Armand B. Pepperman

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

Abstract. The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid β-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 (FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid.

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA3). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.

Release of atrazine and alachlor from clay‐oxamide controlled‐release formulations

Controlled-release herbicide formulations have been shown to decrease the leaching potential of several herbicides under laboratory and -eld conditions. The utility and efficacy of these formulations may be improved by combining several herbicides and a fertilizer source in a single formulation. The objective of these studies was to develop granular alginate formulations that were composed of a combination of the herbicides atrazine and alachlor with the slow-release nitrogen source oxamide (ethanediamide). Controlled release of the herbicides was obtained by addition of selected minerals, including calcium bentonite, -ne- grind bentonite, montmorillonite K10, kaolinite and iron (III) oxide. A formula- tion without clay was used as a comparison. The formulations tested had herbicide active ingredient contents ranging from D0E02 to 0E54% and a nitro- gen content of 21%. Release of the herbicides was studied by equilibrating the formulations with deionized water on a rotary shaker at 200 rev min~1 and sam- pling at regular time intervals up to 104 hours. The minerals used in the di†erent formulations inNuenced the herbicide active ingredient composition, as well as the release properties of the individual formulations. The atrazine content of the formulations decreased in the order calcium bentonite ( -ne-grind bentonite ( kaolinite ( montmorillonite \ iron oxide ( no clay. For alachlor the content decreased in the order of calcium bentonite ( -ne-grind bentonite ( montmorillonite ( iron oxide ( kaolinite ( no clay. Controlled release of atrazine (i.e. reduction in release rate) varied in the order calcium bentonite ( iron oxide ( montmorillonite ( -ne-grind bentonite \ kaolinite ( no clay, and for alachlor -ne-grind bentonite ( calcium bentonite ( montmorillonite ( no clay \ kaolinite \ iron oxide. A certain per- centage of the applied active ingredient of both alachlor and atrazine was not recovered. From 5 to 27% of the active ingredient was not released, with the greatest retention by the bentonite formulations. Release of nitrogen was not strongly inNuenced by mineral type, although a trend indicated greater release with formulations containing kaolinite. 1998 SCI ( Pestic. Sci., 53, 233E240 (1998)

Comparison of the seed germination effects of synthetic analogs of strigol gibberellic acid, cytokinins, and other plant growth regulators

Four synthetic multiring analogs of strigol, a naturally occurring sesquiterpene lactone that promotes germination of dormant seeds ofStriga (witchweed), were found to stimulate germination of dormantLactuca (lettuce) seeds. The effects on light-sensitive and light-insensitive lettuce seeds were concentration-dependent and exceeded those produced by equimolar (0.1 mM) solutions of gibberellic acid. Strigol and epistrigol promoted lettuce seed germination to a lesser degree than did the synthetic analogs. The strigol group compounds had minimal effect on the germination of monocot seeds. The results indicate that the synthetic strigol analogs have plant growth regulatory activity in dormant seeds of genera beyondStriga in which germination stimulation by strigol and the synthetic analogs was first demonstrated.

Mobility of atrazine from alginate controlled release formulations

Abstract Controlled release (CR) formulations can significantly influence the fate of herbicides in the environment. The influence of alginate‐encapsulated CR formulations on the mobility of atrazine (6‐chloro‐N‐ethyl‐N´‐ (l‐methylethyl)‐1,3, 5‐triazine‐2,4‐diamine) in two soil profiles was investigated. Two CR formulations were evaluated based on Na‐alginate (1%), kaolin (10%) and Tween 20 (0.5%). The first formulation (CR‐Oil) was prepared with linseed oil (4%) and the second formulation (CR‐N) did not have oil. These formulations were compared to technical grade (TG) atrazine and to a commercial liquid (CL) formulation (Aatrex 4L). Formulations labeled with 14C were applied to packed soil columns containing a surface and a subsoil horizon. The columns were then leached with 3‐cm of 0.01 M CaCl2 per day at a rate of 1.5 cm hr‐1 for a period of ten days. At the termination of the experiment the columns were sectioned in 5‐cm increments and extracted with MeOH. Leachate samples and extracts were analyzed ...

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica).

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.

Physicochemical Properties of Starch in Extruded Rice Flours

ABSTRACT The effects of extruding temperatures and subsequent drying conditions on X-ray diffraction patterns (XRD) and differential scanning calorimetry (DSC) of long grain (LG) and short grain (SG) rice flours were investigated. The rice flours were extruded in a twin-screw extruder at 70–120°C and 22% moisture, and either dried at room temperature, transferred to 4°C for 60 hr, or frozen and then dried at room temperature until the moisture was 10–11%. The dried materials were milled without the temperature increasing above 32°C. XRD studies were conducted on pellets made from extruded and milled flours with particle sizes of 149–248 μm; DSC studies were conducted from the same material. DSC studies showed that frozen materials retrograded more than the flours dried at room temperature. The LG and SG samples had two distinct XRD patterns. The LG gradually lost its A pattern at >100°C, while acquiring V patterns at higher temperatures. SG gradually lost its A pattern at 100°C but stayed amorphous at the...

Identification of a subtilisin-like protease in seeds of developing tung fruits

Summary A subtilisin-like protease was identified in the low-density microsomal fraction of developing tung seeds. The abundance of the protease changed significantly throughout seed development and showed a direct temporal correlation with accumulation of storage oil. Once storage oil synthesis was complete, the abundance of the protease decreased dramatically. Determination of the N-terminal amino acid sequence revealed that the protease sequence began just after a conserved pro-domain cleavage site, suggesting that the protease was fully active in developing tissue. Extraction of microsomal membranes with high salt or pH demonstrated that the protease was not a membrane-anchored protein.

On the extraction of oil from raw comminuted cottonseed kernels with the acetone-hexane-water azeotrope

The effects of particle size on the rate of ex-traction of oil from raw comminuted cottonseed kernels with the acetone-hexane-water azeotrope were examined. Five samples of cottonseed of different average particle sizes and three extrac-tion times were used in the investigation. The particles were comminuted in such a manner as to be, as a first approximation, essentially spher-ical in shape ; the concentration gradient of oil in the particles was found to follow the relationship L =r-A +B where L is the oil content of the extracted seed, r is the particle radius, and A and B are constants. The establishment of the concentration gradient was extremely rapid (within 10 seconds), and the constants A and B assume the same values for each of the three extraction times employed. These two facts in-dicate that mass oil movement in the particles is a dilatation and occurs too rapidly for diffusion phenomena to be evident.