- Email: [email protected]
Enhanced production of total flavones and exopolysaccharides viaVitreoscilla hemoglobin biosynthesis in Phellinus igniarius
Bioresource Technology 102 (2011) 1747–1751
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius Hu Zhu a,b,⇑, Shujing Sun c, Shuaishuai Zhang b a
State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Dongying 257061, PR China Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266555, PR China c College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China b
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 21 August 2010 Accepted 23 August 2010 Available online 19 September 2010 Keywords: Dry mycelial weight Exopolysaccharides Phellinus igniarius Total flavones Vitreoscilla hemoglobin
a b s t r a c t The Vitreoscilla hemoglobin gene (vgb) was expressed by chromosomal integration in Phellinus igniarius to alleviate oxygen limitation and improve metabolites yields during submerged fermentation. Firstly, an expression vector containing vgb was constructed, and transformed into protoplast from P. igniarius. Carbon monoxide difference spectrum absorbance assay showed that vgb was successfully expressed and had biological activity. In shake flasks, the vgb expression enhanced dry mycelial weight 1.32-fold and increased total flavones and exopolysaccharides production 1.78- and 1.33-fold, respectively. When P. igniarius (vgb+) and P. igniarius (vgb) strains were cultured in bioreactor, Vitreoscilla hemoglobin in P. igniarius promoted the mycelia growth from 5.40 to 10.90 g/L and stimulated total flavones and exopolysaccharides synthesis; their maximum productions reached to 11.43 and 1.33 g/L. Furthermore, compared to P. igniarius (vgb), the acetic acid accumulation in P. igniarius (vgb+) cultures decreased from 1.54 and 1.78 to 1.19 and 1.27 g/L in flask and bioreactor, respectively. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phellinus igniarius (L.: Fr.) Quél, a basidiomycete fungus belonging to the family Polyporaceae, is a medicinal mushroom containing many bioactive compounds that have been reported to possess antibacterial, antiviral, antioxidative, antitumor and antimutagenic activities (Liu et al., 2006; Lung et al., 2010; Mo et al., 2003; Shon and Nam, 2004; Song et al., 2008). The fruiting bodies of P. igniarius are used as a folk medicine for a variety of human diseases in several Asian countries. Currently, commercial products from medicinal mushrooms are mostly obtained through the field-cultivation of the fruiting body. However, solid culture does not guarantee a standardized product, with product composition varying from batch to batch (Liu et al., 2009; Rodriguez Estrada et al., 2009; Tang et al., 2009). Accordingly, mushroom submerged fermentation is viewed as a promising alternative for the efficient production of their valuable products (Zhong and Gong, 2005). It is well known that fermentation is a typical biochemical engineering process. In fermentation by aerobic organisms, the supply of oxygen, because of its low solubility in broth, usually becomes a serious limitation, especially during high cell density fermentation. ⇑ Corresponding author at: State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Dongying 257061, PR China. Tel./fax: +86 532 86981566. E-mail address: [email protected] (H. Zhu). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.085
Traditional methods to increase the oxygen supply such as the enhancement of stirring and aeration, and the addition of pure or enriched oxygen are usually expensive and inefficient (Ma et al., 2010). Vitreoscilla hemoglobin (VHb) is an oxygen-binding protein that allows the bacterium to grow aerobically even under microaerophilic conditions. Horng and co-workers demonstrated that the expression of VHb could enhance growth properties (Guo et al., 2009; Horng et al., 2010; Su et al., 2010) and protein (Khleifat et al., 2006; Wei and Chen, 2008) synthesis in recombinant Escherichia coli and Enterobacter aerogenes. Vgb gene, which codes for VHb and has its own ribosomal binding site, has been successfully engineered into various heterologous bacteria (Geckil et al., 2004; Horng et al., 2010; Li et al., 2010; Olano et al., 2008; Wei et al., 1998) and eukaryotes (Sanny et al., 2010; Wu et al., 2003; Zhu et al., 2006b), to meet the industrial purpose of enhancing cell density, protein synthesis, and oxidative metabolism, particularly under oxygen-limited conditions. As far as known, P. igniarius was highly sensitive to oxygen supply for its aerobiosis. With higher cell density and viscosity achieved, oxygen limitation became more severe during late stage of fermentation. Therefore, the vgb gene engineering in P. igniarius serves as a very effective tool for enhancing growth, respiration, and metabolism at low exogenous oxygen concentrations by promoting oxygen delivery. In this study, based on the former research (Xie and Zhu, 2003; Xie et al., 2004; Zhu et al., 2006b), the vgb was successfully introduced into P. igniarius, where it was stably maintained, and
1748
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
expressed. Results indicated that the constitutively expressed VHb improved dry mycelial weight, total flavones and exopolysaccharides production, and decreased acetic acid accumulation under different aeration conditions. This present study would pave the way for modifying P. igniarius genetically and become an example for solving problems in biochemical engineering by genetic manipulation. Meanwhile, this research is also of great importance to genetic engineering of other edible and medical mushroom. 2. Methods 2.1. Strains and media P. igniarius (1511C0001ACCC51328) was purchased from Agricultural Culture Collection of China (Beijing) and grown in solid slant medium (5% (w/v) glucose, 12% (w/v) tryptone, 12% (w/v) yeast extract, 0.15% (w/v) MgSO4, 0.102% (w/v) KH2PO4, 2% (w/v) agar) at 28 °C. For transformation of P. igniarius, liquid medium was inoculated with mycelium and incubated for 3 days at 28 °C. The same medium was used throughout the fermentation experiments. Potassium chloride (0.5 M) was used as an osmotic stabilizer during preparation and regeneration of the protoplasts. Transformants were selected and stored on solid agar medium containing hygromycin at 50 lg/ml (Zhu et al., 2006b). E. coli DH5a (Stratagene, USA) was used for DNA manipulations, which was cultured at 37 °C in LB medium composed of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) NaCl. 2.2. Plasmid construction The plasmid expression vector pVHb was constructed according to the former report (Zhu et al., 2006b). Briefly, intermediate plasmid pBI-vgb was generated by incorporating vgb from plasmid pOK12-vgb digested with BamH I and Sac I into plasmid pBI121 instead of beta-glucuronidase (GUS) gene. Then, plasmid pBI-vgb was cut using Hind III and EcoR I. The reading frame fragment comprising the CaMV35S promoter, Nos terminator, and vgb sequence was recovered. Finally, pVHb was constructed by inserting this fragment excised from pBI-vgb using cohesive-end ligation at the multiple cloning sites of pCAMBIA1300 digested by restriction enzymes Hind III and EcoR I simultaneously. Thus, the resultant plasmid, pVHb, consisted of a pCAMBIA1300 backbone containing the hygromycin B phosphotransferase and vgb genes, each of which was put under the control of CaMV35S promoter and followed by different terminators. 