法尼烯是一种倍半萜化合物,在食品、香料等领域有广泛应用价值。法尼烯合成酶是法尼烯合成途径中的关键限速酶,对法尼烯的合成有重要催化作用。通过生物信息学方法获取了甜橙来源萜类合成酶(farnesene synthase from Citrus sinensis,FSCS)基因Fscs, 通过异源表达对其进行了功能鉴定,研究了重组酶的酶学性质并将其应用于酿酒酵母法尼烯合成。结果显示,在以法尼基焦磷酸为底物时,FSCS可催化其生成α-法尼烯;Mg2+为酶必需的辅因子,K+可促进产物生成,8 mmol/L以上Mn2+对产物生成有抑制作用,最适金属离子条件为30 mmol/L Mg2+, 40 mmol/L K+;最适反应温度为20 ℃,酶对高温耐受性差。将酶学性质应用于酿酒酵母重组表达菌株,最适金属离子条件下法尼烯产量提升了30%,达到43.76 mg/L;温度条件的改变对发酵后期法尼烯积累有一定促进作用。该研究对一种新的萜类合成酶进行了功能表征,并通过酶学性质研究为构建微生物细胞工厂高效生产法尼烯提供了参考。
Farnesene is a sesquiterpene compound which has a wide range of applications in food, fragrance & flavor and other industries. Farnesene synthase is the key enzyme in biosynthesis of farnesene. Its properties significantly affect the formation of farnesene. A farnesene synthase gene from Citrus sinensis (FSCS) was cloned and expressed in Escherichia coli BL21(DE3). The recombinant enzyme was purified and its biochemical characteristics were investigated. Results showed that FSCS could produce α-farnesene by using farnesyl diphosphate as substrate. In addition, Mg2+ was indispensable, and K+ could enhance the activity of FSCS. However, 8 mmol/L or more Mn2+ significantly inhibited its activity. The optimal condition of Mg2+ and K+ were 30 mmol/L and 40 mmol/L, respectively. The optimal temperature was 20℃, and sensitive to higher temperature. Under the optimal conditions in recombinant Saccharomyces cerevisiae, the yield of farnesene was 43.76 mg/L increased by 30%. The results could provide reference for efficient production of farnesene in microbial cell factories.
[1] ZHANG R B,WANG B,GROSSI G,et al.Molecular basis of alarm pheromone detection in aphids[J].Current Biology,2017,27(1):55-61.
[2] SOULEYRE E J F,BOWEN J K,MATICH A J,et al.Genetic control of α-farnesene production in apple fruit and its role in fungal pathogenesis[J].The Plant Journal,2019,100(6):1 148-1 162.
[3] WANG X W,ZENG L T,LIAO Y Y,et al.Formation of α-farnesene in tea (Camellia sinensis) leaves induced by herbivore-derived wounding and its effect on neighboring tea plants[J].International Journal of Molecular Sciences,2019,20(17):4 151-4 165.
[4] TIPPMANN S,SCALCINATI G,SIEWERS V,et al.Production of farnesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQ-controlled feed[J].Biotechnology and Bioengineering,2016,113(1):72-81.
[5] 孙丽超, 李淑英,王凤忠,等.萜类化合物的合成生物学研究进展[J].生物技术通报,2017,33(1):64-75.
SUN L C,LI S Y,WANG F Z,et al.Research progresses in the synthetic biology of terpenoids[J].Biotechnology Bulletin,2017,33(1):64-75.
[6] LANGE B M,AHKAMI A.Metabolic engineering of plant monoterpenes,sesquiterpenes and diterpenes-current status and future opportunities[J].Plant Biotechnology Journal,2013,11(2):169-196.
[7] MILLER D J,ALLEMANN R K.Sesquiterpene synthases:Passive catalysts or active players?[J] Natural Product Reports,2012,29(1):60-71.
[8] GREEN S,FRIEL E N,MATICH A,et al.Unusual features of a recombinant apple α-farnesene synthase[J].Phytochemistry,2007,68(2):176-188.
[9] LIN J Y,WANG D,CHEN X L,et al.An (E,E)-α-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles[J].Plant Biotechnology Journal,2017,15(4):510-519.
[10] PICAUD S,BRODELIUS M,BRODELIUS P E.Expression,purification and characterization of recombinant (E)-β-farnesene synthase from Artemisia annua[J].Phytochemistry,2005,66(9):961-967.
[11] ZHANG X H,NIU M Y,TEIXEIRA DA SILVA J A,et al.Identification and functional characterization of three new terpene synthase genes involved in chemical defense and abiotic stresses in Santalum album[J].BMC Plant Biology,2019,19(1):115.
[12] ASAI T,MATSUKAWA T,KAJIYAMA S.Metabolic changes in Citrus leaf volatiles in response to environmental stress[J].Journal of Bioscience and Bioengineering,2016,121(2):235-241.
[13] 刘宇. 过表达苹果α-法尼烯合酶基因烟草早衰机制研究[D].泰安:山东农业大学,2016.
LIU Y.Study on mechanisms of early senescence of tobacco overexpressing apple α-farnesene synthase gene[D].Taian:Shandong Agricultural University,2016.
[14] CHEN X L,KOLLNER T G,JIA Q D,et al.Terpene synthase genes in eukaryotes beyond plants and fungi:Occurrence in social amoebae[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(43):12 132-12 137.
[15] KIRYU M,HAMANAKA M,YOSHITOMI K,et al.Rice terpene synthase 18 (OsTPS18) encodes a sesquiterpene synthase that produces an antibacterial (E)-nerolidol against a bacterial pathogen of rice[J].Journal of General Plant Pathology,2018,84(3):221-229.
[16] GREEN S,SQUIRE C J,NIEUWENHUIZEN N J,et al.Defining the potassium binding region in an apple terpene synthase[J].Journal of Biological Chemistry,2009,284(13):8 661-8 669.
[17] HUANG M S,ABEL C,SOHRABI R,et al.Variation of herbivore-induced volatile terpenes among Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases,TPS02 and TPS03[J].Plant Physiology,2010,153(3):1 293-1 310.
[18] RUPASINGHE H P V,PALIYATH G,MURR D P.Sesquiterpene α-farnesene synthase:Partial purification,characterization,and activity in relation to superficial scald development in apples[J].Journal of the American Society for Horticultural Science,2000,125(1):111-119.
[19] ISMAIL K S,SAKAMOTO T,HASUNUMA T,et al.Zinc,magnesium,and calcium ion supplementation confers tolerance to acetic acid stress in industrial Saccharomyces cerevisiae utilizing xylose[J].Biotechnology Journal,2014,9(12):1 519-1 525.
[20] BARROS DE SOUZA R,SILVA R K,FERREIRA D S,et al.Magnesium ions in yeast:Setting free the metabolism from glucose catabolite repression[J].Metallomics,2016,8(11):1 193-1 203.
[21] ROLLERO S,BLOEM A,CAMARASA C,et al.Combined effects of nutrients and temperature on the production of fermentative aromas by Saccharomyces cerevisiae during wine fermentation[J].Applied Microbiology and Biotechnology,2015,99(5):2 291-2 304.
[22] 祝霞, 刘琦,赵丹丹,等.酿造条件对酿酒酵母发酵香气的影响[J].食品科学,2019,40(16):115-123.
ZHU X,LIU Q,ZHAO D D,et al.Effect of different winemaking conditions on fermentation aroma production by Saccharomyces cerevisiae[J].Food Science,2019,40(16):115-123.
