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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
The overall objective is to develop fermentation-based bioprocess systems that utilize the renewable agricultural fats, oils and coproducts (AFOC) as feedstocks to ultimately produce value-added bioproducts with enhanced properties and environmental benefits. Specific objectives are to expand the list of bioproducts producible via the fermentation of AFOC, to increase yields, expand variety, and improve properties of the target bioproducts via strain improvement, fermentation manipulation, and post-production modification; and to develop end-product applications for the bioproducts.

1b.Approach (from AD-416)
The capability of various AFOC to support cell growth of microorganisms that produce the target bioproducts -- rhamnolipids (RL), sophorolipids (SL), poly(hydroxyalkanoates) (PHA), gamma-polyglutamic acid and cyanophycin -- will be investigated. Genes needed for efficient production of bioproducts from AFOC will be identified, cloned, characterized and expressed. Chimeric genes and mutants having novel substrate specificity will be generated from PHA synthase genes with different PHA compositional profiles. Genes of enzymes or regulatory proteins involved in the biosynthesis of SL in Candida and RL in Pseudomonas will be cloned and characterized for subsequent protein and metabolic engineering to improve product variety and yield. Fed-batch and continuous culture techniques will be explored to increase the yields of bioproducts from fermentation of AFOC. Sophorolipids will be used to prepare new materials such as gemini surfactants, polymers of SL, value-added fatty acids, and bolaamphiphiles. Reactive functional groups, especially amino groups, will be incorporated to the sidechains of PHA for altered property and subsequent derivatization.

3.Progress Report
Project scientists had variously devised a method to attach charged groups to sophorolipid (SL) biosurfactants derived from fats and oils, thereby increasing the water solubility of the SL by more than 10-fold with no loss of surfactant ability; improved a fermentation protocol to use biodiesel-derived crude glycerol coproduct streams as feeds to produce poly(3-hydroxybutyrate) (PHB) bioplastic at greater than 1 g/L product yields in a shorter timeframe than a previous method; determined that adding levulinic acid - a byproduct of the pulp and paper industry that is typically derived from woody biomass - as a co-substrate with glycerol resulted in the production of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB/V) bioplastic that has superior structural properties to PHB; developed a MALDI/TOF-based analytical method to determine sequence distribution of the repeat-units in poly(hydroxyalkanoate) (PHA) biopolyesters; studied the effects of substrates (glucose versus glycerol) and oxygen content on the production yields of cyanophycin (a poly(amino acids)) biopolymer; continued to construct and test different fusion genes of poly(hydroxyalkanote) biosynthesis to achieve product variability and an improved product yield; subcloned and evaluated expression in Saccharomyces cerevisiae (a common baker’s yeast) of a potential biosynthesis gene for sophorolipid biosurfactants.

1. Modified the property of a biofuel-related bacterium through gene insertion: Ralstonia eutropha (also variously known as Cupriavidus necator, Wautersia eutropha, Hydrogenomonas eutropha, and Alcaligenes eutrophus) is a versatile microorganism being studied for biosynthesis of biodegradable plastic, production of bioenergy, and bioremediation of toxic chemicals. Only a limited number of inefficient and at times cumbersome gene-transfer methods, however, are available for genetically improving the biological properties of this organism towards these applications. To overcome this limitation, scientists at Eastern Regional Research Center, Wyndmoor, PA and at R.W. Holley Center for Agriculture and Health, Ithaca, NY collaborated to develop a gene-transfer method useful for the expression of foreign genes in R. eutropha. The scientists screened several pieces of DNA from a Pseudomonas bacterium and identified one DNA fragment (called P2 promoter) that can direct the activation (i.e., expression) of a foreign gene in R. eutropha and many other Pseudomonas bacteria. Requests and MTAs have been processed to transfer this technology to researchers in biotechnology, biobased products, and bioenergy fields soon after its publication.

Review Publications
Ashby, R.D., Ngo, H., Solaiman, D., Strahan, G.D. 2009. Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13- methyltetradecanoic acid and mixed isostearic acid isomer substrates. Applied Microbiology and Biotechnology. 85:359-370.

Zerkowski, J.A., Nunez, A., Strahan, G.D., Solaiman, D. 2009. Clickable Lipids: Azido and Alkynyl Fatty Acids and Triacylglycerols. Journal of the American Oil Chemists' Society. 86(11):1115-1121.

Solaiman, D., Swingle, B.M. 2010. Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomads. New Biotechnology. 27(1):1-9.

Ashby, R.D., Solaiman, D. 2010. The influence of increasing media methanol concentration on sophorolipid biosynthesis from glycerol-based feedstocks. Biotechnology Letters. 32:1429-1437.

Solaiman, D., Swingle, B.M., Ashby, R.D. 2010. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha. Journal of Microbiological Methods. 82:120-123.

Last Modified: 10/13/2015
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