2.3. Protoplast generation and transformation Protoplast generation and transformation was performed with restriction enzyme-mediated DNA integration (REMI) according to the report from Zhu and co-workers (Zhu and Ma, 2007) and the former research (Zhu and Xie, 2003). Briefly, P. igniarius mycelium was cultivated for 3 days with shaking (150 rpm) at 28 °C. The mycelium was harvested by centrifugation at 10,000g for 5 min, triturated and homogenized using a sterilized glass homogenizer, and then rinsed with osmotic stabilizer (0.5 M KCl). Then, the mycelium was incubated for 3 h at 35 °C in 1 mL 20 mg/mL lywallzyme (Guang Dong Institute of Microbiology, China) containing 0.5 M KCl. After incubation, these protoplasts were washed free of enzyme and transferred to TPB solution (0.6 M KCl, 25 mM CaCl2, H2O). The whole mixture (300 lL) of plasmid and Hind III (150 U) was added to 100 lL of protoplast suspension (105 cells/ mL) in an Eppendorf tube and chilled on ice for 5 min. After that, 20 lL of PEG4000/S was added and mixed briefly, and then the mixture was kept for 5 min on ice. All the following steps were car-
ried out according to the protocol published in the paper (Zhu and Xie, 2003). 2.4. PCR and Southern blotting Both PCR and Southern blotting (Zhu et al., 2006b) were performed to confirm whether the mycelium colonies were transformants, or not. The chromosomal DNAs of the transformants and the nontransgenic mycelium as the control were used as the templates of the PCR to confirm the integration of the pVHb using the vgb gene specific primers (forward primer: 50 -AGGAAGACCCT CATGTTAG-30 , reverse primer: 50 -CAAAGCCCAATTGGACG-30 ). The PCR products were analyzed by the ethidium bromide staining after 1.0% agarose gel electrophoresis. At the same time, Southern blotting using the DIG system (Roche) was strictly operated according to the manufacturer’s directions. 2.5. Carbon monoxide difference spectrum absorbance assay of VHb The activity of the expressed VHb was detected by CO-difference spectra (Horng et al., 2010). P. igniarius (vgb+) and P. igniarius (vgb) mycelia were harvested from culture medium by centrifugation 10,000g for 15 min, resuspended in 5 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 20 mg/mL lywallzyme, and incubated for 3 h at 35 °C. After incubation, these protoplasts were washed free of enzyme, transferred to potassium phosphate buffer, and disrupted on ice by an ultrasonic processor as described (Zhu et al., 2006a). The samples were divided into two aliquots. One was exposed to 20% CO for 2 min and the other to air; then, hemoglobin levels were subsequently obtained by the CO-difference spectra using UV-2450 spectrophotometer (Shimadzu, Japan) (Su et al., 2010). 2.6. Analytical methods of dry mycelial weight, total flavones content, acetic acid and exopolysaccharides production The mycelial biomass is expressed as the gram dry mycelial weight per liter (DMW). The DMW was obtained by centrifuging at 10,000g for 15 min, washing the mycelia three times with distilled water, and drying at 80 °C to a constant weight. Total flavones were assayed using UV colorimetric method as reported elsewhere (Liu et al., 2006). Briefly, reaction mixtures consisted of 100 ll fermentation broth sample, 1 ml 70% ethanol and 1 ml stain reagent I (0.8 g boracic acid and 1.0 g sodium acetate dissolved in 100 ml 70% v/v ethano1). Spectra were determined at the wavelength of 385 nm by comparison with controls in which the sample was replaced by 70% (v/v) ethano1. Sample concentrations were calculated from the standard curve made using the diluted rutin as standard substance. Several dilutions of samples were performed to make sure the readings are within the standard curve range. After the centrifugation of fermentation broth at 10,000g for 15 min, the resulting supernatant was filtered through a Whatman filter paper no. 2 (Whatman International Ltd., Maidstone, UK). The resulting culture filtrate was mixed with four times its volume of absolute ethanol, stirred vigorously, and left overnight at 4 °C. The precipitated exopolysaccharides was centrifuged at 10,000g for 20 min, with the supernatant discarded, and then the precipitated exopolysaccharides was dissolved in water, precipitated with alcohol again, by three times. At last, the precipitated exopolysaccharides was lyophilized and the exopolysaccharides content was measured by a phenol–sulphuric acid method using glucose as the standard (Tang et al., 2009). The residual sugar concentration was analyzed with the dinitrosalicylic acid method (Zhu and Sun, 2009). Acetate was determined by high-pressure liquid chromatography (Alliance, Waters, UK)
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
equipped with 250 4.6 mm C18 column (Waters) 0.05 mol L1 (NH4)H2PO4 (pH 2.6) as a mobile phase.
with
2.7. Batch fermentation at shake-flask and bioreactor scale To confirm the effect of VHb on P. igniarius, submerged fermentation experiments were carried out in shake flasks and bioreactor to compare DMW, total flavones, acetic acid and exopolysaccharides yields between P. igniarius (vgb+) and P. igniarius (vgb). The wild-type and transformant cultures were inoculated into three 250-mL shake flasks, each containing 100 mL medium with tailor-made bungs, and incubated at 28 °C and 150 rpm in an orbital shaker for 8 days. Meanwhile, bioreactor cultivations were performed in a 5-L in situ sterilizable bioreactor (Biotech, Shanghai, China) with an effective working volume of 3 L. The process was monitored and controlled using bioprocess automation software. The 4 days grown wild-type and transformant cultures were transferred to the bioreactor containing liquid medium. The cultivation conditions were at 28 °C, pH 7.0, and 150-rpm agitation. To ensure anaerobic conditions, the bioreactor was sparged with nitrogen at a flow rate of 0.5 volumes per volume per minute. In all experiments, samples were taken at 24-h intervals.
