant proteins. Most of our research has focused on alleviating the metabolic bottlenecks associated with the production of recombinant proteins. We have developed a number of strategies by which the metabolic overloads caused by recombinant protein production can be circumvented.
Another longstanding interest of our laboratory has been the production of biochemicals which can be derived from oxaloacetic acid, such as succinic acid and threonine. Carbon flux at the phosphoenolpyruvic acid node is routed preferentially towards pyruvic acid instead of oxaloacetic acid in most microorganisms and thus the production of biochemicals derived from oxaloacetic acid is problematic. We have shown that the overproduction of pyruvate carboxylase, which converts phosphoenolpyruvic acid to oxaloacetic acid, can dramatically increase the production of both succinic acid and threonine, two very important industrial biochemicals.
Using different metabolic engineering strategies we have developed processes that can be used to produce two other very important industrial biochemicals, lactic acid and pyruvic acid. We are investigating strategies to produce glyceric acid and serine as well as investigating approaches by which acrylic acid might be produced fermentatively. We are also working on processes by which the waste glycerol from biodiesel production can be utilized to produce industrial biochemicals thus generating an important value-added co-product for this industry.
Currently one of our most significant research efforts has involved the production of the biofuel ethanol from lignocellulosic biomass. Recent congressional mandates have called for the production of 36 billion gallons of ethanol per year by 2022 to offset the enormous amount of gasoline that is consumed by the US, which is currently 150 billion gallons per year. Most experts are in agreement that to meet these goals a significant amount of the ethanol will have to be produced from lignocellulosic biomass.
While lignocellulosic biomass is theoretically a great source of sugar from which ethanol can be produced, its use is highly problematic. Due to its complex structure, lignocelluose must be aggressively treated to liberate the sugars. The lignocellulosic hydrolysates that are generated contain both hexoses (glucose, mannose, and galactose) and pentoses (xylose and arabinose) as well as significant amounts of microbial inhibitors, such as acetic acid and furfural.
Most researchers have been focused on the construction of single "do it all" organisms that can consume all of the sugars present in lignocellulosic hydrolysates. In general this approach has not worked because different sugars are metabolized at different rates with glucose being consumed first, because glucose can be metabolized more easily than the other sugars. Fermentation processes to produce ethanol using these single "do it all"; organisms take to long to consume all of the sugars to be economically viable. Our laboratory has developed a consortium approach where a collection of isogenic microorganisms, each of which has been designed to consume only one sugar, acts in concert to quickly and efficiently convert all of the sugars present in lignocellulosic hydrolysates into ethanol or other useful biochemicals. We have also extended our substrate-specific approach to engineer microorganisms that can consume the inhibitor acetic acid and detoxify the lignocellulosic hydrolysate without consuming any of the sugars.
Peptide therapeutics are widely used in medicine. Examples of some of the more recognizable peptide drugs include insulin, glucagon, calcitonin, and oxytocin. Numerous experts have touted peptides as the major drug discovery platform of the future thanks to the ease of performing high throughput screens using combinatorial peptide libraries. Because the binding affinity of peptides for their protein targets are equal to those of antibodies, peptide therapeutics have been considered by many to be logical replacements for antibody drugs, especially since peptides would not cause the immune complications that plague antibody drugs. However, peptide drugs are highly problematic due to their short half-lives; peptides are readily degraded by peptidases and proteases. Typically, peptide drugs have to be utilized at higher doses or more frequently administered to counteract this problem.
While peptide modifications, such as acetylation, amidation, or pegylation, can be used to increase the half-lives of peptide drugs, these strategies have not been found to be universally applicable in extending peptide half-lives. We became interested in the problem of peptide degradation and designed an intracellular molecular genetic screen to determine if other modifications might be able to stabilize peptides. Using this screen we discovered that peptides could be stabilized by attaching protein-stabilizing motifs to either end of the peptide. We have shown that a three amino acid XPP stabilizing group, where X is any amino acid, can increase peptide half lives by 2 to 10 fold, while larger stabilizing groups, such as small four helix bundled proteins, can increase peptide half lives by 10 to 100 fold. Current research in our laboratory involves using the protein motifs we discovered to create new peptide drugs.
We have used the protein stabilizing motifs to stabilize a potential peptide therapeutic that was isolated by phage display, which acts by dissociating immune complexes that cause the proliferation of autoimmune diseases. The stabilized peptide, named NB406, outperformed Enbrel, one of the most widely used drugs for the treatment of rheumatoid arthritis, in an in vivo rabbit arthritis model. Future plans include optimizing the stabilization group that is used for NB406 and conducting additional animal models for other autoimmune diseases, such as atherosclerosis.
We have implemented the intracellular genetic screen we developed to create novel peptide antibiotics that can be used to treat Staphylococcal infections. While our genetic screen could be used to engineer novel peptide antibiotics for a number of different microorganisms, identifying new antibiotics for treating Staphylococcal infections was an obvious first choice due to the increasing problem of drug-resistant Staphylococcus sp. Over 20 promising candidates have been isolated. Interestingly, several of these peptides are arginine rich like some of the naturally occurring peptide antibiotics, such as PR-39 and Bac7. We are in the process of synthesizing the most potent inhibitors so their minimal inhibitory concentrations (MICs) can be determined and compared to amoxicillin and vancomycin, the antibiotics that are routinely used to treat Staphylococcal infections.