13-week dietary study and in vitro and in vivo genotoxicity studies of a structuring fat produced through a microalgal fermentation process.

Microalgae are increasingly being utilized as food ingredients for a variety of applications, including as sources of protein, egg and dairy substitutes, and cooking oils. The dietary safety of a new structuring fat produced using a heterotrophic fermentation process by a strain of Prototheca moriformis was evaluated in a 13-week dietary toxicity study and compared with kokum fat, a structuring fat of similar composition used in the food industry and derived from Garcinia indica seeds. The algal structuring fat was evaluated for its genotoxic potential using both in vitro and in vivo assays. No treatment-related adverse events occurred in rats consuming algal structuring fat or kokum fat in the 13-week study; no treatment-related effects were reported for body weight, food consumption, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, or histopathology. While statistically significant effects occurred in some parameters, none were dose-related or considered adverse. Overall, the NOAELs for the algal structuring fat and the kokum fat were 100 000 ppm, the highest concentrations tested. The algal structuring fat was not mutagenic in the bacterial reverse mutation assay in the Salmonella typhimurium or Escherichia coli strains tested and was not clastogenic in the in vivo mouse bone marrow chromosome aberration assay.

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids.

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs.

Enhanced production of heterologous macrolide aglycones by fed-batch cultivation of Streptomyces coelicolor.

A media development program for the enhanced production of macrolide aglycones by Streptomyces coelicolor is described. Shake flask studies utilizing a yeast extract and a bakers' yeast increased production by 200% and 80%, respectively. However, ammonia generation and high pH were identified as potential problems in these enriched media. Studies in pH-controlled fermentors revealed that production stage pH significantly affects macrolide titers, with low pH (5.5) being more productive than high pH (6.5). Implementation of glucose feeding in shake flask cultures reduced ammonia generation and controlled production stage pH, resulting in significantly enhanced productivities. The combined effects of media supplementation and glucose feeding resulted in a three to five-fold overall improvement in total macrolide aglycone titers, and is the first reported high-level (>1 g/l) production of recombinant polyketides in a heterologous host.

Production of a novel FK520 analog in Streptomyces hygroscopicus: improving titer while minimizing impurities.

FK520, also called ascomycin, is an immunosuppressive agent produced by Streptomyces hygroscopicus. Engineering the polyketide synthase genes of the parent strain generated novel FK520 analogs with the potential for improved in vivo stability. By replacing the acyl transferase (AT) domain in the polyketide synthase module 8 with an AT specific for methylmalonyl CoA (the rapamycin AT 3), the strain produced 13-desmethoxy-13-methyl-FK520 (13dmmFK520). Process development and scale-up studies of this recombinant S. hygroscopicus strain producing 13dmmFK520 are described here. Production kinetics and compound stability in fermentation broth were significantly different compared to the native FK520. Fermentation of the new strain resulted in the synthesis of a contaminating substance that co-purified with the 13dmmFK520. To optimize 13dmmFK520 production and to facilitate purification, growth parameters and media development were examined. Although a medium was identified that increased product titers by ca. 300%, the ratio of impurity to product was doubled. Lower dissolved oxygen (20% compared to 50% and 80%) increased titers by 20% with no appreciable effect on the concentration of impurity. Increasing the fermentation pH from 6.0 to 6.5 did not change the 13dmmFK520 titer, but reduced the impurity-to-product ratio by approximately 450%.

Modulation of epothilone analog production through media design.

Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of L-serine with either acetate or propionate yields epothilone H(1) or H(2) as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with a genetically modified epothilone PKS in various media.

Suspension cultivation of Mycobacterium ulcerans for the production of mycolactones.

Mycolactones are polyketide toxins produced by Mycobacterium ulcerans, the causative agent of the tropical skin disease known as Buruli ulcer. Development of novel therapeutic agents from mycolactones has been hindered by the difficulty of producing sufficient amounts of material. Here, we describe the successful adaptation of M. ulcerans to suspension cultivation and the development of a fed-batch fermentation process that was scaled up to 150 l. In addition to producing mycolactones A and B, a number of new mycolactone-related compounds were also observed.

Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.

Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering.

Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

Process development and metabolic engineering for the overproduction of natural and unnatural polyketides.

Polyketide natural products are a rich source of bioactive substances that have found considerable use in human health and agriculture. Their complex structures require that they be produced via fermentation processes. This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production. Classical strain improvement procedures, process development methods, and metabolic engineering approaches are described. The elucidation of molecular mechanisms that underlie polyketide biosynthesis has played an important role in each of these areas over the past few years.