3. Results and discussion 3.1. Transformation of protoplasts with REMI and identification of vgb insertional transformants The expression of vgb gene in bacteria was studied extensively (Horng et al., 2010; Li et al., 2010; Su et al., 2010; Yu et al., 2002). However, up to now, there were scarce reports on genetic transformation of vgb gene in (Lin et al., 2004; Sanny et al., 2010; Wu et al., 2003) fungi, and above all basidiomycetes (Zhu et al., 2006b). In this study, when the REMI was carried out using the restriction enzyme Hind III which digested pVHb at the unique site, genetic transformation of protoplasts generated from P. igniarius was successfully implemented with the expression plasmid pVHb, and the transformation yield was estimated as 1.1 102 transformants per microgram of plasmid DNA. Among transgenic mycelium colonies, ten candidates randomly selected were further verified the VHb insertion into the host chromosomes with PCR using the vgb-specific primers. PCR products of 1.3 kb in size show that the exogenous gene has been integrated into the chromosome of the host strain. To determine VHb gene insertion copy number in genome DNA, three transformants detected by PCR were analyzed by Southern blotting using VHb gene as the probe. The different Southern hybridization profiles of the three picked transformants indicated that the DNA was inserted into different sites in the genome. Two hybridizing bands were detected in transformant P1, indicating two insertions within the P1 genome. Transformants P2 and P3 showed only one hybridizing band, which implied that there was only one copy of the target gene. Hybridizing signal did not appear in the control strain (data not shown).
1749
vgb gene driven by CaMV35S promoter could be stably expressed throughout the cultivation. 3.3. Comparison of DMW, total flavones and exopolysaccharides production between P. igniarius (vgb+) and P. igniarius (vgb) It was reported that the expression of VHb increased the cell density and enhanced the yield of antibiotics, or altered metabolite production (Guo et al., 2009; Horng et al., 2010; Olano et al., 2008; Zhu et al., 2006b). To verify the effect of VHb expression on mycelia growth and metabolite, P. igniarius (vgb+) and P. igniarius (vgb) were grown in the shake-flask and bioreactor under microaerobic conditions. The growth curve of P. igniarius with or without VHb expression was monitored by DMW. Fig. 1A shows that, in flask and bioreactor cultures, DMWs were enhanced by 1.32- and 2.01-fold in VHb-expressed strain compared with non-VHb-expressed stain at stationary phase. The maximum dry weights of mycelium were 11.91 and 10.93 g/L, respectively. These results indicated that the function of active VHb protein in this modified strain could improve cell growth and final DMW at the hypoxic state, as compared to the wild-type strain. At the same time, this metabolic effect of intracellular VHb was seen more clearly in metabolites production. As shown in Fig. 1B, compared with P. igniarius (vgb), constitutive VHb expression in P. igniarius stimulated an increase in total flavones synthesis from 9.67 to 17.21 g/ L in shaken culture. P. igniarius (vgb+) strain (11.43 g/L) had 1.40fold higher total flavones content than P. igniarius (vgb) strain (8.14 g/L) in bioreactor fermentation. The trends of exopolysaccharides production variation were the same under different culture conditions. The VHb expression increased final exopolysaccharides harvest (Fig. 1C). The maximum exopolysaccharides concentration (1.18 g/L) could be obtained in flask culture. The kinetics of exopolysaccharides accumulation in the bioreactor was the same as in flask culture. The maximal exopolysaccharide production could reach to 1.33 g/L. All these results indicated that VHb expression facilitated metabolites production including total flavones content and exopolysaccharides accumulation in P. igniarius. Substantial differences were seen in the late stage of cultivation. The transformant exhibited higher cell viability and metabolic ability than the wild-type strain. Furthermore, significant differences in specific growth rates were observed at the late exponential phase. Furthermore, when cultivated under oxygen-limited conditions, VHbexpressing P. igniarius had higher specific growth rates and metabolic adaptation compared to a control strain without vgb gene. Of course, the accumulation of these metabolites not only correlates with cell growth but also with alteration of biosynthesis pathways. The vgb gene expression and hemoglobin synthesis in response to oxygen levels promoted total flavones and exopolysaccharides production. Because the molecular mechanism of the influence of VHb on aerobic metabolism is not known in Vitreoscilla or in any other organism, the presence and function of VHb in enhancing respiration and formation of ATP has been considered to be the reason for the improvement in this system (Zhu et al., 2006b). 3.4. The expression of VHb decreased the concentration of acetic acid
3.2. Carbon monoxide difference spectrum absorbance assay of VHb The activity of the integrated VHb was demonstrated by CO-difference spectrum since an absorption peak at 419 nm results from absorbance due to CO binding to VHb (Horng et al., 2010). This peak was present in P. igniarius (vgb+), but absent in P. igniarius (vgb). These results indicated the recombinant strain bearing VHb gene could express functional VHb in P. igniarius, which suggested that CaMV35S promoter was capable of driving the expression of heterologous genes in P. igniarius, moreover, also indicated
It is inevitable that acetic acid, lactate, ethanol and other secretion products gradually accumulate in the fermentation process. Excessive acetic acid not only inhibits microbial growth but also induces cell death in fungi at both high and low temperatures. As shown in Fig. 1D, accumulation of acetic acid was observed from the beginning of the submerged cultures of P. igniarius (vgb+) and P. igniarius (vgb). In flask and bioreactor cultures, the maximum concentration of acetic acid produced by non-VHb-expressed stain was 1.54 and 1.78 g/L. In comparison, the acetic acid accumulation
1750
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
Fig. 1. Comparison of growth profiles (A), total flavones content (B), exopolysaccharides production (C), and acetic acid accumulation (D) between vgb () and vgb+ (—) strains in flask (dj) and bioreactor (N) cultivations under oxygen-limiting conditions. The mycelial biomass is expressed as the gram dry mycelial weight per liter (DMW). The data were the mean of three repeats.
in P. igniarius (vgb+) cultures decreased to 1.19 and 1.27 g/L, respectively. Acetic acid started to accumulate considerably after consuming 40 g/L of glucose, and it retarded cell growth and lowered the rates of glucose uptake and metabolites production in the later stages of fermentation. After glucose depletion, acetic acid concentration decreased gradually because acetic acid was used as the main carbon source. VHb protein not only enhanced metabolites synthesis but also obviously decreased acetic acid accumulation during fermentation by P. igniarius. Probably, TDNA insertions maybe result in inactivation of some genes, which altered biosynthesis pathway of acetic acid. 4. Conclusions In this study, the chromosomal integration of vgb into P. igniarius was described. Expression of the VHb in P. igniarius (vgb+) increased the yield of total flavones and exopolysaccharides which are accompanied with faster growth of the strain during fermentation. The information obtained from the current research is helpful for the hyperproduction of metabolites from higher fungi. Furthermore, with the development of modern genetic technology, genetic strategies increasingly focus on solving chemical engineering problems in biochemical engineering. Acknowledgements This work was financially supported by the Natural Science Foundation of China (No. 30800775) and the Fundamental Research Funds for the Central Universities (No. 27R0904083A).
