Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum
Alsaker K.V.,Northwestern University |
Alsaker K.V.,Microbia |
Paredes C.,Northwestern University |
Papoutsakis E.T.,Northwestern University |
Papoutsakis E.T.,University of Delaware
Biotechnology and Bioengineering | Year: 2010
Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional-stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cell physiology. Ontological analysis demonstrated that stress by all three metabolites resulted in upregulation of genes related to post-translational modifications and chaperone activity, and downregulation of the translationmachinery genes. Motility genes were downregulated by acetate-stress only. The general metabolite stress included upregulation of numerous stress genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated expression of the butyryl-CoA- and butyrate-formation genes, while butyrate stress downregulated expression of acetate-formation genes. Pyrimidine-biosynthesis genes were downregulated by most stresses, but purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for thiamine and histidine biosynthesis. Methionine-biosynthesis genes were upregulated by acetate stress, indicating a possibly conserved stress response mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression was upregulated by acetate stress. Butyrate stress upregulated many iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related to cellular repair from oxidative stress, such as perR and superoxide dismutases. Butanol stress upregulated the glycerol metabolism genes glpA and glpF. Surprisingly, metabolite stress had no apparent effect on the expression of the sporulation-cascade genes. It is argued that the list of upregulated genes in response to the three metabolite stresses includes several genes whose overexpression would likely impart tolerance, thus making the information generated in this study, a valuable source for the development of tolerant recombinant strains. © 2009 Wiley Periodicals, Inc.
Zelle R.M.,Technical University of Delft |
Harrison J.C.,Microbia |
Pronk J.T.,Technical University of Delft |
Van Maris A.J.A.,Technical University of Delft
Applied and Environmental Microbiology | Year: 2011
Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2. The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylasenegative (Pyc-) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvatecarboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc- S. cerevisiae background. When PDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO2 atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h-1). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2δ pdc2δ strain grown under a CO2 atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C4-dicarboxylic acid production. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
News Article | September 22, 2010
The chemical giant Royal DSM has agreed to purchase the Lexington, MA-based bio-manufacturing firm Microbia from Ironwood Pharmaceuticals, according to a press release. DSM, based in Heerlen, The Netherlands, has not disclosed terms of the deal. Xconomy was unable to immediately reach representatives from Microbia and DSM about the transaction. Microbia (formerly Microbia Precision Engineering) designs microbes used to make chemicals from renewable sources as opposed to traditional petroleum-based methods. Despite its innovative bio-manufacturing technology and its collaborations with major companies such as DuPont (NYSE:DD) and Teva Pharmaceutical Industries (NASDAQ:TEVA), Microbia hasn’t managed to become profitable. Microbia spun out from Cambridge, MA-based Ironwood (NASDAQ:IRWD) in 2006 as an independent subsidiary of Ironwood. Ironwood has since been a majority owner of Microbia, though it sold a minority stake in the business in September 2006 to the British food and beverage ingredients maker Tate & Lyle. “We are thrilled to join DSM. Being part of a large global organization, and a world leader in industrial biotechnology, vitamins and carotenoid development, manufacturing and sales will expedite our ability to leverage our proprietary bio-based technology platform,” Kevin Madden, the chief scientist at Microbia, said in a statement. Ironwood’s sale of the Microbia subsidiary to DSM might be healthy for its own bottom line. Microbia’s unprofitable business was blamed for $1 million in losses on Ironwood’s balance sheet in 2009, according to its annual financial statement. DSM said the purchase of Microbia advances its efforts to develop natural carotenoids, which are valuable compounds used as ingredients for foods and nutritional products. Ironwood, which went public in January 2010, is dedicated to developing drugs such as its lead compound linaclotide for irritable bowel syndrome, while its Microbia subsidiary has been focused on providing microbes and bioprocessing technology to chemical and drug companies and developing natural carotenoids internally. We’ll update this story when we learn of DSM’s plans for Microbia.
