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Woburn, MA, United States

Modular Genetics, Inc. | Date: 2014-03-14

Engineered polypeptides useful in synthesizing acyl amino acids are provided. Also provided are methods of making acyl amino acids using engineered polypeptides.

Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 750.00K | Year: 2014

This Small Business Innovation Research Phase II project is aimed at optimizing production of a bio-surfactant in preparation for commercial launch of the product. During Phase I, the company developed an engineered microorganism that synthesizes the surfactant, and a key customer confirmed the identity and purity of a sample of the surfactant. During Phase II, synthetic biology methods will be used to increase the efficiency of the microorganism producing the surfactant. In addition, multiple samples of purified surfactant will be shipped to customers for evaluation. Customer feedback will identify any product features that require modification and will result in development of a detailed product specification, which will include metrics such as: purity, color, acceptable variation in composition and molecular weight, etc. The objectives of this Phase II project are to optimize surfactant characteristics and microbial production efficiency so that the surfactant can be profitably manufactured and sold for use in consumer products formulations. The broader impact/commercial potential of this project is that it should enable the company to demonstrate that synthetic biology methods can be used to increase the efficiency of production of a bio-surfactant so the surfactant can be sold as a commercial product. Progress toward that goal should enable the company to attract a partner, for example a large chemical company, who will agree to collaborate on commercialization of the bio-surfactant. If the bio-surfactant can be made and sold profitably, the company will be positioned to fund future research and development aimed at commercial launch of additional bio-surfactants. Benefits to society are that chemicals produced using this technology will be manufactured using domestically grown renewable raw materials, which do not compete with food. Furthermore, the energy required to produce these chemicals is low since the fermentation reaction is performed near ambient temperature. The chemicals are inherently safer than traditional chemicals because toxic solvents are not used, and the surfactants are biodegradable and do not contribute to increased greenhouse gas accumulation. These bio-surfactants will initially be used in personal care products, such as body washes and shampoos. However, the surfactant market is large and diverse, creating an opportunity for use of bio-surfactants in products as varied as laundry detergent, paints and coatings, and floatation-agents used in the mining industry to purify valuable minerals.

Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE I | Award Amount: 225.00K | Year: 2016

The broader impact/commercial potential of this Small Business Innovation Research Phase I project would be commercialization of a surfactant (acyl ethanolamine) made from renewable raw materials, which do not compete with food sources. Surfactants are the bubbly components of cleaning products that give them their cleansing power. Surfactants are manufactured from petroleum or from seed oils, such as palm oil. The use of those raw materials increases greenhouse gas pollution and contributes to deforestation of rainforests. Society is demanding environmentally sustainable (greener) products with reduction or removal of toxicity. The demand for greener chemicals creates an opportunity to replace todays surfactants with greener alternatives. The surfactant chemicals produced in this project are inherently safer than traditional chemicals because toxic solvents are not used, and the surfactants are biodegradable and produced from renewable raw materials such as sugars, and as a result do not contribute to increased greenhouse gas accumulation. The acyl ethanolamine surfactant is designed to replace current commercial surfactants, which are contaminated, during the current manufacturing process, with the carcinogen 1,4 dioxane. The surfactant produced by the effort described here will not contain any 1,4 dioxane. Successful completion of this project will demonstrate a new technology for the production of nonionic surfactants. This is significant since nonionic surfactants represent about 40% of the $30 billion surfactant market.

The technical objective of this Phase I research project is to construct a Bacillus strain that produces an acyl amino alcohol surfactant, namely, acyl ethanolamine. Certain naturally existing peptide synthetase enzymes catalyze the linkage of particular amino acids to other particular amino acids. In addition, certain peptide synthetase enzymes catalyze the linkage of particular fatty acids to particular amino acids. Past work demonstrated that this system can be engineered to catalyze the creation of unique molecules, such as acyl glycinate (fatty acid linked to glycine). During enzymatic synthesis of acyl glycinate, glycine is covalently attached to the synthetase via a thioester bond. Product release is catalyzed by a thioesterse domain. Release by a thioesterase results in production of fatty acid linked to the amino acid glycine. Certain naturally occurring peptide synthetase enzymes use reductase domains to release products. We hypothesize that release of acyl glycine via a reductase domain will result in the synthesis of acyl ethanolamine, rather than acyl glycine. The objective of this Phase I project is to create a chimeric peptide synthetase enzyme that converts acyl glycine into acyl ethanolamine during the process of release of the product from the enzyme.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2003

DESCRIPTION (provided by applicant): Our revised project High Throughput Bioengineering of Detoxification Enzymes focuses on organophosphate hydrolases, which have immediate value for mediation or detection of chemical warfare agents (CW; i.e., nerve gases), and long term value for dealing with contaminating pesticides in humans and the environment. Two candidate organophosphate hydrolases have been structurally determined; a TIM barrel-like, dimeric organophosphate hydrolase (OPH) from Pseudomonas diminuta, and a 13-propeller peptide diisopropylfluoro-phosphatase (DFPase) from Lo/igo vu/garis (squid). Each has some proven, but inefficient, effect against organophosphate CW agents and pesticides, and modification of certain residues has been shown to increase their ability to hydrolyze some of these agents [1, 3, 3b, 11-15]. We propose to use our patented technologies for seamless gene assembly (TOPPs) to generate a large library of designed, substrate-specific substitutions (DPSSs). By making multiple modifications in 24-25 residues shown lining the active sites of both OPH and DFPase [1,2,3], we will generate at least 1,000 modified genes for each in 6 months. By screening the expressed genes for their enzyme kinetics with 3-4 substrates (including pesticides and surrogate CW agents), data will be available for designing new modifications in these sets. Preliminary data have already been integrated into our iterative, primer design, and we are preparing to automate the whole process. Our Phase I work will be to expand enormously the number of mutants available for both enzymes, and we believe this approach is a novel way to produce the desired improvements. Phase II aims will likely include i) verification of enzyme activities in the resource library, ii) further analyses of their stability, optimal conditions of assay and other substrates (by us or by collaborators), and iii) finally adding adaptors and modifications to these enzymes for use as biosensors or as a detoxification and/or decontamination tools.

Marti M.E.,Iowa State University | Marti M.E.,Selcuk University | Colonna W.J.,Iowa State University | Patra P.,Columbia University | And 7 more authors.
Enzyme and Microbial Technology | Year: 2014

•Surfactants were produced microbially on biological substrate and purified to >90% purity.•Surface active properties of biosurfactants indicated they could be utilized as oil dispersants.•Toxicity of biosurfactants to fish larvae in water and in saline varied from moderate to high. Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were ~30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (DOR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10g/L, respectively. However, CMC values were higher, 0.02 and 0.4g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per trillion (ppt) Instant Ocean® sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean® solutions containing 12ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis, which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate. © 2013 Elsevier Inc. Source

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