异源表达透明颤菌血红蛋白基因提高灵芝中灵芝酸的积累

摘要/Abstract

摘要： 灵芝中异源表达透明颤菌血红蛋白基因可提高灵芝酸的产量。我们构建了表达载体PJW-EXP-VGB, 载体上的灵芝三磷酸甘油醛脱氢酶基因启动子用于驱动透明颤菌血红蛋白基因在灵芝中的转录。将PJW-EXP-VGB转入到灵芝原生质体后，获得了转基因工程菌株。进一步分析了转基因菌株中透明颤菌血红蛋白的活性和透明颤菌血红蛋白表达对总灵芝酸产量，中间代谢产物羊毛甾醇积累以及灵芝酸合成途径上关键基因表达水平的影响。CO-差异波谱分析表明透明颤菌血红蛋白基因能在灵芝中成功表达且具有生物活性。在转基因灵芝细胞中，总灵芝酸的产量达到0.78 mg/100mg细胞干重，比野生型灵芝细胞高了1.5倍；羊毛甾醇的最大积累量比野生型灵芝低1.28倍；灵芝酸合成途径中关键酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶基因、鲨烯合酶基因、羊毛甾醇合酶基因的表达水平相比野生型菌株分别提升了1.6，1.5，1.6倍。结果表明异源表达透明颤菌血红蛋白基因是提高灵芝中灵芝酸的有效方法。

关键词: 灵芝, 灵芝酸, 透明颤菌血红蛋白, 中间代谢产物

Abstract: The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lingzhi to enhance antitumor ganoderic acid (GA) production. The VHb gene (Vitreoscilla hemoglobin, vgb), under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G .lingzi. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription levels of GA biosynthesis genes were investigated. In VHb-expressing G. lingzhi, the maximum crude concentrations of total individual GA was 0.78 mg/100 mg dry weight, which is 1.5-fold higher than that in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr), squalene synthase (sqs), and lanosterol synthase (ls) genes were up-regulated by 1.6-, 1.5- and 1.6 -fold respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

Key words: Ganoderma lingzhi, Ganoderic acid, Vitreoscilla hemoglobin, intermediate metabolite

中图分类号:

Q819

王俊杰徐军伟. 异源表达透明颤菌血红蛋白基因提高灵芝中灵芝酸的积累[J]. 食品科学.

参考文献

[1] SANODIYA B S, THAKUR G S, BAGHEL R K, et al. Ganoderma lucidum: a potent pharmacological macrofungus[J]. Biotechnol, 2009, 10(8), 717–742.
[2] BISHOP K S, KAO C H J, XU Y, et al. From 2000 years of Ganoderma lucidum to recent developments in nutraceuticals[J]. Phytochemistry, 2015, 114(SI), 56–65.
[3] XU J W, ZHAO W, ZHONG J J. Biotechnological production and application of ganoderic acids[J]. Biotechnol, 2010, 87(2), 457–466.
[4] ZHONG J J, TANG Y J. Submerged cultivation of medicinal mushrooms for production of valuable bioactive metabolites[J]. Biotechnolm, 2004, 87, 25–59.
[5] UPADHYAY M, SHRIVASTAVA B, Jain A et al. Production of ganoderic acid by Ganoderma lucidum RCKB-2010 and its therapeutic potential. Microbiol[J]. 2014, 64(2), 839–846.
[6] WEI Z H, DUAN Y Y, QIAN Y Q et al. Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods[J]. Bioprocess Biosyst. Eng, 2014, 37(9), 1789–1797.
[7] XU J W, XU YN, ZHONG J J. Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum. Microbiol[J]. Biotechnol, 2010, 85(4), 941–948.
[8] LIANG C X, LI Y B, XU J W et al. Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction[J]. Biotechnol, 2010, 86(5), 1367–1374.
[9] REN A, QIN L, SHI L et al. Methyl jasmonate induces ganoderic acid biosynthesis in the basidiomycetous fungus Ganoderma lucidum[J]. Technol, 2010, 101(17), 6785–6790.
[10] REN A, LI XB, MIAO Z G, et al. Transcript and metabolite alterations increase ganoderic acid content in Ganoderma lucidum using acetic acid as an inducer. Biotechnol[J]. Lett, 2014, 36(12), 2529–2536.
[11] LI H J, ZHANG D H, HAN L L, et al. Further improvement in ganoderic acid production in static liquid culture of Ganoderma lucidum by integrating nitrogen limitation and calcium ion addition[J]. Bioprocess Biosyst. Eng, 2016, 39(1), 75–80.
[12] TANG Y J, ZHANG W, ZHONG J J. Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum[J]. Bioresour. Technol, 2009, 100(5), 1852–1859.
[13] ZHU L W, ZHONG J J, TANG Y J. Multi-fed batch culture integrated with three-stage light irradiation and multiple additions of copper ions for the hyperproduction of ganoderic acid and ganoderma polysaccharides by the medicinal mushroom Ganoderma lucidum[J]. Process Biochem, 2010, 45(12), 1904–1911.
[14] TANG Y J, ZHONG J J. Role of oxygen supply in submerged fermentation of Ganoderma lucidum for production of ganoderma polysaccharide and ganoderic acid[J]. Enzyme Microb. Technol, 2003, 32(3-4), 478–484.
[15] ZHANG W X, TANG Y J, ZHONG J J. Impact of oxygen level in gaseous phase on gene transcription and ganoderic acid biosynthesis in liquid static cultures of Ganoderma lucidum[J]. Bioprocess Biosyst. Eng, 2010, 33(6), 683–690.
[16] STARK B C, PAGILLA K R, DIKSHIT K L. Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation[J]. Appl. Microbiol. Biotechnol, 2015, 99(4), 1627–1636.
[17] ZHANG S, WANG J, WEI Y, et al. Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation[J]. J. Biotechnol, 2014, 187, 147–153.
[18] WANG S, LIU F, HOU Z, et al. Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) [J]. World J. Microbiol. Biotechnol, 2014, 30(4), 1369–1376.
[19] ZHANG S, WANG J, WEI Y, et al. Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation[J]. J. Biotechnol, 2014, 187, 147–153.
[20] LI H J, ZHANG D H, YUE T H, et al. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene[J]. J. Biotechnol, 2016, 217, 132–137.
[21] SUEN Y L, TANG H, HUANG J, et al. Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin[J]. J. Agric. Food. Chem, 2014, 62(51), 12392–12398.
[22]ZHU H, SUN S, ZHANG S. Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius. Bioresour[J]. Technol, 2011, 102(2), 1747–1751.
[23] FREY A D, KALLIO P T. Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology[J]. FEMS Microbiol. Rev, 2003, 27(4), 525–545.
[24] ZHANG L, LI Y, WANG Z, et al. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering[J]. Biotechnol. Adv, 2007, 25(2), 123–136.
[25] CHEN S, XU J, LIU C, et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat. Commun, 2012, 3, 913.
[26] SHI L, FANG X, LI M, et al. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum[J]. World J. Microbiol. Biotechnol., 2012, 28(1), 283–291.
[27]YU X, JI S L, HE Y L, et al. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher basidiomycetes) [J]. Int. J. Med. Mushrooms, 2014, 16(2), 161–168.
[28] YOU B J, LEE M H, TIEN N, et al. A novel approach to enhancing ganoderic acid production by Ganoderma lucidum using apoptosis induction[J]. PLoS One, 2013, 8(1), e53616.