Yeast terminator engineering： from mechanism exploration to artificial design

doi:10.12211/2096-8280.2020-009

Abstract

Abstract:

The complexity of biological systems poses challenges for the construction of biological components. The discovery of new control parts and the design of stable and adjustable parts have become one of the important contents of synthetic biology. Acted as a genetic part independently of the coding gene to terminate transcription,a terminator is an important biological part for tuning gene expression when designing synthetic gene networks. The activity of terminator can be strong or weak, thus the choice of terminator will directly affect the amount of mRNA produced. With the gradual clarification of the structure and function on terminator and in-depth analysis of the mechanism of transcription termination, terminator engineering has been rapidly developed to construct shorter, controllable and designable terminators. In this review, the progress about the structural discovery, functional characterization, and transcription termination mechanism of terminator in Saccharomyces cerevisiae are systematically summarized. The artificial design of terminator and its application in the field of fine regulation of pathway engineering are discussed. The challenges and possible solutions for terminator engineering, potential and significance for developing terminator engineering in non-model yeast are also prospected. This review provides a theoretical guidance for researchers to develop synthetic biological elements and optimization of heterologous synthetic pathways.

Key words: Saccharomyces cerevisiae, terminator, mRNA processing, transcription termination, metabolic pathway regulation

摘要：

生物系统的复杂性为生物元器件的构建提出了挑战，新型控制元件的发现和稳定可调节的元件设计成为合成生物学的重要内容之一。终止子作为基因元件独立于编码基因行使终止转录的功能，是一种重要的合成生物学控制元件。研究表明终止子作用有强有弱，终止子的选择会直接影响mRNA的量，并且随着终止子结构与功能的逐渐清晰和对转录终止机理的深入解析，以短小、可控、可设计为特征的终止子工程得以快速发展。本文以常规酿酒酵母为基础，系统总结了酿酒酵母中终止子元件在结构发现、功能表征、转录终止机理方面的最新研究进展，并讨论了终止子的人工设计及在途径工程精细调控领域的应用情况，展望了终止子工程面临的挑战、可能的解决途径及在非模式酵母中发展的潜力和意义。这为研究人员开发合成生物学元件和异源合成途径优化提供了科学的理论参考。

关键词: 酿酒酵母, 终止子, mRNA加工, 转录终止, 代谢途径调控

CLC Number:

SHENG Yue, ZHANG Genlin. Yeast terminator engineering： from mechanism exploration to artificial design[J]. Synthetic Biology Journal, 2020, 1(6): 709-721.

盛月, 张根林. 酵母终止子工程：从机理探索到人工设计[J]. 合成生物学, 2020, 1(6): 709-721.

