Roberto E. Armenta

Use of raw glycerol to produce oil rich in polyunsaturated fatty acids by a thraustochytrid

Glucose is the typical carbon source for producing microbial polyunsaturated fatty acids (PUFA) with single cell microorganisms such as thraustochytrids. We assessed the use of a fish oil derived glycerol by-product (raw glycerol), produced by a fish oil processing plant, as a carbon source to produce single cell oil rich in polyunsaturated fatty acids (PUFA), notably docosahexaenoic acid (DHA). These results were compared to those obtained when using analytical grade glycerol, and glucose. The thraustochytrid strain tested produced similar amounts of oil and PUFA when grown with both types of glycerol, and results were also similar to those obtained using glucose. After 6 days of fermentation, approximately 320 mg/g of oil, and 145 mg/g of PUFA were produced with all carbon sources tested. All oils produced by our strain were 99.95% in the triacylglycerol form. To date, this is the first report of using raw glycerol derived from fish oil for producing microbial triglyceride oil rich in PUFA.

Stability studies on astaxanthin extracted from fermented shrimp byproducts.

To the best of our knowledge, stability studies on astaxanthin contained in carotenoproteins extracted from lactic acid fermented shrimp byproduct have never been reported. Carotenoprotein powder, containing 1% free astaxanthin, was subjected to oxidation factors of illumination, oxygen availability, and temperature, using synthetic astaxanthin as a control. The individual effects as well as first and second degree interactions were studied on natural and synthetic free astaxanthin stability. Air and full light were the two individual factors with the highest effects on astaxanthin oxidation. Sixty-two and 46% natural and synthetic astaxanthin, respectively, oxidized when exposed to air for 8 weeks of storage, whereas 35 and 28% of natural and synthetic astaxanthin, respectively, oxidized under full light. Ninety-seven and 88% of natural and synthetic astaxanthin, respectively, oxidized under a combination of full light, air, and 45 degrees C at 8 weeks of storage. Storage in the dark, nonoxygen, and 25 degrees C were the treatments that efficiently minimized astaxanthin oxidation. Natural astaxanthin from fermented shrimp byproduct presented moderate stability levels. Although natural astaxanthin oxidized faster than the synthetic pigment, its stability may improve by antioxidant and polymer addition.

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass.

A High-Throughput Screen for the Identification of Improved Catalytic Activity: β-Carotene Hydroxylase

Astaxanthin is a natural product of immense value. Its biosynthesis has been investigated extensively and typically requires the independent activity of two proteins, a β-carotene ketolase and β-carotene hydroxylase. Rational engineering of this pathway has produced limited success with respect to the biological production of astaxanthin. Random mutagenesis of the β-carotene ketolase has also been pursued. However, to date, no suitable method has been developed for the investigation of the β-carotene hydroxylase because β-carotene and zeaxanthin cannot be differentiated visually, unlike β-carotene and canthaxanthin. Thus, random mutagenesis and efficient selection of improved β-carotene hydroxylase clones is not feasible. Presented here are the steps required for the efficient generation of a β-carotene hydroxylase random mutagenesis library in Escherichia coli. Subsequently presented is a novel high-throughput screening method for the rapid identification of clones with enhanced β-carotene hydroxylase activity. The validity of the presented method is confirmed by functional expression of the mutated proteins, combined with accurate quantification of produced carotenoids. The developed method has potential applications in the development of biological systems for improved carotenoid biosynthesis, as well as robust astaxanthin production.