Development and scale-up of a fed-batch process for the production of capsular polysaccharide from Haemophilus influenzae.

The production of the capsular polysaccharide, polyribosylribitolphosphate, from Haemophilus influenzae type b is important for the production of effective conjugate vaccines. Factors limiting the production of this polysaccharide from H. influenzae type b in liquid culture were investigated. A fed-batch fermentation was developed that increased cell density and PRP titer approximately four fold when compared to the batch fermentation. This fed-batch process was successfully scaled from the 1.5 l development scale to the 500 l manufacturing scale. The maximum cell density in the 500 l fermentation was 6 g dry cell weight per liter and the PRP concentration was 1.3 g l(-1).

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogs in Streptomyces coelicolor: understanding precursor effects.

A fermentation process employing precursor-directed biosynthesis is being developed for the manufacture of 6-deoxyerythronolide B (6-dEB) analogues. Through a plasmid-based system in Streptomyces coelicolor, 6-dEB synthesis is catalyzed by 6-dEB synthase (DEBS). 6-dEB synthesis is abolished by inactivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by providing synthetic activated diketides. Because of its inherent catalytic flexibility, the KS1-deficient DEBS is capable of utilizing unnatural diketides to form various 13-substituted 6-dEBs. Here we characterize process variables associated with diketide feeding in shake-flask experiments. 13-R-6-dEB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R-group present in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.

A randomized trial of nortriptyline for smoking cessation.

BACKGROUND: Smoking cessation rates with current therapy are suboptimal. One class of drugs that may improve cessation is the tricyclics. OBJECTIVE: To add nortriptyline hydrochloride to a behavioral smoking cessation program to enhance cessation rates and reduce withdrawal symptoms. SUBJECTS AND METHODS: We conducted a randomized, double-blind, placebo-controlled trial at an affiliated Department of Veterans Affairs Medical Center and an Army Medical Center. Subjects were aged 18 through 70 years, smoked 10 or more cigarettes per day, and were without current major depression. Nortriptyline hydrochloride or matched placebo was started at 25 mg before bed 10 days prior to quit day and titrated to 75 mg/d or to the maximal tolerated dose. The behavioral intervention consisted of 2 group sessions and 12 individual follow-up visits. Withdrawal symptoms were measured using a daily diary, and smoking cessation was defined as self-reported abstinence, expired carbon monoxide of 9 ppm or less, and a 6-month urine cotinine level of less than 50 ng/mL. RESULTS: A total of 214 patients were randomized (108 to nortriptyline and 106 to placebo). There was a significant reduction in several withdrawal symptoms including anxious/tense, anger/irritability, difficulty concentrating, restlessness, and impatience by day 8 after quit day in the nortriptyline group. The cessation rate at 6 months was 15 (14%) of 108 and 3 (3%) of 106, respectively (P = .003; absolute difference, 11%; 95% confidence interval, -18% to -4%). Nortriptyline caused frequent adverse effects, including dry mouth (64%) and dysgeusia (20%). CONCLUSIONS: We conclude that nortriptyline led to an increased short-term cessation rate compared with placebo. In addition, there were significant, but relatively small, reductions in withdrawal symptoms. Nortriptyline may represent a new therapeutic approach to smoking cessation.

Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity.

Four different insect cell lines that can be used as hosts for baculovirus infection were assayed for the presence of endogenous exoglycosidases. All four cell lines, derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, or Malacosoma disstria, contained N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, and sialidase activities. Exoglycosidase activities were found in cell lysates as well as cell-free supernatants from uninfected and wild-type baculovirus infected cells. Oligosaccharide analysis of cellular glycoproteins using lectins recognizing Gal beta 1, 3GalNAc, Gal beta 1, 4GlcNAc, and NeuAc alpha 2,6Gal demonstrated that only Gal beta 1,3GalNAc was present. The demonstration that these cells contain exoglycosidases raises the possibility that the oligosaccharides of baculovirus-expressed glycoproteins are subject to enzymatic degradation.

Health care costs of veterans with multiple sclerosis: implications for the rehabilitation of MS. VA Multiple Sclerosis Rehabilitation Study Group.