Figures/Tables 4

References 125

1	温栾, 卢俊南, 钟伟, 等. 合成生物学设计技术[J]. 中国细胞生物学学报, 2019,41(11): 2060-2071.
	WEN L, LU J N, ZHONG W, et al. Design technology in synthetic biology[J]. Chinese Journal of Cell Biology, 2019, 41(11): 2060-2071.
2	赵禹, 赵雅坤, 刘士琦, 等. 非常规酵母的分子遗传学及合成生物学研究进展[J].微生物学报,2020,60(8): 1574-1591.
	ZHAO Y, ZHAO Y K, LIU S Q, et al. Progress in molecular genetics and synthetic biology of unconventional yeast[J]. Acta Microbiologica Sinica, 2020,60(8):1574-1591.
3	邢玉华, 谭俊杰, 李玉霞, 等. 合成生物学的关键技术及应用进展[J]. 中国医药生物技术, 2012, 7(5): 357-363.
	XIN Y H, TAN J J, LI Y X, et al. Key technology and application progress of synthetic biology[J]. Chinese Medical Biotechnology, 2012, 7(5): 357-363.
4	赵国屏. 合成生物学:开启生命科学"会聚"研究新时代[J]. 中国科学院院刊, 2018, 33(11): 1135-1149.
	ZHAO G P. Synthetic biology:unsealing the convergence era of life science research[J]. Bulletin of Chinese Academy of Sciences, 2018,33(11): 1135-1149.
5	HEIDI R, NICHOLAS M, ALPER H S. The synthetic biology toolbox for tuning gene expression in yeast[J]. FEMS Yeast Research, 2014, 15(1): 1-12.
6	FISCHER V, SCHUMACHER K, TORA L, et al. Global role for coactivator complexes in RNA polymerase II transcription[J]. Transcription, 2019, 10(1): 29-36.
7	LAWSON M R, BERGER J M. Tuning the sequence specificity of a transcription terminator[J]. Current Genetics, 2019, 65(3): 729-733.
8	JEONG S W, LANG W H, REEDER R H. The release element of the yeast polymerase I transcription terminator can function independently of Reb1p.[J]. Molecular & Cellular Biology, 1995, 15(11): 5929-5936.
9	JEONG S W, LANG W H, REEDER R H. The yeast transcription terminator for RNA polymerase I is designed to prevent polymerase slippage.[J]. Journal of Biological Chemistry, 1996, 271(27): 16104-16110
10	CHOO K B, WU S M, HUNG L, et al. Effect of vector type, host strains and transcription terminator on heterologous gene expression in yeast[J]. Biochemical and Biophysical Research Communications, 1986, 140(2): 602-608.
11	WITTMANN A, SUESS B. Engineered riboswitches: expanding researchers′ toolbox with synthetic RNA regulator[J]. FEBS Letters, 2012, 586(15): 2076-2083.
12	BRENT R, PTASHNE M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene[J]. Nature, 1985, 314(5995): 198.
13	GUO Z, SHERMAN F. Signals sufficient for 3′-end formation of yeast mRNA[J]. Molecular & Cellular Biology, 1996, 16(6): 2772-2776.
14	THOMPSON C M, KOLESKE A J, CHAO D M, et al. A multisubunit complex associated with the RNA polymerase Ⅱ CTD and TATA-binding protein in yeast[J]. Cell, 1993, 73(7): 1361-1375.
15	GUO Z, SHERMAN F. 3′-end-forming signals of yeast mRNA[J]. Molecular & Cellular Biology, 1995, 15(11): 5983-5990.
16	RYAN K, CALVO O, MANLEY J L. Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease[J]. RNA, 2004, 10(4): 565-573.
17	SHI Y S, MANLEY J L. The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site[J]. Genes & Development, 2015, 29(9): 889-897.
18	CHAN S L, HUPPERTZ I, YAO C G, et al. CPSF30 and Wdr33 directly bind to AAUAA in mammalian mRNA 3′ processing[J]. Genes & Development, 2014, 28(21): 2370-2380.
19	WHITELAW E, PROUDFOOT N. Alpha-thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3′ end processing in the human alpha 2 globin gene[J]. The EMBO Journal, 1986, 5(11): 2915-2922.
20	GIBBS M D, REEVES R A, ANWAR S, et al. A yeast intron as a translational terminator in a plasmid shuttle vector[J]. FEMS Yeast Research, 2004, 4(6): 573-577.
21	DICHTL B. Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage andpolyadenylation factor[J]. European Molecular Biology Organization Journal, 2001, 20(12): 3197-3209.
22	CHEN S, HYMAN L E. A specific RNA-protein interaction at yeast polyadenylation efficiency elements[J]. Nucleic Acids Research, 1998, 26(21): 4965-4974.
23	BROCKMAN J, SINGH P, LIU D, et al. PACdb: PolyA cleavage site and 3′-UTR database[J]. Bioinformatics, 2005, 21(18): 3691-3693.
24	KELLER W, MINVIELLE-SEBASTIA L. A comparison of mammalian and yeast pre-mRNA 3′-end processing[J]. Current Opinion in Cell Biology, 1997, 9(3): 329-336.