References Geckil, H., Barak, Z., Chipman, D.M., Erenler, S.O., Webster, D.A., Stark, B.C., 2004. Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene. Bioprocess Biosyst. Eng. 26, 325–330. Guo, X., Zou, X., Sun, M., 2009. Effects of phytohormones on mycelial growth and exopolysaccharide biosynthesis of medicinal mushroom Phellinus linteus. Bioprocess Biosyst. Eng. 32, 701–707. Horng, Y.T., Chang, K.C., Chien, C.C., Wei, Y.H., Sun, Y.M., Soo, P.C., 2010. Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P promoter in Escherichia coli. Lett. Appl. Microbiol. 50, 158–167. Khleifat, K.M., Abboud, M.M., Al-Mustafa, A.H., Al-Sharafa, K.Y., 2006. Effects of carbon source and Vitreoscilla hemoglobin (VHb) on the production of betagalactosidase in Enterobacter aerogenes. Curr. Microbiol. 53, 277–281. Li, M., Wu, J., Lin, J., Wei, D., 2010. Expression of Vitreoscilla hemoglobin enhances cell growth and dihydroxyacetone production in Gluconobacter oxydans. Curr. Microbiol. (Epub ahead of print). Lin, Y.H., Li, Y.F., Huang, M.C., Tsai, Y.C., 2004. Intracellular expression of Vitreoscilla hemoglobin in Aspergillus terreus to alleviate the effect of a short break in aeration during culture. Biotechnol. Lett. 26, 1067–1072. Liu, Y.F., Yang, Y., Jia, W., Zhang, J.S., Tang, Q.J., Tang, C.H., 2006. Determination of total flavones in the medicinal mushroom Phellinus. Acta Edulis Fungi 13, 49– 52. Liu, Q.N., Liu, R.S., Wang, Y.H., Mi, Z.Y., Li, D.S., Zhong, J.J., Tang, Y.J., 2009. Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides. Bioresour. Technol. 100, 3644–3649. Lung, M.Y., Tsai, J.C., Huang, P.C., 2010. Antioxidant properties of edible basidiomycete Phellinus igniarius in submerged cultures. J. Food Sci. 75, E18–24. Ma, X., Fan, D., Shang, L.A., Cai, Q., Chi, L., Zhu, C., Mi, Y., Luo, Y.E., 2010. Oxygen transfer rate control in the production of human-like collagen by recombinant Escherichia coli. Biotechnol. Appl. Biochem. 55, 169–174. Mo, S.Y., Yang, Y.C., Shi, J.G., 2003. Studies on chemical constitutes of Phellinus igniarius. Zhongguo Zhong Yao Za Zhi 28, 339–341. Olano, C., Lombo, F., Mendez, C., Salas, J.A., 2008. Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab. Eng. 10, 281–292.
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751 Rodriguez Estrada, A.E., Jimenez-Gasco Mdel, M., Royse, D.J., 2009. Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay. Bioresour. Technol. 100, 5270–5276. Sanny, T., Arnaldos, M., Kunkel, S.A., Pagilla, K.R., Stark, B.C., 2010. Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl. Microbiol. Biotechnol. doi:10.1007/s00253-010-2817-7. Shon, Y.H., Nam, K.S., 2004. Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea. Biotechnol. Lett. 26, 159–163. Song, T.Y., Lin, H.C., Yang, N.C., Hu, M.L., 2008. Antiproliferative and antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linnearus: Fries) Quelet. J. Ethnopharmacol. 115, 50–56. Su, Y., Li, X., Liu, Q., Hou, Z., Zhu, X., Guo, X., Ling, P., 2010. Improved polygamma-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. Bioresour. Technol. 101, 4733–4736. Tang, Y.J., Zhang, W., Zhong, J.J., 2009. Performance analyses of a pH-shift and DOTshift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum. Bioresour. Technol. 100, 1852–1859. Wei, X.X., Chen, G.Q., 2008. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol. 436, 273–287. Wei, M.L., Webster, D.A., Stark, B.C., 1998. Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size. Biotechnol. Bioeng. 57, 477–483.
1751
Wu, J.M., Hsu, T.A., Lee, C.K., 2003. Expression of the gene coding for bacterial hemoglobin improves beta-galactosidase production in a recombinant Pichia pastoris. Biotechnol. Lett. 25, 1457–1462. Xie, B.G., Zhu, H., 2003. Isolation and optimization of regeneration condition of Tremella fuciformis protoplasts. Mycosystema 22, 574–578. Xie, B.G., Zhu, H., Jiang, Y.J., Rao, Y.B., Zheng, J.G., 2004. Genetic transformation markers of Tremella fuciformis. J. Agric. Biotechnol. 12, 610–611. Yu, H., Shi, Y., Zhang, Y., Yang, S., Shen, Z., 2002. Effect of Vitreoscilla hemoglobin biosynthesis in Escherichia coli on production of poly(beta-hydroxybutyrate) and fermentative parameters. FEMS Microbiol. Lett. 214, 223–227. Zhong, J.J., Gong, H.G., 2005. Hydrodynamic shear stress affects cell growth and metabolite production by medicinal mushroom Ganoderma lucidum. Chin. J. Chem. Eng. 13, 426–428. Zhu, Z.P., Ma, H.L., 2007. Separation and regeneration of protoplast from Phellinus igniarius. Zhongguo Zhong Yao Za Zhi 32, 2232–2235. Zhu, H., Sun, S.J., 2009. Effect of constant glucose feeding on the production of exopolysaccharides by Tremella fuciformis spores. Appl. Biochem. Biotechnol. 152, 366–371. Zhu, H., Xie, B.G., 2003. Isolation and optimization of regeneration condition of Tremella fuciformis protoplasts. Mycosystema 22, 574–578. Zhu, H., Pan, R.J., Wang, T.W., Shen, Y.L., Wei, D.Z., 2006a. Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltransferase. Appl. Microbiol. Biotechnol. 72, 1033–1038. Zhu, H., Wang, T.W., Sun, S.J., Shen, Y.L., Wei, D.Z., 2006b. Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis. Appl. Microbiol. Biotechnol. 72, 770–776.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius Hu Zhu a,b,⇑, Shujing Sun c, Shuaishuai Zhang b a
State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Dongying 257061, PR China Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266555, PR China c College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China b
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 21 August 2010 Accepted 23 August 2010 Available online 19 September 2010 Keywords: Dry mycelial weight Exopolysaccharides Phellinus igniarius Total flavones Vitreoscilla hemoglobin