News Article | February 23, 2009
Microbia envisions a future in which specialty chemicals we take for granted, like the beta-carotene that goes in dietary supplements, will come from renewable sources instead of the usual petrochemicals. It won’t wean the world off oil, but it could enable this Lexington, MA-based company and its partners to claim they’re helping to green up the planet, all the while pursuing a $200 million market opportunity. I heard the Microbia story last week during a conversation with CEO Richard Bailey. It’s kind of a twisting tale that begins with basic research at the Whitehead Institute for Biomedical Research in Cambridge and gave birth to a couple different companies along the way. Before diving into all that, here’s the gist: Microbia now fashions itself as an industrial biotech company that has found ways to finesse yeast, bacteria and other fermentation materials into pumping out big-time yields of special chemicals. These new versions of carotenoid chemicals, like beta-carotene and canthaxanthin (used to make cheese look orange and salmon look pink, among other things), come from renewable sources instead of petrochemical derivatives. The first two renewable products could arrive on the market in 2010 and generate as much as $200 million in revenue within the next three years, Bailey says. And he thinks Microbia can do it more cheaply, as long as oil is $50 a barrel or higher. (The price dipped to $39 a barrel a couple days after we talked.) “Petroleum won’t be here forever,” Bailey says. “Green chemistry does sell, but it has to come at price parity,” to the existing methods. How Microbia has gotten itself into this position is an interesting story about how science can lead down unpredictable paths. It was started in 1998 by scientists at the Whitehead Institute. The early work was heavy on how yeast strains become pathogenic, or diseased. This led to insights into how to turn yeast into a cheap, effective factory for all sorts of chemicals, Bailey says. The company put that knowledge to work via fee-based contracts with other companies, which reduced its cash burn for years, while another side of Microbia focused on using the knowledge to develop its own drugs. Bailey, a veteran of Monsanto’s nutrition division, came on board as general manager to run the industrial side of the company back in 2002. At that time, the place had great science but needed some business strategy, in his view. “There was a lot of work on science; I’d argue too much,” Bailey says. By 2006, as the drug development programs approached the hugely expensive pivotal trial stage, the industrial contracts couldn’t pay the bills anymore. “Even if we got paid by the wheelbarrow of cash every day, it wouldn’t be sufficient,” Bailey says. So management … Next Page »
News Article | March 17, 2015
Montreal, QC – Prevetec Microbia announced a new $4.7 million financing round on March 12. Telesystem, Groupe Jafaco Gestion, and Desjardins-Innovatech came in with 36% of the funding while the remaining 64% was brought in by the Canadian venture capital fund, VVC, which focuses on animal health. The company develops and markets products that help prevent bacterial infections and improve performance in food animal production. “We wish to thank our long-time shareholders for their support in this key phase of our Company’s commercial development and welcome our new investor. Our mission is to bring multiple innovative products to market in the coming years and collaborate efficiently with our key partners,” said Michel Fortin, President and CEO of Prevtec. Prevtec was founded by Dr John M. Fairbrother and Dr Eric Nadeau as an offshoot company of the Faculté de medicine vétérinaire at the Université de Montréal, where Dr Fairbrother still works. The company strives to be a world leader in the development and commercialization of antibiotic alternatives that prevent bacterial infection and improve food animal performance. Prevtec plans to use the most recent round of funding in the European certification process, in order to follow through with its strategic plan to have 4 of its products certified and on sale in 12 countries and on 3 continents by 2015. Prevtec’s centerpiece is a live bacterial swine vaccine called Coliprotec®F4. It is an oral vaccine, pending market authorization, that the company says helps prevent post-weaning diarrhea in pigs caused by the E.coli bacteria. This is first vaccine of its kind fully developed and produced in Canada. In the last decade, post-weaning diarrhea in piglets has caused a multitude of problems. It delays growth, is harmful to performance and can cause a mortality rate of up to 20% in affected herds. This all can lead to high costs for swine producers, not least of which come in the form of antibiotic injections into the animals. Prevtec’s vaccine can be administered orally and reduces stress on the animal. Also, when taking into account the fact that consumers and health professionals are ever more wary of the use of antibiotics in food production, the playing field may be ripe for Prevtec’s invention.