25	MACDONALD C C, JOSÉ-LUIS R. Reexamining the polyadenylation signal: were we wrong about AAUAAA? [J]. Molecular & Cellular Endocrinology, 2002, 190(1): 1-8.
26	TULLER T, RUPPIN E, KUPIEC M. Properties of untranslated regions of the S. cerevisiae genome[J]. BMC Genomics, 2009, 10(1): 391.
27	CURRAN K A, MORSE N J, MARKHAM K A, et al. Short synthetic terminators for improved heterologous gene expression in yeast[J]. ACS Synthetic Biology, 2015, 4(7): 824-832.
28	MACPHERSON M, SAKA Y. Short synthetic terminators for assembly of transcription units in vitro and stable chromosomal integration in yeast S. cerevisiae [J]. ACS Synthetic Biology, 2017, 6(1): 130-138.
29	SEOANE S, GUIARD B, RODRÍGUEZ-TORRES A M, et al. Effects of splitting alter native KlCYC1 3′-UTR regions on processing: metabolic consequences and biotech nological applications[J]. Journal of Biotechnology, 2005, 118(2): 149-156.
30	YAMANISHI M, ITO Y, KINTAKA R, et al. A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a ‘Terminatome' toolbox[J]. ACS Synthetic Biology, 2013, 2(6): 337-347.
31	YAMANISHI M, KATAHIRA S, MATSUYAMA T. TPS1 Terminator increases mRNA and protein yield in a Saccharomyces cerevisiae expression system[J]. Bioscience Biotechnology and Biochemistry,2011, 75(11): 2234-2236.
32	ITO Y, KITAGAWA T, YAMANISHI M, et al. Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae [J]. Scientific Reports, 2016, 6(1): 197-214.
33	WEI L N, WANG Z X, ZHANG G L, et al. Characterization of terminators in Saccharomyces cerevisiae and exploration factors affecting their strength[J]. ChemBioChem, 2017, 18: 2422-2427.
34	KECMAN T, KUŚ K, HEO D H, et al. Elongation/termination factor exchange mediated by PP1 phosphatase orchestrates transcription termination[J]. Cell Reports, 2018, 25(1): 259-269.
35	CAMBRAY C, GUIMARAES J C, MUTALIK V K, et al. Measurement and modeling of intrinsic transcription terminators[J]. Nucleic Acids Research, 2013,41(9): 5139-5148.
36	LEAVITT J M, ALPER H S. Advances and current limitations in transcript-level control of gene expression[J]. Current Opinion in Biotechnology, 2015, 34(3): 98-104.
37	SOUTOURINA J. Transcription regulation by the Mediator complex[J]. Nature Reviews Molecular Cell Biology, 2017, 19(4): 262-274.
38	PORRUA O, LIBRI D. Transcription termination and the control of the transcriptome: why, where and how to stop[J]. Nature Reviews Molecular Cell Biology, 2015, 16(3): 190-202.
39	PROUDFOOT N. New perspectives on connecting messenger RNA 3′ end formation to transcription[J]. Current Opinion in Cell Biology, 2004, 16(3): 272-278.
40	HARLEN K M, CHURCHMAN L S. The code and beyond: transcription regulation by the RNA polymerase Ⅱ carboxy-terminal domain[J]. Nature Reviews Molecular Cell Biology, 2017, 18(4): 263-273.
41	STEURER B, JANSSENS R C, GEVERTS B, et al. Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA polymerase Ⅱ[J]. Proceedings of the National Academy of Ences, 2018, 115(19): E4368-E4376.
42	EATON J D, DAVIDSON L, BAUER D L V, et al. Xrn2 accelerates termination by RNA polymerase Ⅱ, which is underpinned by CPSF73 activity[J]. Genes & Development, 2018, 32(2): 127.
43	PUIG S, PÉREZ-ORTÍN J, MATALLANA E. Transcriptional and structural study of a region of two convergent overlapping yeast genes[J]. Current Microbiology, 1999, 39(6): 369-373.
44	GRZYBOWSKA E A, WILCZYNSKA A, SIEDLECKI J A. Regulatory functions of 3′UTRs.[J]. Biochemical & Biophysical Research Communications, 2001, 288(2): 291-295.
45	DICHTL B. Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination[J]. The EMBO Journal, 2014, 21(15): 4125-4135.
46	ITO Y, YAMANISHI M, IKEUCHI A, et al. Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae [J]. Journal of Biotechnology, 2013, 168(4): 486-492.
47	CURRAN K A, KARIM A S, GUPTA A, et al. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications[J]. Metabolic Engineering, 2013, 19(Complete): 88-97.
48	KELLER W, BIENROTH S, LANG K M, et al. Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3′ processing signal AAUAAA[J]. The EMBO Journal,1991, 10(13):4241-4249.
49	TATOMER D C, ELROD N D, LIANG D, et al. The Integrator complex cleaves nascent mRNAs to attenuate transcription[J]. Genes & Development, 2019, 33(21/22): 1525-1538.