a b s t r a c t The Vitreoscilla hemoglobin gene (vgb) was expressed by chromosomal integration in Phellinus igniarius to alleviate oxygen limitation and improve metabolites yields during submerged fermentation. Firstly, an expression vector containing vgb was constructed, and transformed into protoplast from P. igniarius. Carbon monoxide difference spectrum absorbance assay showed that vgb was successfully expressed and had biological activity. In shake flasks, the vgb expression enhanced dry mycelial weight 1.32-fold and increased total flavones and exopolysaccharides production 1.78- and 1.33-fold, respectively. When P. igniarius (vgb+) and P. igniarius (vgb) strains were cultured in bioreactor, Vitreoscilla hemoglobin in P. igniarius promoted the mycelia growth from 5.40 to 10.90 g/L and stimulated total flavones and exopolysaccharides synthesis; their maximum productions reached to 11.43 and 1.33 g/L. Furthermore, compared to P. igniarius (vgb), the acetic acid accumulation in P. igniarius (vgb+) cultures decreased from 1.54 and 1.78 to 1.19 and 1.27 g/L in flask and bioreactor, respectively. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phellinus igniarius (L.: Fr.) Quél, a basidiomycete fungus belonging to the family Polyporaceae, is a medicinal mushroom containing many bioactive compounds that have been reported to possess antibacterial, antiviral, antioxidative, antitumor and antimutagenic activities (Liu et al., 2006; Lung et al., 2010; Mo et al., 2003; Shon and Nam, 2004; Song et al., 2008). The fruiting bodies of P. igniarius are used as a folk medicine for a variety of human diseases in several Asian countries. Currently, commercial products from medicinal mushrooms are mostly obtained through the field-cultivation of the fruiting body. However, solid culture does not guarantee a standardized product, with product composition varying from batch to batch (Liu et al., 2009; Rodriguez Estrada et al., 2009; Tang et al., 2009). Accordingly, mushroom submerged fermentation is viewed as a promising alternative for the efficient production of their valuable products (Zhong and Gong, 2005). It is well known that fermentation is a typical biochemical engineering process. In fermentation by aerobic organisms, the supply of oxygen, because of its low solubility in broth, usually becomes a serious limitation, especially during high cell density fermentation. ⇑ Corresponding author at: State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Dongying 257061, PR China. Tel./fax: +86 532 86981566. E-mail address: [email protected] (H. Zhu). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.085
Traditional methods to increase the oxygen supply such as the enhancement of stirring and aeration, and the addition of pure or enriched oxygen are usually expensive and inefficient (Ma et al., 2010). Vitreoscilla hemoglobin (VHb) is an oxygen-binding protein that allows the bacterium to grow aerobically even under microaerophilic conditions. Horng and co-workers demonstrated that the expression of VHb could enhance growth properties (Guo et al., 2009; Horng et al., 2010; Su et al., 2010) and protein (Khleifat et al., 2006; Wei and Chen, 2008) synthesis in recombinant Escherichia coli and Enterobacter aerogenes. Vgb gene, which codes for VHb and has its own ribosomal binding site, has been successfully engineered into various heterologous bacteria (Geckil et al., 2004; Horng et al., 2010; Li et al., 2010; Olano et al., 2008; Wei et al., 1998) and eukaryotes (Sanny et al., 2010; Wu et al., 2003; Zhu et al., 2006b), to meet the industrial purpose of enhancing cell density, protein synthesis, and oxidative metabolism, particularly under oxygen-limited conditions. As far as known, P. igniarius was highly sensitive to oxygen supply for its aerobiosis. With higher cell density and viscosity achieved, oxygen limitation became more severe during late stage of fermentation. Therefore, the vgb gene engineering in P. igniarius serves as a very effective tool for enhancing growth, respiration, and metabolism at low exogenous oxygen concentrations by promoting oxygen delivery. In this study, based on the former research (Xie and Zhu, 2003; Xie et al., 2004; Zhu et al., 2006b), the vgb was successfully introduced into P. igniarius, where it was stably maintained, and
1748
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
expressed. Results indicated that the constitutively expressed VHb improved dry mycelial weight, total flavones and exopolysaccharides production, and decreased acetic acid accumulation under different aeration conditions. This present study would pave the way for modifying P. igniarius genetically and become an example for solving problems in biochemical engineering by genetic manipulation. Meanwhile, this research is also of great importance to genetic engineering of other edible and medical mushroom. 2. Methods 2.1. Strains and media P. igniarius (1511C0001ACCC51328) was purchased from Agricultural Culture Collection of China (Beijing) and grown in solid slant medium (5% (w/v) glucose, 12% (w/v) tryptone, 12% (w/v) yeast extract, 0.15% (w/v) MgSO4, 0.102% (w/v) KH2PO4, 2% (w/v) agar) at 28 °C. For transformation of P. igniarius, liquid medium was inoculated with mycelium and incubated for 3 days at 28 °C. The same medium was used throughout the fermentation experiments. Potassium chloride (0.5 M) was used as an osmotic stabilizer during preparation and regeneration of the protoplasts. Transformants were selected and stored on solid agar medium containing hygromycin at 50 lg/ml (Zhu et al., 2006b). E. coli DH5a (Stratagene, USA) was used for DNA manipulations, which was cultured at 37 °C in LB medium composed of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) NaCl. 2.2. Plasmid construction The plasmid expression vector pVHb was constructed according to the former report (Zhu et al., 2006b). Briefly, intermediate plasmid pBI-vgb was generated by incorporating vgb from plasmid pOK12-vgb digested with BamH I and Sac I into plasmid pBI121 instead of beta-glucuronidase (GUS) gene. Then, plasmid pBI-vgb was cut using Hind III and EcoR I. The reading frame fragment comprising the CaMV35S promoter, Nos terminator, and vgb sequence was recovered. Finally, pVHb was constructed by inserting this fragment excised from pBI-vgb using cohesive-end ligation at the multiple cloning sites of pCAMBIA1300 digested by restriction enzymes Hind III and EcoR I simultaneously. Thus, the resultant plasmid, pVHb, consisted of a pCAMBIA1300 backbone containing the hygromycin B phosphotransferase and vgb genes, each of which was put under the control of CaMV35S promoter and followed by different terminators. 