Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

Starter culture for the production of ‘soyiru’

Bacillus subtilis or licheniformis facilitated production of soyiru with the best results being given by using both together. Fermentation employing Streptococcus enterococcus was unsuccessful.H.A. Suberu is with the Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria. J.A. Akinyanju is with the Department of Biological Sciences, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria.     

Characteristics of an aqueous two-phase system for α-amylase production

The characteristics of an aqueous two-phase system for the overproduction of extracellular enzyme through α-amylase fermentation by Bacillus amyloliquefaciens were investigated. With higher molecular weight of polyethylene glycol (PEG) or lower molecular weight of dextran, the partition coefficient of α-amylase was increased. α-Amylase biosynthesis was increased when PEG 6000 was included in the medium compared to the medium without PEG. Phosphate addition to the PEG-dextran system improved the partition coefficient of α-amylase, but deactivated α-amylase severely. By using sodium sulfate instead of phosphate, α-amylase deactivation was negligible, and high partitioning of the enzyme in the top phase was obtained.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Comparison of expression systems for the production of human interferon-α2b

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 μg/L of culture, while the amount of nuclear expression in the plant was 4.46 μg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Bioengineering of microorganisms for C₃ to C₅ alcohols production

Production of renewable fuels and chemicals is an absolute requirement for the sustainability of societies. This fact has been neglected during the past century as cheap and abundant, yet not renewable, sources of hydrocarbons were available. Since fossil fuel availability is decreasing, biological production of fuels and chemicals has been proposed to be a potential alternative to fossil sources. Higher alcohols (from C₃ to C₅) are useful substitutes for gasoline because of their high energy density and low hygroscopicity and are important feedstocks for other chemicals. Some Clostridia species are known to naturally ferment sugars to isopropanol and 1-butanol. However, other C₃ to C₅ alcohols are not produced in large quantities by natural microorganisms. A non-fermentative strategy to produce a broad range of higher alcohols has been devised using the ubiquitous keto acid biosynthetic pathways. This review provides a current overview of these different strategies.     

The production of papain—an agricultural industry for tropical America

In tropical America there is very little interest in the cultivation ofCarica papaya for the production of papain. This article, dealing primarily with field production and processing of crude papain, demonstrates that where coffee is grown commercially, papain may be produced profitably.     

A high-throughput screen for the engineered production of β-lactam antibiotics

High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of β-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.     

γ-ray induced mutagenesis ofCellulomonas biazotea for improved production of cellulases

A rifampin-resistant mutant ofCellulomonas biazotea secreted elevated levels of cellulasesin vivo. The cellulase production in the mutant was not inhibited in the presence of 5% glucose, cellobiose or glycerol in the solid medium. The mutant exhibited approximately two- to three-fold enhanced product yields and productivity of cellular β-glucosidase over the wild parent in shake-flask culture studies when grown on either cellulosic or lignocellulosic substrates. Extracellular production of filter paper cellulase (FPase) and endo-glucanase (CMCase) were also significantly (p≤0.05) altered. During growth of the mutant on α-cellulose, the maximum volumetric productivities for CMCase, FPase and β-glucosidase were 52, 23.3, and 15.2 IUL⁻¹ h⁻¹,i.e 118, 121, and 229% their respective values for the parental strain. Some enzyme properties of the mutant cellulases were altered. Mutant-derived cellulases produced higher yields of glucose arising by degradation of bagasse, wheat straw, and α-cellulose (1.53-, 1.57-, and 1.75-fold, respectively).     

Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.     

Nutritional status of Aureobasidium sp. ATCC 20524 for the production of β-fructofuranosidase

Sucrose at 10 to 20% (w/v) was the best carbon source for the production of -fructofuranosidase by Aureobasidium sp. ATCC 20524. At higher concentrations, it arrested growth. Glucose and fructose were also good carbon sources for the enzyme production. Yeast extract at 1.5 to 2% (w/v) was the best nitrogen source for the enzyme production and for cell growth. Addition of NaNO₃ (1 to 2%, w/v) and MgSO₄·7H₂O (0.5 to 1.5%, w/v) to the cultivation medium increased the intracellular enzymatic activity. The total enzymatic activity and cell growth reached 1.2×10⁴ U/flask and 2.5 g dry cell/flask, respectively after 48 h.Sachio Hayashi, Yoshihiko Shimokawa, Masaharu Nonoguchi, Yoshiyuki Takasaki and Kiyohisa Imada are with the Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Nishi, Gakuen Kibanadai, Miyazaki, 889-21 Japan. Hideo Ueno is with the Nippon Oligo Corporation, 588 Izumisawa, Jyohana-chyo, Tonami-gun, Toyama, 939-18, Japan.