50	CHIU A C, SUZUKI H I, WU X B, et al. Transcriptional pause sites delineate stable nucleosome-associated premature polyadenylation suppressed by U1 snRNP[J]. Molecular Cell,2018, 69(4): 648-663, e7.
51	DEZAZZO J D, IMPERIALE M J. Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit[J]. Molecular & Cellular Biology, 1989, 9(11): 4951-4961.
52	SHAW G, KAMEN R. A conserved adenine uridine sequence from the 3′untranslated region of granulocyte-monocyte colony stimulating factor messenger RNA mediates selective messenger RNA degradation[J]. Cell, 1986, 46: 659-668.
53	WENG L, LI Y, XIE X, et al. Poly(A) code analyses reveal key determinants for tissue-specific mRNA alternative polyadenylation[J]. RNA, 2016, 22(6): 813-821
54	MANDEL D R, TONG L, BAI Y. Protein factors in pre-mRNA 3′-end processing[J]. Cellular & Molecular Life Sciences, 2007, 65(7/8): 1099-1122.
55	CHAN S, CHOI E A, SHI Y. Pre-mRNA 3′-end processing complex assembly and function[J]. RNA, 2011,2(3): 321-335.
56	ERIKA L P, JOEL H G, SUSAN D, et al. Ipa1 Is an RNA polymerase II elongation factor that facilitates termination by maintaining levels of the poly(A) site endonuclease Ysh1[J]. Cell Reports, 2019, 26(7): 1919-1933.
57	TIAN B, MANLEY J L. Alternative cleavage and polyadenylation: the long and short of it[J]. Trends in Biochemical Sciences, 2013, 38(6): 312-320.
58	DAFNE C D G, KENSEI N, MANLEY J L, et al. Mechanisms and consequences of alternative polyadenylation[J]. Molecular Cell, 2011, 43(6): 853-866.
59	VIPHAKONE N, VOISINET-HAKIL F, MINVIELLE S L. Molecular dissection of mRNA poly(A) tail length control in yeast[J]. Nucleic Acids Research, 2008, 36(7): 2418-2433.
60	PORRUA O, HOBOR F, BOULAY J, et al. In vivo SELEX reveals novel sequence and structural determinants of Nrd1-Nab3-Sen1-dependent transcription termination.[J].The EMBO Journal, 2014, 31(19): 3935-3948.
61	SIERECKI E. The Mediator complex and the role of Protein-Protein interactions in the gene regulation machinery[J]. Seminars in Cell and Developmental Biology, 2020, 99: 20-30.
62	LEMAY J F, MARGUERAT S, LAROCHELLE M, et al. The Nrd1-like protein Seb1 coordinates cotranscriptional 3′ end processing and polyadenylation site selection[J]. Genes & Development, 2016, 30(13): 1558-1572.
63	GRZECHNIK P, GDULA M R, PROUDFOOT N J. Pcf11 orchestrates transcription termination pathways in yeast[J]. Genes & Development, 2015, 29(8): 849-861.
64	NEMEC C M, YANG F, GILMORE J M, et al. Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(20): E3944-E3953.
65	LUNDE B M, REICHOW S, KIM M, et al. Cooperative interaction of transcription termination factors with the RNA polymerase Ⅱ C-terminal domain[J]. Nature Structural & Molecular Biology, 2010,17(10): 1195-1201.
66	KUMAR R, REYNOLDS D M, SHEVCHENKO A, et al. Forkhead transcription factors, Fkh1p and Fkh2p, collaborate with Mcm1p to control transcription required for M-phase[J]. Current Biology, 2000, 10(15): 896-906.
67	ZHANG Z, KLATT A, HENDERSON A J, et al. Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression[J]. Genes & Development, 2007, 21(13): 1609-1614.
68	ZHANG Z, GILMOUR D S. Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript[J]. Molecular Cell, 2006, 21(1): 65-74.
69	NOBLE C G, HOLLINGWORTH D, MARTIN S R, et al.Key features of the interaction between Pcf11 CID and RNA polymerase II CTD[J]. Nature Structural & Molecular Biology, 2005, 12(2): 144-151
70	ZHANG Z. CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3′-end processing factor, Pcf11[J]. Genes & Development, 2005, 19(13): 1572-1580.
71	KAMIENIARZ-GDULA K, GDULA M R, PANSER K, et al. Selective roles of vertebrate PCF11 in premature and full-length transcript termination[J]. Molecular Cell, 2019,74(1): 158-172.e9.
72	RAPHAËL H, FRÉDÉRIQUE M, NICOLAS V, et al. An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination[J].Nucleic Acids Research. 2012, 40(3): 1226-1239.
73	LUNDE B M, REICHOW S L, KIM M,et al. Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain[J]. Nature Structural & Molecular Biology, 2010, 17(10): 1195-1201.