2.3. Protoplast generation and transformation Protoplast generation and transformation was performed with restriction enzyme-mediated DNA integration (REMI) according to the report from Zhu and co-workers (Zhu and Ma, 2007) and the former research (Zhu and Xie, 2003). Briefly, P. igniarius mycelium was cultivated for 3 days with shaking (150 rpm) at 28 °C. The mycelium was harvested by centrifugation at 10,000g for 5 min, triturated and homogenized using a sterilized glass homogenizer, and then rinsed with osmotic stabilizer (0.5 M KCl). Then, the mycelium was incubated for 3 h at 35 °C in 1 mL 20 mg/mL lywallzyme (Guang Dong Institute of Microbiology, China) containing 0.5 M KCl. After incubation, these protoplasts were washed free of enzyme and transferred to TPB solution (0.6 M KCl, 25 mM CaCl2, H2O). The whole mixture (300 lL) of plasmid and Hind III (150 U) was added to 100 lL of protoplast suspension (105 cells/ mL) in an Eppendorf tube and chilled on ice for 5 min. After that, 20 lL of PEG4000/S was added and mixed briefly, and then the mixture was kept for 5 min on ice. All the following steps were car-
ried out according to the protocol published in the paper (Zhu and Xie, 2003). 2.4. PCR and Southern blotting Both PCR and Southern blotting (Zhu et al., 2006b) were performed to confirm whether the mycelium colonies were transformants, or not. The chromosomal DNAs of the transformants and the nontransgenic mycelium as the control were used as the templates of the PCR to confirm the integration of the pVHb using the vgb gene specific primers (forward primer: 50 -AGGAAGACCCT CATGTTAG-30 , reverse primer: 50 -CAAAGCCCAATTGGACG-30 ). The PCR products were analyzed by the ethidium bromide staining after 1.0% agarose gel electrophoresis. At the same time, Southern blotting using the DIG system (Roche) was strictly operated according to the manufacturer’s directions. 2.5. Carbon monoxide difference spectrum absorbance assay of VHb The activity of the expressed VHb was detected by CO-difference spectra (Horng et al., 2010). P. igniarius (vgb+) and P. igniarius (vgb) mycelia were harvested from culture medium by centrifugation 10,000g for 15 min, resuspended in 5 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 20 mg/mL lywallzyme, and incubated for 3 h at 35 °C. After incubation, these protoplasts were washed free of enzyme, transferred to potassium phosphate buffer, and disrupted on ice by an ultrasonic processor as described (Zhu et al., 2006a). The samples were divided into two aliquots. One was exposed to 20% CO for 2 min and the other to air; then, hemoglobin levels were subsequently obtained by the CO-difference spectra using UV-2450 spectrophotometer (Shimadzu, Japan) (Su et al., 2010). 2.6. Analytical methods of dry mycelial weight, total flavones content, acetic acid and exopolysaccharides production The mycelial biomass is expressed as the gram dry mycelial weight per liter (DMW). The DMW was obtained by centrifuging at 10,000g for 15 min, washing the mycelia three times with distilled water, and drying at 80 °C to a constant weight. Total flavones were assayed using UV colorimetric method as reported elsewhere (Liu et al., 2006). Briefly, reaction mixtures consisted of 100 ll fermentation broth sample, 1 ml 70% ethanol and 1 ml stain reagent I (0.8 g boracic acid and 1.0 g sodium acetate dissolved in 100 ml 70% v/v ethano1). Spectra were determined at the wavelength of 385 nm by comparison with controls in which the sample was replaced by 70% (v/v) ethano1. Sample concentrations were calculated from the standard curve made using the diluted rutin as standard substance. Several dilutions of samples were performed to make sure the readings are within the standard curve range. After the centrifugation of fermentation broth at 10,000g for 15 min, the resulting supernatant was filtered through a Whatman filter paper no. 2 (Whatman International Ltd., Maidstone, UK). The resulting culture filtrate was mixed with four times its volume of absolute ethanol, stirred vigorously, and left overnight at 4 °C. The precipitated exopolysaccharides was centrifuged at 10,000g for 20 min, with the supernatant discarded, and then the precipitated exopolysaccharides was dissolved in water, precipitated with alcohol again, by three times. At last, the precipitated exopolysaccharides was lyophilized and the exopolysaccharides content was measured by a phenol–sulphuric acid method using glucose as the standard (Tang et al., 2009). The residual sugar concentration was analyzed with the dinitrosalicylic acid method (Zhu and Sun, 2009). Acetate was determined by high-pressure liquid chromatography (Alliance, Waters, UK)
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
equipped with 250 4.6 mm C18 column (Waters) 0.05 mol L1 (NH4)H2PO4 (pH 2.6) as a mobile phase.
with
2.7. Batch fermentation at shake-flask and bioreactor scale To confirm the effect of VHb on P. igniarius, submerged fermentation experiments were carried out in shake flasks and bioreactor to compare DMW, total flavones, acetic acid and exopolysaccharides yields between P. igniarius (vgb+) and P. igniarius (vgb). The wild-type and transformant cultures were inoculated into three 250-mL shake flasks, each containing 100 mL medium with tailor-made bungs, and incubated at 28 °C and 150 rpm in an orbital shaker for 8 days. Meanwhile, bioreactor cultivations were performed in a 5-L in situ sterilizable bioreactor (Biotech, Shanghai, China) with an effective working volume of 3 L. The process was monitored and controlled using bioprocess automation software. The 4 days grown wild-type and transformant cultures were transferred to the bioreactor containing liquid medium. The cultivation conditions were at 28 °C, pH 7.0, and 150-rpm agitation. To ensure anaerobic conditions, the bioreactor was sparged with nitrogen at a flow rate of 0.5 volumes per volume per minute. In all experiments, samples were taken at 24-h intervals.
