2012 Annual Report
1a.Objectives (from AD-416):
The long term goal of the project is to develop commercially viable integrative bioprocesses encompassing biological and bio/chemical technologies to produce environmentally friendly biobased materials and chemicals using agricultural fats (i.e., tallow, lard, chicken fats, etc.), oils (i.e., soybean oil, cuphea oil, sunflower oil, etc.) and coproducts as feedstocks.
1: Develop biocatalysts and bioprocesses that enable the commercial production of new biobased products from agricultural lipids and related coproducts such as bioglycerol.
1A: Improve the efficiency of feedstocks utilization and enable the production of new varieties of bioproducts in high yields through metabolic engineering of microorganisms.
1B: Systematic optimization of bioprocesses to improve yields and economics of biobased products.
2: Develop new commercially viable applications for sophorolipids, rhamnolipids, cyanophycin and gamma-polyglutamic acid.
2A: Increase the values of product components.
2B: Develop biomaterials for end-use products.
1b.Approach (from AD-416):
Microorganisms capable of producing new variants of rhamnolipids (RLs) and sophorolipids (SLs) will be created by introducing mutated genes that originally encode the synthesis of native RL and SL. Increasing the RL yields in non-pathogenic P. chlororaphis by metabolic engineering will be attempted by increasing the copy-number and transcription level of the responsible genes (i.e., rhlAB), or by ‘knocking out’ the gene(s) responsible for diverting precursors away from RL biosynthesis. We will also enhance the value of P. chlororaphis by introducing another gene, rhlC, to allow the biosynthesis of dirhamnose lipids. The efficiency of RL- and SL-producing microorganisms to utilize soy molasses (SM) will be improved by introducing genes encoding enzymes that breakdown the complex sugars in SM. We will also express in Candida bombicola (an SL-producing yeast) and P. chlororaphis the genes that are used for glycerol metabolism to improve the organisms’ efficiency of using bioglycerol. To enhance colonization and biofilm formation for higher RL yields from P. chlororaphis, we will experiment with attaching a sponge-like adapter to the impellers of a fermenter and then use minimal aeration (slow impeller rotation, reduced air introduction, or both). Separately, process optimization will be carried out for the fermentative synthesis of SL, gamma-polyglutamic acid (PGA) and cyanophycin (Cp) using such low-cost feedstocks as crude glycerine from biodiesel production, soy molasses and meat and bone meal hydrolysates. Fermentation parameters such as carbon source content and concentration, feed rate, aeration, pH etc. will be varied and optimized using Response Surface Methodology (RSM). To increase the values of bio-product subcomponents or for application in end-use products, biological and chemical syntheses will be performed to obtain the followings for subsequent characterization: .
1)novel epoxy and polyhydroxy fatty acids by using functionalized fatty acids (i.e., vernolic acid, ricinoleic acid, lesquerellic acid) as fermentative substrates,.
2)cyanophycin-derived biosurfactants by linking fatty acids to the polar dipeptide unit of Cp,.
3)terminally unsaturated fatty acids by elimination reactions on hydroxy fatty acids,.
4)biopolymer/biosurfactants composites by solution-casting of PHA and SL or RL,.
5)oligoamides by converting the hydroxy fatty acids to amino fatty acids and self-coupling them, and.
6)water-soluble SLs by linking charged amino acids to the sugar headgroups.
Progress was made toward achieving the project objectives of developing commercially viable biocatalysts, bioprocesses, and new biobased products through genetic improvement, fermentation manipulation, and value-added chemical modification of biodetergents and bioplastics. To this end, project scientists synthesized biobased materials possessing detergent property by linking together the components of a “green” bioproduct called cyanophycin and the common agricultural products (i.e., fatty acids); produced a bioplastic (i.e., gamma-PGA) by fermentation to be characterized and sent to a collaborator at NCAUR for reaction with starch to produce new bioplastics; produced new sophorolipid biodetergents from oxygen-enriched fatty acids for structural and detergency characterization; collaboratively developed a bioprocess model of an industrial scale fermentative production of biodetergent (i.e., sophorolipids) from common agro-feedstocks (i.e., glucose and sunflower oil); introduced genes (called alpha-galactosidases) capable of breaking down the sugars of soy molasses into the bioplastic- and biodetergent-producing bacteria to make them grow better in soy molasses; and continued the cloning from a little-known yeast of a new gene capable of linking a sugar and a fatty acid together to make biodetergents.
Genes useful for making biodetergents. Biodetergents such as sophorolipids and rhamnolipids can be produced by microorganisms using renewable feedstocks from agriculture. They are biodegradable, eco-friendly, and sustainable. There is a need, however, to make them cheaper and functionally better. One way to achieve these goals is to genetically engineer microorganisms for high-yield production and/or new product capability. To this end, genes responsible for making the biodetergents are required. ARS researchers at Wyndmoor, Pennsylvania have cloned these genes from selected bacteria and yeast. In one case, one such gene is introduced into a non-pathogenic bacterium resulting in the production of a modified biodetergent. In another instance, a different gene from yeast was cloned, characterized, and put in another common yeast to make biodetergent and sugar-sterol complexes with nutraceutical potential. Invention disclosures had been approved by ARS National Life Sciences Patent Committee for the filing of two (2) patent applications, and manuscripts had been prepared pending submission per OTT release.
Ashby, R.D., Solaiman, D., Strahan, G.D., Zhu, C., Tappel, R.C., Nomura, C.T. 2012. Glycerine and levulinic acid: renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers. Bioresource Technology. 118:272-280.
Solaiman, D., Ashby, R.D., Zerkowski, J.A. 2012. Substrate preference and oxygen requirement for cyanophycin synthesis by recombinant Escherichia coli. Biocatalysis and Agricultural Biotechnology. 1(1):9-14.
Lee, J.H., Ashby, R.D., Needleman, D.S., Lee, K., Solaiman, D. 2012. Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans. Applied Microbiology and Biotechnology. DOI: 10.1007/s00253-012-4133-x.
Zerkowski, J.A., Solaiman, D. 2012. Omega-functionalized fatty acids, alcohols, and ethers via olefin metathesis. Journal of the American Oil Chemists' Society. 89(7):1325-1332.