74	NOBLE C G, BARBARA B, TAYLOR I A.Structure of a nucleotide-bound Clp1-Pcf11 polyadenylation factor[J]. Nucleic Acids Research, 2007, 35(1): 87-99.
75	SINGH N, MA Z, GEMMILL T, et al. The Ess1 prolyl isomerase is required for transcription termination of small noncoding RNAs via the Nrd1 pathway[J].Molecular Cell, 2009, 36(2): 255-266.
76	E- J CHO, KOBOR M S, KIM M, et al. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain[J]. Genes & Development, 2002, 15(24): 3319-3329.
77	JONES J C, PHATNANI H P, HAYSTEAD T A,et al. C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats[J]. Journal of Biological Chemistry, 2004, 279(24): 24957-24964.
78	OSTAPENKO D, SOLOMON M J. Phosphorylation by cak1 regulates the C-terminal domain kinase Ctk1 in Saccharomyces cerevisiae [J]. Molecular and Cellular Biology, 2005, 25(10): 3906-3913.
79	XIAO T J, HALL H, KIZER K O, et al. Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast[J]. Genes & Development, 2003, 17(5): 654-663.
80	EATON J D, DAVIDSON L, BAUER D L V, et al. Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity[J]. Genes & Development, 2018, 32(2): 127-139.
81	FONG N, BRANNAN K, ERICKSON B, et al. Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase Ⅱ termination suggest widespread kinetic competition[J]. Molecular Cell, 2015, 60(2): 256-267.
82	NICOLAS V, FLORENCE V H, LIONEL M S. Molecular dissection of mRNA poly(A) tail length control in yeast[J]. Nucleic Acids Research, 2008, 36(7): 2418-33.
83	WEISS E A, GILMARTIN G M, NEVINS J R. Poly(A) site efficiency reflects the stability of complex formation involving the downstream element.[J]. The EMBO Journal, 1991, 10(1): 215-219.
84	FATICA A, MORLANDO M, BOZZONI I. Yeast snoRNA accumulation relies on a cleavage‐dependent/polyadenylation‐independent 3′‐processing apparatus[J]. The EMBO Journal, 2014, 19(22): 6218-6229.
85	EGECIOGLU D E, HENRAS A K, CHANFREAU G F. Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome[J]. RNA, 2006, 12(1): 26-32.
86	GHAZAL G, GAGNON J, JACQUES P E, et al. Yeast rNase Ⅲ triggers polyadenylation-independent transcription termination[J]. Molecular Cell, 2009, 36(1): 99-109.
87	MATA J. Genome-wide mapping of polyadenylation sites in fission yeast reveals widespread alternative polyadenylation[J]. RNA Biology, 2013, 10(8): 1407-1414.
88	YAMANAKA S, YAMASHITA A, HARIGAYA Y, et al. Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells.[J]. The EMBO Journal, 2010, 29(13): 2173-2181.
89	MORALES J C, RICHARD P, ROMMEL A, et al. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair[J]. Nucleic Acids Research, 2014, 42(8): 4996-5006.
90	QIU H, HU C, HINNEBUSCH A G. Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters[J]. Molecular Cell, 2009, 33(6): 752-762.
91	MIRNA J, HINZE H, HOLZER H. Inactivation of Yeast Enzymes by proteinase A and B and carboxypeptidase Y from yeast[J]. Hoppe-Seyler´s Zeitschrift für Physiologische Chemie, 1976, 357(1): 735-740.
92	HOUSELEY J, RUBBI L, GRUNSTEIN M, et al. A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster[J]. Molecular Cell, 2008, 32(5): 685-695.
93	STEVEN P, MARCIN G. FRACZEK, JIAN WU, et al . A resource for functional profiling of noncoding RNA in the yeast Saccharomyces cerevisiae[J]. RNA, 2017, 23(8): 1166-1171.
94	RUSSO P, SHERMAN F. Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae [J]. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86(21): 8348-8352.
95	EMBO R. Yeast Trf5p is a nuclear poly(A) polymerase[J]. The EMBO Reports, 2006, 7(2): 205-211.
96	CARROLL K L, PRADHAN D A, GRANEK J A, et al. Identification of cis elements directing termination of yeast nonpolyadenylated snoRNA transcripts[J]. Molecular and Cellular Biology, 2004, 24(14): 6241-6252.
97	WILSON S M. Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae [J]. The Journal of Cell Biology, 1994, 127(5): 1173-1184.
98	STEINMETZ E J, CONRAD N K, BROW D A, et al. RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts[J]. Nature, 2001, 413(6853): 327-331.