3. Results and discussion 3.1. Transformation of protoplasts with REMI and identification of vgb insertional transformants The expression of vgb gene in bacteria was studied extensively (Horng et al., 2010; Li et al., 2010; Su et al., 2010; Yu et al., 2002). However, up to now, there were scarce reports on genetic transformation of vgb gene in (Lin et al., 2004; Sanny et al., 2010; Wu et al., 2003) fungi, and above all basidiomycetes (Zhu et al., 2006b). In this study, when the REMI was carried out using the restriction enzyme Hind III which digested pVHb at the unique site, genetic transformation of protoplasts generated from P. igniarius was successfully implemented with the expression plasmid pVHb, and the transformation yield was estimated as 1.1 102 transformants per microgram of plasmid DNA. Among transgenic mycelium colonies, ten candidates randomly selected were further verified the VHb insertion into the host chromosomes with PCR using the vgb-specific primers. PCR products of 1.3 kb in size show that the exogenous gene has been integrated into the chromosome of the host strain. To determine VHb gene insertion copy number in genome DNA, three transformants detected by PCR were analyzed by Southern blotting using VHb gene as the probe. The different Southern hybridization profiles of the three picked transformants indicated that the DNA was inserted into different sites in the genome. Two hybridizing bands were detected in transformant P1, indicating two insertions within the P1 genome. Transformants P2 and P3 showed only one hybridizing band, which implied that there was only one copy of the target gene. Hybridizing signal did not appear in the control strain (data not shown).
1749
vgb gene driven by CaMV35S promoter could be stably expressed throughout the cultivation. 3.3. Comparison of DMW, total flavones and exopolysaccharides production between P. igniarius (vgb+) and P. igniarius (vgb) It was reported that the expression of VHb increased the cell density and enhanced the yield of antibiotics, or altered metabolite production (Guo et al., 2009; Horng et al., 2010; Olano et al., 2008; Zhu et al., 2006b). To verify the effect of VHb expression on mycelia growth and metabolite, P. igniarius (vgb+) and P. igniarius (vgb) were grown in the shake-flask and bioreactor under microaerobic conditions. The growth curve of P. igniarius with or without VHb expression was monitored by DMW. Fig. 1A shows that, in flask and bioreactor cultures, DMWs were enhanced by 1.32- and 2.01-fold in VHb-expressed strain compared with non-VHb-expressed stain at stationary phase. The maximum dry weights of mycelium were 11.91 and 10.93 g/L, respectively. These results indicated that the function of active VHb protein in this modified strain could improve cell growth and final DMW at the hypoxic state, as compared to the wild-type strain. At the same time, this metabolic effect of intracellular VHb was seen more clearly in metabolites production. As shown in Fig. 1B, compared with P. igniarius (vgb), constitutive VHb expression in P. igniarius stimulated an increase in total flavones synthesis from 9.67 to 17.21 g/ L in shaken culture. P. igniarius (vgb+) strain (11.43 g/L) had 1.40fold higher total flavones content than P. igniarius (vgb) strain (8.14 g/L) in bioreactor fermentation. The trends of exopolysaccharides production variation were the same under different culture conditions. The VHb expression increased final exopolysaccharides harvest (Fig. 1C). The maximum exopolysaccharides concentration (1.18 g/L) could be obtained in flask culture. The kinetics of exopolysaccharides accumulation in the bioreactor was the same as in flask culture. The maximal exopolysaccharide production could reach to 1.33 g/L. All these results indicated that VHb expression facilitated metabolites production including total flavones content and exopolysaccharides accumulation in P. igniarius. Substantial differences were seen in the late stage of cultivation. The transformant exhibited higher cell viability and metabolic ability than the wild-type strain. Furthermore, significant differences in specific growth rates were observed at the late exponential phase. Furthermore, when cultivated under oxygen-limited conditions, VHbexpressing P. igniarius had higher specific growth rates and metabolic adaptation compared to a control strain without vgb gene. Of course, the accumulation of these metabolites not only correlates with cell growth but also with alteration of biosynthesis pathways. The vgb gene expression and hemoglobin synthesis in response to oxygen levels promoted total flavones and exopolysaccharides production. Because the molecular mechanism of the influence of VHb on aerobic metabolism is not known in Vitreoscilla or in any other organism, the presence and function of VHb in enhancing respiration and formation of ATP has been considered to be the reason for the improvement in this system (Zhu et al., 2006b). 3.4. The expression of VHb decreased the concentration of acetic acid
3.2. Carbon monoxide difference spectrum absorbance assay of VHb The activity of the integrated VHb was demonstrated by CO-difference spectrum since an absorption peak at 419 nm results from absorbance due to CO binding to VHb (Horng et al., 2010). This peak was present in P. igniarius (vgb+), but absent in P. igniarius (vgb). These results indicated the recombinant strain bearing VHb gene could express functional VHb in P. igniarius, which suggested that CaMV35S promoter was capable of driving the expression of heterologous genes in P. igniarius, moreover, also indicated
It is inevitable that acetic acid, lactate, ethanol and other secretion products gradually accumulate in the fermentation process. Excessive acetic acid not only inhibits microbial growth but also induces cell death in fungi at both high and low temperatures. As shown in Fig. 1D, accumulation of acetic acid was observed from the beginning of the submerged cultures of P. igniarius (vgb+) and P. igniarius (vgb). In flask and bioreactor cultures, the maximum concentration of acetic acid produced by non-VHb-expressed stain was 1.54 and 1.78 g/L. In comparison, the acetic acid accumulation
1750
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751
Fig. 1. Comparison of growth profiles (A), total flavones content (B), exopolysaccharides production (C), and acetic acid accumulation (D) between vgb () and vgb+ (—) strains in flask (dj) and bioreactor (N) cultivations under oxygen-limiting conditions. The mycelial biomass is expressed as the gram dry mycelial weight per liter (DMW). The data were the mean of three repeats.
in P. igniarius (vgb+) cultures decreased to 1.19 and 1.27 g/L, respectively. Acetic acid started to accumulate considerably after consuming 40 g/L of glucose, and it retarded cell growth and lowered the rates of glucose uptake and metabolites production in the later stages of fermentation. After glucose depletion, acetic acid concentration decreased gradually because acetic acid was used as the main carbon source. VHb protein not only enhanced metabolites synthesis but also obviously decreased acetic acid accumulation during fermentation by P. igniarius. Probably, TDNA insertions maybe result in inactivation of some genes, which altered biosynthesis pathway of acetic acid. 4. Conclusions In this study, the chromosomal integration of vgb into P. igniarius was described. Expression of the VHb in P. igniarius (vgb+) increased the yield of total flavones and exopolysaccharides which are accompanied with faster growth of the strain during fermentation. The information obtained from the current research is helpful for the hyperproduction of metabolites from higher fungi. Furthermore, with the development of modern genetic technology, genetic strategies increasingly focus on solving chemical engineering problems in biochemical engineering. Acknowledgements This work was financially supported by the Natural Science Foundation of China (No. 30800775) and the Fundamental Research Funds for the Central Universities (No. 27R0904083A).
