Submerged Fermentation of the Medicinal Mushroom Ganoderma lucidum for Production of Polysaccharide and Ganoderic Acid

Jian-Jiang Zhong, Ya-Jie Tang, and Qing-Hua Fang

State Key Laboratory of Bioreactor Engineering (China), East China University of Science and Technology, Shanghai 200237, China (email: [email protected])

Effects of oxygen supply and lactose feeding on submerged cultures of Ganoderma lucidum were investigated. A higher KL2l value led to a higher biomass density and a higher productivity of both intracellular polysaccharide and ganoderic acid. In a stirred bioreactor, at an initial KLa of 78.2 h'1, a maximal cell concentration of 15.6 g/L by dry weight (DW) and a maximal intracellular polysaccharide (IPS) production of 2.2 g/L were obtained. An increase of initial KLa led to a higher production and productivity of ganoderic acid (GA), and the GA production and productivity at an initial KL& value of 96.0 h"1 was 1.8-fold those at an initial KLa value of 16.4 h'1. In shake flasks, lactose feeding enhanced a-phosphoglucomutase activity, EPS production, phosphoglucose isomerase activity, and lactate accumulation. The information obtained may be useful for efficient large-scale production of these valuable bioactive products by the submerged cultures.

© 2004 American Chemical Society

Mushrooms are abundant sources of a wide range of useful native products and new compounds with interesting biological activities. The obstacles in large-scale fermentation of mushroom include slow growth rate, low productivity, and difficulty in scale-up. In contrast to various studies on fermentation of conventional filamentous microorganisms such as streptomycetes and fungi, until now there are few investigations on the development of mushroom culture processes (7).

Ganoderma lucidum (Leyss.:Fr.) Karst is one of the most famous traditional Chinese medicinal mushrooms used as a health food and medicine in the Far East for more than 2000 years. Polysaccharides produced by G. lucidum are a type of carcinostatic agent, which have antitumor (2) and hypoglycemic activities (3). The higher fungus also produces ganoderic acids with various biological functions such as cytotoxicity to hepatoma cells, inhibition of cholesterol synthesis and stimulation of platelet aggregation (1,4), as well as new interesting biological activities including anti-tumor and anti-HIV-1 (5,6). Because it usually takes several months to cultivate the mushroom and the product yield is low in soil cultivation, submerged culture of G. lucidum is viewed as a promising alternative for efficient production of valuable polysaccharides (7,8) and ganoderic acids (9,10). However, there is scarce information on the simultaneous production of polysaccharide and ganoderic acid by submerged cultivation (11-13).

Oxygen (02) affects cell growth, cellular morphology, nutrient uptake, and metabolite biosynthesis. Ishmentskii et al. (14) reported that a high 02 transfer rate could reduce, enhance, or have no effect on the production of pullulan, depending on the strain ploid of Pullaria (Aureobasidium) pullulans. In the submerged fermentation of Monascus ruber, Hajjaj et al. (15) reported that improving the 02 supply increased the cell yield and production of red pigment and citrinin. Yoshida et al. (16,17) showed the significance of 02 transfer in submerged cultures of a mushroom Lentinus edodes. Until now, there is lack of reports on effects of 02 supply on simultaneous production of Ganoderma polysaccharide and ganoderic acid. One aim of this work was to study this factor.

In our preliminary work, lactose was found to be a favorable carbon source for the submerged fermentation of G. lucidum. Based on previous reports on EPS biosynthesis in bacteria (18-20), a possible pathway for our case is proposed in Fig.l. Enzymes leading to EPS formation can roughly be divided into four groups: enzymes responsible for the initial metabolism of a carbohydrate; enzymes involved in sugar nucleotide synthesis and interconversion; glycosyltransferases that form the repeating unit attached to the glycosyl carrier lipid; and translocases and polymerases that form the polymer. The phosphoglucose isomerase (PGI, Enzyme 8 in Fig.l) and a-phosphoglucomutase (a-PGM, Enzyme 3 in Fig.l) are the enzymes at the branch point between Embden-Meyerhof-Parnas (EMP) pathway and later part of EPS biosynthesis (Fig. 1). In another aspect, substrate feeding is a useful strategy to enhance cell density and process productivity. It is interesting and important to understand the

Pgm Pathway

Fig. 1. Proposed pathway for EPS biosynthesis by Ganoderma lucidum 1, p-galactosidase; 2, glucokinase; 3, a-phosphoglucomutase (a-PGM); 4, UDP-glucose pyrophosphorylase; 5, UDP-galactose-4-epimerase; 6, TDP-glucose pyrophosphorylase; 7, TDP-rhamnose biosynthetic system; 8, phophoglucose isomerase (PGI); 9, 6-phosphofructokinase; 10, fructose bisphosphatase; 11, fructose-1,6-bisphosphate aldolase; 12, triose phosphate isomerase; 13, a-galactose kinase; 14, galactose-6-phosphate isomerase; 15, tagatose-6-phosphate kinase; 16, tagatose-1,6-bisphosphate aldolase; 17, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase; 18, phosphoglyceromutase, enolase, and pyruvate kinase; and 19, lactate dehydrogenase. Abbreviations: DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-biphosphate; G 3-P, glyceraldehyde 3-phosphate; P, phosphate; 3-PGA, 3-phosphoglyceric acid; TBP, fagatose 1,6-bisphosphate; TDP, thymidine diphosphate; TDP-4K-6D-manose, TDP-4-keto-6-deoxymannose; UDP, uridine diphosphate.

Fig. 1. Proposed pathway for EPS biosynthesis by Ganoderma lucidum 1, p-galactosidase; 2, glucokinase; 3, a-phosphoglucomutase (a-PGM); 4, UDP-glucose pyrophosphorylase; 5, UDP-galactose-4-epimerase; 6, TDP-glucose pyrophosphorylase; 7, TDP-rhamnose biosynthetic system; 8, phophoglucose isomerase (PGI); 9, 6-phosphofructokinase; 10, fructose bisphosphatase; 11, fructose-1,6-bisphosphate aldolase; 12, triose phosphate isomerase; 13, a-galactose kinase; 14, galactose-6-phosphate isomerase; 15, tagatose-6-phosphate kinase; 16, tagatose-1,6-bisphosphate aldolase; 17, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase; 18, phosphoglyceromutase, enolase, and pyruvate kinase; and 19, lactate dehydrogenase. Abbreviations: DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-biphosphate; G 3-P, glyceraldehyde 3-phosphate; P, phosphate; 3-PGA, 3-phosphoglyceric acid; TBP, fagatose 1,6-bisphosphate; TDP, thymidine diphosphate; TDP-4K-6D-manose, TDP-4-keto-6-deoxymannose; UDP, uridine diphosphate.

responses of the product biosynthesis and related enzymes' activities to sugar feeding in G. lucidum fermentation. Another aim of this work was to investigate the effects of lactose feeding on the EPS production and the activities of related enzymes.

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