99	ZHANG W, LIU H, LI X, et al. Production of naringenin from D-xylose with co-culture of E. coli and S. cerevisiae [J]. Engineering in Life Sciences, 2017, 17: 1021-1029.
100	LIAN J, SI T, NAIR N U, ZHAO H. Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains[J]. Metabolic Engineering, 2014, 24: 139-149.
101	RENDA B A, HAMMERLING M J, BARRICK J E. Engineering reduced evolutionary potential for synthetic biology[J]. Molecular Biosystems, 2014, 10(7): 1668-1678.
102	CURRAN K A, KARIM A S, GUPTA A, et al. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications[J]. Metabolic Engineering, 2013, 19: 88-97.
103	LEBER C, SILVA N A D. Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids[J]. Biotechnology & Bioengineering, 2014, 111(2).
104	ITO Y, YAMANISHI M, IKEUCHI A, et al. Combinatorial screening for transgenic yeasts with high cellulase activities in combination with a tunable expression system[J]. PLoS One, 2015, 10(12): e0144870.
105	CHEN Y J, LIU P, NIELSEN A A K, et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints[J]. Nature Methods, 2013, 10(7): 659-664.
106	WANG Z X, WEI L N, SHENG Y, et al. Yeast synthetic terminators: fine regulation of strength through linker sequences[J]. ChemBioChem, 2019, 20(18): 2383-2389.
107	CHEN Y, XIAO W H, WANG Y, et al. Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering[J]. Microbial Cell Factories, 2016, 15(1): 113.
108	JOSEPH V G, ZARMIK M, XIAO C F, et al. Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast[J]. Cell, 2014, 156(4): 812-824.
109	MISCHO H E, PROUDFOOT N J. Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast [J]. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2013,1829(1): 174-185.
110	LIAN J Z, SI T, NAIR N U, et al. Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains.[J]. Metabolic Engineering,2014, 24: 139-149.
111	SILVA N A DA, SRIKRISHNAN S. Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae [J]. FEMS Yeast Research, 2012,12(2): 197-214.
112	HONG S P, SEIP J, WALTERS P D, et al. Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter[J]. Yeast, 2012, 29(2): 59-72.
113	LI X, WANG Z X, ZHANG G l, et al. Improving lycopene production in Saccharomyces cerevisiae through optimizing pathway and chassis metabolism[J]. Chemical Engineering Science, 2019, 193: 364-369.
114	AHMED M S, IKRAM S, RASOOL A, et al. Design and construction of short synthetic terminators for β-amyrin production in Saccharomyces cerevisiae [J]. Biochemical Engineering Journal, 2019, 146: 105-116.
115	XIE Z X, LIU D, LI B Z, et al. Design and chemical synthesis of eukaryotic chromosomes[J]. Chemical Society Reviews, 2017, 46(23): 7191-7207.
116	SONG H, DING M Z, JIA X Q, et al. Synthetic microbial consortia: from systematic analysis to construction and applications[J]. Chemical Society Reviews, 2014, 43(20): 6954-6981.
117	ZHANG Y, GU L F, HOU Y F, et al. Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeting alternative polyadenylation[J]. Cell Research, 2015, 25(7): 864-876.
118	ZHANG X, ZUO X, YANG B, et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation[J]. Cell, 2014, 158(3): 607-619.
119	XUE Y C, ZHOU Y, WU T B, et al. Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping[J]. Molecular Cell,2009, 36(6): 996-1006.
120	XIAO R, TANG P, YANG B, et al. Nuclear matrix factor hnRNP U/SAF-A exerts a global control of alternative splicing by regulating U2 snRNP maturation[J]. Molecular Cell. 2012, 45(5): 656-668.
121	FENG S, XU M, LIU F, et al. Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe [J]. BMC Plant Biology, 2019, 19(1): 365.
122	PICELLI S, FARIDANI O R, BJÖRKLUND A K, et al. Full-length RNA-seq from single cells using Smart-seq2[J]. Nature Protocols, 2014, 9(1): 171-181.
123	PRIELHOFER R, BARRERO J J, STEUER S, et al. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris [J]. BMC Systems Biology, 2017, 11(1): 123.
124	SARAYA R, KRIKKEN A M, VEENHUIS M, et al. Novel genetic tools for Hansenula polymorpha [J]. FEMS Yeast Research,2012,12(3): 271-278.
125	LARROUDE M, ROSSIGNOL T, NICAUD J M, et al. Synthetic biology tools for engineering Yarrowia lipolytica [J]. Biotechnology Advances, 2018, 36(8): 2150-2164.