References Geckil, H., Barak, Z., Chipman, D.M., Erenler, S.O., Webster, D.A., Stark, B.C., 2004. Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene. Bioprocess Biosyst. Eng. 26, 325–330. Guo, X., Zou, X., Sun, M., 2009. Effects of phytohormones on mycelial growth and exopolysaccharide biosynthesis of medicinal mushroom Phellinus linteus. Bioprocess Biosyst. Eng. 32, 701–707. Horng, Y.T., Chang, K.C., Chien, C.C., Wei, Y.H., Sun, Y.M., Soo, P.C., 2010. Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P promoter in Escherichia coli. Lett. Appl. Microbiol. 50, 158–167. Khleifat, K.M., Abboud, M.M., Al-Mustafa, A.H., Al-Sharafa, K.Y., 2006. Effects of carbon source and Vitreoscilla hemoglobin (VHb) on the production of betagalactosidase in Enterobacter aerogenes. Curr. Microbiol. 53, 277–281. Li, M., Wu, J., Lin, J., Wei, D., 2010. Expression of Vitreoscilla hemoglobin enhances cell growth and dihydroxyacetone production in Gluconobacter oxydans. Curr. Microbiol. (Epub ahead of print). Lin, Y.H., Li, Y.F., Huang, M.C., Tsai, Y.C., 2004. Intracellular expression of Vitreoscilla hemoglobin in Aspergillus terreus to alleviate the effect of a short break in aeration during culture. Biotechnol. Lett. 26, 1067–1072. Liu, Y.F., Yang, Y., Jia, W., Zhang, J.S., Tang, Q.J., Tang, C.H., 2006. Determination of total flavones in the medicinal mushroom Phellinus. Acta Edulis Fungi 13, 49– 52. Liu, Q.N., Liu, R.S., Wang, Y.H., Mi, Z.Y., Li, D.S., Zhong, J.J., Tang, Y.J., 2009. Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides. Bioresour. Technol. 100, 3644–3649. Lung, M.Y., Tsai, J.C., Huang, P.C., 2010. Antioxidant properties of edible basidiomycete Phellinus igniarius in submerged cultures. J. Food Sci. 75, E18–24. Ma, X., Fan, D., Shang, L.A., Cai, Q., Chi, L., Zhu, C., Mi, Y., Luo, Y.E., 2010. Oxygen transfer rate control in the production of human-like collagen by recombinant Escherichia coli. Biotechnol. Appl. Biochem. 55, 169–174. Mo, S.Y., Yang, Y.C., Shi, J.G., 2003. Studies on chemical constitutes of Phellinus igniarius. Zhongguo Zhong Yao Za Zhi 28, 339–341. Olano, C., Lombo, F., Mendez, C., Salas, J.A., 2008. Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab. Eng. 10, 281–292.
H. Zhu et al. / Bioresource Technology 102 (2011) 1747–1751 Rodriguez Estrada, A.E., Jimenez-Gasco Mdel, M., Royse, D.J., 2009. Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay. Bioresour. Technol. 100, 5270–5276. Sanny, T., Arnaldos, M., Kunkel, S.A., Pagilla, K.R., Stark, B.C., 2010. Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl. Microbiol. Biotechnol. doi:10.1007/s00253-010-2817-7. Shon, Y.H., Nam, K.S., 2004. Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea. Biotechnol. Lett. 26, 159–163. Song, T.Y., Lin, H.C., Yang, N.C., Hu, M.L., 2008. Antiproliferative and antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linnearus: Fries) Quelet. J. Ethnopharmacol. 115, 50–56. Su, Y., Li, X., Liu, Q., Hou, Z., Zhu, X., Guo, X., Ling, P., 2010. Improved polygamma-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. Bioresour. Technol. 101, 4733–4736. Tang, Y.J., Zhang, W., Zhong, J.J., 2009. Performance analyses of a pH-shift and DOTshift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum. Bioresour. Technol. 100, 1852–1859. Wei, X.X., Chen, G.Q., 2008. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol. 436, 273–287. Wei, M.L., Webster, D.A., Stark, B.C., 1998. Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size. Biotechnol. Bioeng. 57, 477–483.
1751
Wu, J.M., Hsu, T.A., Lee, C.K., 2003. Expression of the gene coding for bacterial hemoglobin improves beta-galactosidase production in a recombinant Pichia pastoris. Biotechnol. Lett. 25, 1457–1462. Xie, B.G., Zhu, H., 2003. Isolation and optimization of regeneration condition of Tremella fuciformis protoplasts. Mycosystema 22, 574–578. Xie, B.G., Zhu, H., Jiang, Y.J., Rao, Y.B., Zheng, J.G., 2004. Genetic transformation markers of Tremella fuciformis. J. Agric. Biotechnol. 12, 610–611. Yu, H., Shi, Y., Zhang, Y., Yang, S., Shen, Z., 2002. Effect of Vitreoscilla hemoglobin biosynthesis in Escherichia coli on production of poly(beta-hydroxybutyrate) and fermentative parameters. FEMS Microbiol. Lett. 214, 223–227. Zhong, J.J., Gong, H.G., 2005. Hydrodynamic shear stress affects cell growth and metabolite production by medicinal mushroom Ganoderma lucidum. Chin. J. Chem. Eng. 13, 426–428. Zhu, Z.P., Ma, H.L., 2007. Separation and regeneration of protoplast from Phellinus igniarius. Zhongguo Zhong Yao Za Zhi 32, 2232–2235. Zhu, H., Sun, S.J., 2009. Effect of constant glucose feeding on the production of exopolysaccharides by Tremella fuciformis spores. Appl. Biochem. Biotechnol. 152, 366–371. Zhu, H., Xie, B.G., 2003. Isolation and optimization of regeneration condition of Tremella fuciformis protoplasts. Mycosystema 22, 574–578. Zhu, H., Pan, R.J., Wang, T.W., Shen, Y.L., Wei, D.Z., 2006a. Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltransferase. Appl. Microbiol. Biotechnol. 72, 1033–1038. Zhu, H., Wang, T.W., Sun, S.J., Shen, Y.L., Wei, D.Z., 2006b. Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis. Appl. Microbiol. Biotechnol. 72, 770–776.