Abstract: 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.
 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.  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.  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.  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.  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.  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.  MILLER D J,ALLEMANN R K.Sesquiterpene synthases:Passive catalysts or active players?[J] Natural Product Reports,2012,29(1):60-71.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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 Saccharomycescerevisiae[J].Food Science,2019,40(16):115-123.