终止子类型	名称	FI值	活性	参考文献
天然	IRS4t	1.3501	弱	[30]
	DNM1t	0.6579	弱	[30]
	NFT1t	0.1944	弱	[30]
	HOG1t	3.3178	强	[33]
	SSD1t	3.2127	强	[33]
	TPS1t	3.0941	强	[33]
	ATP5t	1.9912	中	[33]
	CYC1t	1.7559	中	[33]
	SIR2t	1.6812	中	[33]
	PGK1t	1.5185	弱	[33]
	TDH3t	1.4914	弱	[33]
	SLX5t	1.3802	弱	[33]
人工	T-Guo1	2.2000		[13，27]
	T0-T10	0.2~2.4400		[28]
	Tsynth1-30	0.03~3.7000		[27]
	T-1316	3.5270	强	[106]
	T-1299	3.5124	强	[106]
	T-1281	3.4866	强	[106]
	T-195	2.9325	中	[106]
	T-154	2.9370	中	[106]
	T-a9	2.9405	中	[106]
	T-509	2.6149	弱	[106]
	T-414	2.4048	弱	[106]
	T-d11	2.3648	弱	[106]

终止子类型	名称	FI值	活性	参考文献
天然	IRS4t	1.3501	弱	[30]
	DNM1t	0.6579	弱	[30]
	NFT1t	0.1944	弱	[30]
	HOG1t	3.3178	强	[33]
	SSD1t	3.2127	强	[33]
	TPS1t	3.0941	强	[33]
	ATP5t	1.9912	中	[33]
	CYC1t	1.7559	中	[33]
	SIR2t	1.6812	中	[33]
	PGK1t	1.5185	弱	[33]
	TDH3t	1.4914	弱	[33]
	SLX5t	1.3802	弱	[33]
人工	T-Guo1	2.2000		[13，27]
	T0-T10	0.2~2.4400		[28]
	Tsynth1-30	0.03~3.7000		[27]
	T-1316	3.5270	强	[106]
	T-1299	3.5124	强	[106]
	T-1281	3.4866	强	[106]
	T-195	2.9325	中	[106]
	T-154	2.9370	中	[106]
	T-a9	2.9405	中	[106]
	T-509	2.6149	弱	[106]
	T-414	2.4048	弱	[106]
	T-d11	2.3648	弱	[106]

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