We retrospectively determined health care costs among veterans with multiple sclerosis (MS) and correlated the costs with neurologic dysfunction. Total health care costs for the 165 patients averaged $35,000/year. VA benefits and homecare together accounted for 85% of the total costs. Total health care costs correlated with two measures of neurologic dysfunction, the Expanded Disability Status Scale (EDSS) (r = 0.61, p < 0.001) and the Incapacity Status Scale (ISS) (r = 0.64, p < 0.001). The costs of VA benefits, homecare, and hospitalizations also correlated with the EDSS, ISS, and other measures of neurologic dysfunction whereas the cost of outpatient clinic visits did not. In a period of three years, there were 40 hospitalizations, at a total cost of $412,800, that were potentially preventable with appropriate outpatient management. Improving selfcare and avoiding preventable hospitalizations might lower the considerable health care costs of MS.

Production of a discrete, heterogeneous population of beta-galactosidase polypeptides using baculovirus expression vectors.

Gel electrophoresis analysis of immunoprecipitated beta-galactosidase and polyhedrin-beta-galactosidase expressed in Spodoptera frugiperda cells infected with recombinant Autograph californica nuclear polyhedrosis virus revealed the existence of a population of discrete beta-galactosidase polypeptides. Several of the polypeptides observed in the fusion protein expression experiments exhibit a consistent pattern of slightly greater molecular weight when compared to the nonfusion beta-galactosidase that is compatible with the hypothesis that these fusion protein fragments retain the N-terminal polyhedrin residues. Pulse-chase experiments showed that overall beta-galactosidase degradation occurred at a negligible rate compared to the synthesis rate at 96 h postinfection, yet the fragments are observed for short pulse times. Degradation of several different beta-galactosidase polypeptides was observed 24 h postinfection. Ribonucleic acid hybridization analysis of lacZ transcripts shows significant heterogeneity that may result from premature transcription termination. Although a proteolytic origin cannot be excluded, the data assembled suggest that premature termination of transcription or translation is the likely cause for the heterogeneous population of immunoreactive peptides observed. Many discrete forms of beta-galactosidase polypeptides were also observed in studies with Escherichia coli, indicating that production of these heterogeneous forms is not a consequence of heterologous expression of the enzyme.

Modeling the population dynamics of baculovirus-infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields.

The insect cell-baculovirus model presented here is capable of simulating cell population dynamics, extracellular virion densities, and heterologous product titers in reasonable agreement with experimental data for a wide rang of multiplicities of infection (MOI) and times of infection. The model accounts for the infection of a single cell by multiple virions and the consequences on the time course of infection. The probability of infection by more than one virion was approximated using the Poisson distribution, which proved to be a refinement over second-order kinetics. The model tracks initiation and duration of important events in the progression of infected cell development (virus replication, recombinant protein synthesis, and cell lysis) for subpopulations delineated by the time and extent of their initial infection. The model suggests infection strategies, weighing the importance of MOI and infection time. Maximum product titers result from infection in the early exponential growth phase with low MOI.

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.

Factors influencing recombinant protein yields in an insect cell-bacuiovirus expression system: multiplicity of infection and intracellular protein degradation.

The insect cell (Sf9)-baculovirus (AcNPV) expression system was employed for the synthesis of beta-galactosidase, a model heterologous protein. In the recombinant virus studied, the lacZ gene is fused to a portion of the polyhedrin structural gene and is under the control of the polyhedrin promoter. The effect of the multiplicity of infection (MOI) on product titer was determined by infecting cells with MOI values ranging from 0 to 100 and monitoring the production of beta-galactosidase with time. The relationship between final product titer and MOI was dependent on the growth phase of the cells prior to infection. The final product titer from cells infected in the early exponential phase was relatively independent of MOI. For cells infected in late-exponential phase there was a logarithmic relationship between the final beta-galactosidase titer and the MOI used, with the highest MOI studied resulting in greatest protein synthesis. The synthesis and degradation rates of beta-galactosidase were investigated by a pulse-chase technique using L-[(35)S]-methionine. At 24 h postinfection, the degradation rate is of the same order of magnitude as the synthesis rate. However, the synthesis rate of beta-galactosidase increases dramatically at 96 h postinfection. During this later period, the degradation rate is negligible. Although degradation of recombinant protein occurs in this system, degradation activity declines as infection proceeds and is insignificant late in intention when recombinant protein expression is intense.

The effect of pH on the production of pertussis toxin by Bordetella pertussis.

The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields.