中国科学院微生物研究所,中国微生物学会
文章信息
- 张慧杰, 廖思敏, 凌小翠, 冯家勋, 秦秀林. 2022
- ZHANG Huijie, LIAO Simin, LING Xiaocui, FENG Jiaxun, QIN Xiulin.
- 毕赤酵母截短PGK1启动子与不同终止子组合调控外源基因表达
- Truncated PGK1 promoter is paired with varied terminators to regulate heterologous gene expression in Pichia pastoris
- 微生物学报, 62(7): 2642-2657
- Acta Microbiologica Sinica, 62(7): 2642-2657
-
文章历史
- 收稿日期:2021-10-28
- 修回日期:2022-01-15
- 网络出版日期:2022-06-27
巴斯德毕赤酵母(Pichia pastoris,syn. Komagataella phaffii)具有高等真核表达系统的诸多优点,还可实现高密度发酵,且分泌到胞外的内源蛋白少[ 1],易于胞外目的蛋白分离纯化,已成为诸多药品、抗体和外源蛋白的表达平台[ 2– 3],被广泛地应用于基础研究和工业生产。随着代谢工程和合成生物学的快速发展[ 4– 5],毕赤酵母已成为新一代的细胞工厂,而控制基因表达对于优化代谢途径和合成基因网络调控至关重要。因此,挖掘毕赤酵母表达系统的基因表达调控工具迫在眉睫。
启动子、终止子和转录因子等是代谢途径和合成基因网络高效表达所需优化的重要调控元件。其中,启动子和终止子分别位于基因编码框上、下游并调控基因的转录速率和mRNA稳定性,它们的强度(活性)与靶基因编码的蛋白表达量直接相关。因此,通过启动子和终止子的改造,可以获得基因表达调控的有力工具。
启动子中含有转录因子(TFS)和其他转录调节因子结合的位点,在调控基因表达时起到关键作用,使其成为代谢工程和合成生物学工具包中的重要元件之一。在毕赤酵母中,通过改造获得序列更短、活性更高的启动子,可以提高重组菌的α-淀粉酶基因的表达水平[ 6]和南极假丝酵母来源的脂肪酶基因的表达量[ 7]。近期,Hansenula polymorpha来源的甲醇诱导型启动子PMOX也成功用于毕赤酵母表达系统中,并用于调控基因表达。Vogl等[ 8]筛选并构建了毕赤酵母的双向启动子(bidirectional promoter,BDP)文库,应用BDP文库对靶基因进行精确调控,优化了多基因的共表达,从而实现了紫杉二烯和β-胡萝卜素复杂合成代谢途径在酵母细胞中的整合。
终止子也是调控基因表达的重要组件,负责基因的转录终止、释放新生mRNA、调节mRNA的稳定性和半衰期。终止子主要通过调控mRNA的稳定性和半衰期,从而间接调节靶蛋白产量[ 9],终止子改造也逐渐应用到基因调控和代谢工程领域[ 10]。Rajkumar等[ 11]表征了30多个组成型和诱导型启动子、终止子,并将这些启动子和终止子进行组合,调控报告基因gfp的表达,构建了可应用于马克斯克鲁维酵母(Kluyveromyces marxianus)多基因调控的工具。在酿酒酵母(Saccharomyces cerevisiae)中,将终止子DIT1突变后,其活力增加了500%[ 12]。Yamanishi等[ 13]以PTDH3为启动子和gfp为报告基因,检测了5 302个终止子的强度,这些终止子强度是对照终止子TPGK1的0.036到2.52倍,可作为酿酒酵母代谢工程改造的基因调控工具。为了挖掘可用于调控毕赤酵母中外源基因表达水平的终止子序列,Ito等[ 10]系统鉴定了72个终止子的强度,包括内源性的、酿酒酵母来源的和人工合成的终止子,这些终止子调控基因表达的强调可达到对照的17倍。
毕赤酵母中参与甲醇利用途径(methanol utilization pathway,MUT)、糖酵解途径(glycolysis pathway)和活性氧(reactive oxygen species,ROS)防御相关基因的表达量较高[ 14],这些基因的启动子和终止子可作为调控外源基因表达的有用工具。本研究对磷酸甘油酸激酶(phosphoglycerate kinase,PGK1)基因的启动子进行截短并对其强度进行鉴定。然后,选择了上述途径相关基因的终止子和毕赤酵母表达系统常用终止子进行强度表征。这9个终止子是:醇氧化酶(alcohol oxidase,ID:8201223)基因终止子TAOX1、醇脱氢酶(alcohol dehydrogenase,ID:8200841)基因终止子TADH2、甲醛脱氢酶(formaldehyde dehydrogenase,ID:8199001)基因终止子TFLD1、甲醇脱氢酶(formate dehydrogenase,ID:8200284)基因终止子TFDH1、过氧化氢酶(catalase,ID:8198267)基因终止子TCAT1、3-磷酸甘油醛脱氢酶(glyceraldehyde 3-phosphate dehydrogenase,ID:8198905)基因终止子TGAP、磷酸甘油酸激酶(phosphoglycerate kinase,ID:8197742)基因终止子TPGK1、转录增强因子(transcription elongation factor 1,ID:8198713)基因终止子TTEF1和酿酒酵母细胞色素C (cytochrome C,ID:853507)基因终止子TCYC1。将3个强度的启动子分别与不同终止子进行组合,选取其中6个组合(调控基因表达量最高和最低相比达到10倍),用于调控呋喃果糖苷酶(β-fructofuranosidases,β-Ffase)基因的表达。本工作构建的启动子-终止子组合可以实现目的基因的不同水平的表达,可作为毕赤酵母代谢途径优化和靶基因精确调控的有力工具。
1 材料与方法 1.1 菌株和培养基毕赤酵母(Pichia pastoris)野生型菌株GS115保藏于本实验室。大肠杆菌(Escherichia coli)菌株Trans1-T1用于基因克隆。
LLB培养基(g/L):胰蛋白胨10,酵母提取物5,NaCl 10,使用时添加25 μg/mL Zeocin。固体培养基添加1.5%琼脂。
YPD培养基(g/L):胰蛋白胨20,酵母提取物10,葡萄糖20。
MD培养基:1.34%酵氮源基础(yeast nitrogen base,YNB),0.000 04%生物素,葡萄糖10 g/L,1.5%琼脂。
BMDY培养基(g/L):酵母提取物10,胰蛋白胨20,1.34% YNB,葡萄糖10,0.000 04%生物素,100 mmol/L K2HPO4/KH2PO4。
BMD培养基:1.34% YNB,10 g/L葡萄糖,4×10–5%生物素,100 mmol/L磷酸钾,pH 6。
1.2 材料和试剂质粒小规模提取试剂盒、通用型DNA纯化回收试剂盒、RNA提取试剂盒均购自TIANGEN公司;2×Phanta® Max Master Mix、2×Taq Plus Master Mix、Exnase Ⅱ工具酶均购自Vazyme公司;限制性内切酶和1 kb DNA Marker购自Thermo Fisher Scientific公司;T4 DNA Ligase、PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time)购自TaKaRa公司;ZeocinTM购自Invitrogen公司。
1.3 引物和质粒本研究所用引物见 表 1,均由生工生物工程(上海)股份有限公司合成。
Primer names | Primer sequences (5ʹ→3ʹ) | Application |
PGK1-F | TCGACTAGTAGTTGGGTATTCAAATAGTTGACTT | Amplification of the PGK1 promoter |
PGK1-R | AGAATGCGGCCGCTTTCGTAATCAATTGGGCTATG | |
PP-F | AACTACCGCATTAGGATCTTCG | Amplification of PPP |
PD-F | ACTAGTCGATCGCATTTTGGCCTCA | Amplification of PPD |
GFP-F | AATGCGGCCGCATGTCTAAAGGTGAAGAATTATTC | Amplification of the egfp gene |
GFP-R | CCCAAGCTTTTATTTGTACAATTCATCCATACC | |
BF-F | AATGCGGCCGCATGCCCGTAGATTCTTCTCATAAG | Amplification of the BF gene |
BF-R | CCCAAGCTTTCACCTGATCGCTATGCATG | |
TADH2-F | CCCAAGCTTGCCGAATAGTTTGTATACGTCTT | Amplification of the ADH2 terminator |
TADH2-R | CGCGGATCCTTTTAAAATTGAACGGCGACC | |
TCAT1-F | CCCAAGCTTGCTAACTATATTTATTATTAATTAA | Amplification of the CAT1 terminator |
TCAT1-R | CGCGGATCCGATTGTGACCTTTGTCTCTAT | |
TCYC1-F | CCCAAGCTTCACGTCCGACGGCGGC | Amplification of the CYC1 terminator |
TCYC1-R | CGCGGATCCAGCTTGCAAATTAAAGCCTTCGAG | |
TFDH1-F | CCCAAGCTTTTGAAATGTATTTAATTTGATATTA | Amplification of the FDH1 terminator |
TFDH1-R | CGCGGATCCACGATGTACAATCTGAGCTTG | |
TFLD1-F | CCCAAGCTTGTGTATAGTCAATAATAGCCGGAGT | Amplification of the FLD1 terminator |
TFLD1-R | CGCGGATCCATTAACTAAGAACAGCTTTTCCCG | |
TGAP-F | CCCAAGCTTATCGATTTGTATGTGAAATAGCTG | Amplification of the GAP terminator |
TGAP-R | CGCGGATCCGTTCAATTATAGATCCACGAGTG | |
TPGK1-F | CCCAAGCTTTTAGTTCATATAGTTTGAATTCTGA | Amplification of the PGK1 terminator |
TPGK1-R | CGCGGATCCCCGGTCCAGGCCATCA | |
TTEF1-F | GGATGAATTGTACAAATAAAAGCTTATTGCTTGAAGCTTTAATTTATTT | Amplification of the TEF1 terminator |
TTEF1-R | TGAAGCTATGGTGTGTGGGGGATCCACAGATTCATTGACTCTATGATCTC | |
RT-GAP-F | CGGCATCTTCAGTGTAACCC | Amplification of the GAP gene |
RT-GAP-R | GGCTTTCCGTGTCCCAAC | |
RT-GFP-F | GGCTGACAAACAAAAGAATGG | Amplification of the egfp gene |
RT-GFP-R | GGATAAGGCAGATTGAGTGGAT | |
RT-LacZ-F | ATACTGTCGTCGTCCCCTCAAAC | Amplification of the lacZ gene |
RT-LacZ-R | CGGATTCTCCGTGGGAACAA | |
RT-BF-F | TGACCTCATCACCTGGAAAGA | Amplification of the β-Ffase gene |
RT-BF-R | TGTGGTGGTCGCTTGTCAG |
本研究构建的质粒见 表 2。
Plasmids | Description | Sources or references |
pGHg | The egfp gene expressing plasmid, PGAP-egfp-ZeoR-His4 | [ 15] |
pPHg | Plasmid containing egfp gene under the control of the promoter PPGK1, PPGK1-egfp-ZeoR-His4 | This study |
pPPHg | Plasmid containing egfp gene under the control of the truncated promoter PPP, PPP-egfp-ZeoR-His4 | This study |
pPEHg | Plasmid containing egfp gene under the control of the truncated promoter PPE, PPE-egfp-ZeoR-His4 | This study |
pPGHg | Plasmid containing egfp gene under the control of the truncated promoter PPG, PPG-egfp-ZeoR-His4 | This study |
pPDHg | Plasmid containing egfp gene under the control of the truncated promoter PPD, PPD-egfp-ZeoR-His4 | This study |
pPHL | Plasmid containing lacZ gene under the control of the promoter PPGK1, PPGK1-lacZ-ZeoR-His4 | This study |
pPPHL | Plasmid containing lacZ gene under the control of the truncated promoter PPP, PPP- lacZ -ZeoR-His4 | This study |
pPEHL | Plasmid containing lacZ gene under the control of the truncated promoter PPE, PPE- lacZ -ZeoR-His4 | This study |
pPGHL | Plasmid containing lacZ gene under the control of the truncated promoter PPG, PPG- egfp -ZeoR-His4 | This study |
pPDHL | Plasmid containing lacZ gene under the control of the truncated promoter PPD, PPD- 1acZ -ZeoR-His4 | This study |
pG-TPGK1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TPGK1, PPG-egfp-TPGK1-ZeoR-His4 | This study |
pG-TAOX1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TAOX1, PPG-egfp-TAOX1-ZeoR-His4 | This study |
pG-TTEF1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TTEF1, PPG-egfp- TTEF1-ZeoR-His4 | This study |
pG-TADH2 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TADH2, PPG-egfp-TADH2-ZeoR-His4 | This study |
pG-TGAP | Plasmid containing egfp gene under the control of the truncated promoter PG with terminator TGAP, PPG-egfp-TGAP-ZeoR-His4 | This study |
pG-TCYC1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TCYC1, PPG-egfp-TCYC1-ZeoR-His4 | This study |
pG-TFLD1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TFLD1, PPG-egfp-TFLD1-ZeoR-His4 | This study |
pG-TCAT1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TCAT1, PPG-egfp-TCAT1-ZeoR-His4 | This study |
pG-TFDH1 | Plasmid containing egfp gene under the control of the truncated promoter PPG with terminator TFDH1, PPG-egfp-TFDH1-ZeoR-His4 | This study |
pE-TPGK1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TPGK1, PPE-egfp-TPGK1-ZeoR-His4 | This study |
pE-TAOX1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TAOX1, PPE-egfp-TAOX1-ZeoR-His4 | This study |
pE-TTEF1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TTEF1, PPE-egfp-TTEF1-ZeoR-His4 | This study |
pE-TADH2 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TADH2, PPE-egfp-TADH2-ZeoR-His4 | This study |
pE-TGAP | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TGAP, PPE-egfp-TGAP-ZeoR-His4 | This study |
pE-TCYC1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TCYC1, PPE-egfp-TCYC1-ZeoR-His4 | This study |
pE-TFLD1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TFLD1, PPE-egfp-TFLD1-ZeoR-His4 | This study |
pE-TCAT1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TCAT1, PPE-egfp-TCAT1-ZeoR-His4 | This study |
pE-TFDH1 | Plasmid containing egfp gene under the control of the truncated promoter PPE with terminator TFDH1, PPE-egfp-TFDH1-ZeoR-His4 | This study |
pD-TPGK1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TPGK1, PPD-egfp-TPGK1-ZeoR-His4 | This study |
pD-TAOX1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TAOX1, PPD-egfp-TAOX1-ZeoR-His4 | This study |
pD-TTEF1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TTEF1, PPD-egfp-TTEF1-ZeoR-His4 | This study |
pD-TADH2 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TADH2, PPD-egfp-TADH2-ZeoR-His4 | This study |
pD-TGAP | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TGAP, PPD-egfp- TGAP-ZeoR-His4 | This study |
pD-TCYC1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TCYC1, PPD-egfp-TCYC1-ZeoR-His4 | This study |
pD-TFLD1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TFLD1, PPD-egfg-TFLD1 -ZeoR-His4 | This study |
pD-TCAT1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TCAT1, PPD-egfp-TCAT1-ZeoR-His4 | This study |
pD-TFDH1 | Plasmid containing egfp gene under the control of the truncated promoter PPD with terminator TFDH1, PPD-egfp-TFDH1-ZeoR-His4 | This study |
pD-BF-TG | Plasmid containing PoFF32A gene under the control of the truncated promoter PPD with terminator TGAP, PPD-BF-TGAP-ZeoR-His4 | This study |
pD-BF-TP | Plasmid containing PoFF32A gene under the control of the truncated promoter PPD with terminator TPGK1, PPD-BF-TPGK1-ZeoR-His4 | This study |
pD-BF-TT | Plasmid containing PoFF32A gene under the control of the truncated promoter PPD with terminator TTEF1, PPD-BF-TTEF1-ZeoR-His4 | This study |
pG-BF-TA | Plasmid containing PoFF32A gene under the control of the truncated promoter PPG with terminator TAOX1, PPG-BF-TAOX1-ZeoR-His4 | This study |
pE-BF-TA | Plasmid containing PoFF32A gene under the control of the truncated promoter PPE with terminator TAOX1, PPD-BF-TAOX1-ZeoR-His4 | This study |
pD-BF-TA | Plasmid containing PoFF32A gene under the control of the truncated promoter PPD with terminator TAOX1, PPD-BF-TAOX1-ZeoR-His4 | This study |
1.4 截短启动子调控报告基因表达的毕赤酵母重组菌的构建
以毕赤酵母GS115基因组总DNA为模板,PCR扩增启动子PPGK1,将PPGK1克隆至载体pGHg[ 15]的SpeⅠ/NotⅠ位点,替换PGAP1启动子,获得酵母增强型绿色荧光蛋白基因egfp表达质粒pPHg。利用PCR和酶切的方法,简单快速地将启动子PPGK1序列进行截短。首先PCR扩增5ʹ端截短启动子PPP (1.3 kb),用PPP替换载体pPHg中的PPGK1启动子构建质粒pPPHg。然后,质粒pPPHg经Cla Ⅰ酶切后自连,获得含截短启动子PPE (1.1 kb)的质粒pPEHg;质粒pPEHg经Xho Ⅰ酶切后自连,获得含截短启动子PPG (866 bp)的质粒pPGHg。根据已发表的截短的PGK1启动子PPD3序列[ 6],用引物PD-F/ PGK1-PCR扩增获得截短启动子PPD (与PPD3序列比对,缺失第380 bp位碱基C)。将PPD替换载体pPHg中的PPGK1构建质粒pPDHg。将重组质粒pPHg、pPPHg、pPEHg、pPGHg、pPDHg分别用Sal Ⅰ线性化后电击法转化菌株GS115,构建表达报告基因egfp的重组毕赤酵母。
用Not Ⅰ/Hind Ⅲ双酶切质粒pGHL[ 15],获得lacZ基因片段并克隆到质粒pPHg、pPPHg、pPEHg、pPGHg、pPDHg的Not Ⅰ/Hind Ⅲ位点,替换其中的egfp基因,构建lacZ基因的表达质粒pPHL、pPPHL、pPEHL、pPGHL、pPDHL。将重组质粒分别用Sal Ⅰ线性化后电击法转化GS115,构建表达报告基因lacZ的重组毕赤酵母。本研究所构建质粒见 表 2。
1.5 不同启动子和终止子组合调控外源基因表达重组菌构建为了便于终止子的克隆,先构建质粒pPGHG,在终止子5ʹ和3ʹ端分别加上Hind Ⅲ和BamH Ⅰ酶切位点,便于后续终止子替换。首先,以PPG为启动子,构建含不同终止子的系列egfp表达载体pG-Tx (Tx代表终止子:TADH2,TFLD1,TPGK1,TGAP,TCAT1,TCYC1,TFDH1或TTEF1)。以pGHG为模板,用引物TCYC1-F/TCYC1-R扩增TCYC1序列;其他7个终止子用GS115总DNA为模板扩增获得。最后,将8个终止子序列分别替换载体pPGHG上的TAOX1,构建系列重组质粒pG-Tx。用PPE启动子替换质粒pG-TT上的PPG启动子,构建含不同终止子的系列载体pE-Tx。用PPD启动子替换质粒pG-TT上的PPG启动子,构建含不同终止子的载体pD-Tx。质粒pG-Tx经Sal Ⅰ线性化后电击法转化GS115,构建重组菌G-ADH2、G-FLD1、G-PGK1、G-GAP、G-CAT1、G-CYC1、G-FDH1、G-TEF1。
以质粒pPIC9k-PoFF32A[ 16]为模板,用引物BF-F/BF-R扩增含信号肽编码序列的呋喃果糖苷酶(β-Ffase)基因PoFF32A序列,将该序列克隆到pD-Tx (Tx=TGAP1、TPGK1和TTEF1)、pG-TAOX1、pE-TAOX1和pD-TAOX1质粒的Not Ⅰ/ Hind Ⅲ位点,替换原质粒上的egfp,分别构建PoFF32A表达质粒pD-BF-TG、pD-BF-TP、pD-BF-TT、pG-BF-TA、pE-BF-TA和pD-BF-TA。将重组质粒经Sal Ⅰ线性化后电击法转化GS115,构建表达β-Ffase基因并使产物分泌的重组毕赤酵母菌株:PD-TG、PD-TP、PD-TT、PG-TA、PE-TA和PD-TA。
1.6 重组毕赤酵母的yEGFP荧光强度测定毕赤酵母重组菌经48孔深孔板高能量培养[ 17],BMD培养基28、340 r/min培养36 h后,取30 μL菌液至装有170 μL PBS的96孔酶标板中,使用多功能酶标仪(BioTek Synergy H1)检测GFP荧光强度(激发波长:485 nm,发射波长:515 nm,增益:90)。检测GFP荧光强度时,以不表达egfp的重组菌G/PEH为对照除去背景干扰。荧光强度F/OD600 (RFU/OD600)为荧光值与对应细胞密度OD600的比值,以荧光强度表征启动子或终止子强度。
1.7 重组毕赤酵母中基因转录水平的检测将重组毕赤酵母菌在YPD液体培养基中培养36 h后,用RNA提取试剂盒提取重组毕赤酵母菌的总RNA,然后用PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time)试剂盒反转录获得cDNA。最后用SYBR® Premix Ex TaqⅡ (Tli RNaseH Plus)(2×)进行RT-qPCR,以GAPDH基因作为内参基因,RT-qPCR所用引物见 表 1。
1.8 重组毕赤酵母摇瓶培养产β-Ffase将重组毕赤酵母菌株在YPD平板培养,28培养到长出单菌落;挑取单菌落于含有5 mL YPD培养基的试管中活化过夜;以4%的接种量接种于含50 mL BMDY培养基的500 mL三角瓶中,250 r/min、28培养60 h,每隔12 h取样测定OD600和β-Ffase活力,补加葡萄糖至终浓度为1%、pH试纸测定pH值并用5 mol/L KOH调pH至6.0左右。
1.9 呋喃果糖苷酶酶活检测重组毕赤酵母的呋喃果糖苷酶(β-Ffase)酶活测定采用DNS法[ 16],以0.2 mol/L柠檬酸-磷酸缓冲液(pH 5.5)稀释粗酶液,以20%蔗糖为底物,于40反应15 min。每分钟从蔗糖底物中释放出1 μmol还原糖所需的酶量定义为1个酶活力单位。
重组毕赤酵母的β-半乳糖苷酶(β-galactosidase,Gal)酶活力测定参照毕赤酵母表达系统操作手册(Pichia expression kit,Invitrogen)。酶活力单位指在28条件下,每分钟水解1 nmol ONPG (o-nitrophenyl-β-D-galactopyranoside)所需的酶量。
2 结果与分析 2.1 截短PGK1启动子构建及强度表征毕赤酵母3-磷酸甘油酸激酶(3-phosphoglycerate kinase,PGK1)基因启动子(PPGK1)是组成型强度中等的启动子[ 18],其序列较长(约2 kb)不便于表达框的构建和启动子的改造。因此,在避免启动子活性降低的情况下,截短了PPGK1序列,构建了启动子PPP、PPE、PPG和PPD ( 图 1A)。
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图 1 PPGK1截短启动子构建及其在毕赤酵母中驱动egfp基因表达的强度的表征 Figure 1 Construction of the truncated PPGK1 variants and characterization of their strength in driving the expression of gene egfp in P. pastoris. A: schematic representation of the construction of the truncated promoters; B: initial characterization of the truncated promoters using egfp as reporter gene. |
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利用启动子PPP、PPE、PPG和PPD驱动基因egfp在毕赤酵母中的表达,通过检测毕赤酵母重组菌的yEGFP比荧光强度表征对应启动子强度。含不同启动子重组菌的比荧光强度与egfp的mRNA水平相关,说明重组菌之间绿色荧光蛋白水平的差异是由mRNA水平的差异引起的,且启动子的强度由强至弱依次为:PPD、PPE、PPP、PPGK1、PPG。与野生型启动子PPGK1相比,PPG启动子强度下降了30%;PPP、PPE、PPD启动子强度分别提高了约10%、40%、90%;4个截短启动子的强度是启动子PPGK1的70%–190%;这4个启动子调控表达的yEGFP荧光强度,强启动子PPD是弱启动子PPG的2.7倍;在不同启动子调控下,yEGFP荧光强度与egfp的mRNA水平相关( 图 1B)。
以β-半乳糖苷酶(β-galactosidase, Gal)基因(lacZ)为第二报告基因,对截短启动子可靠性和适用性进行验证。在启动子PPG、PPP、PPE、PPD的调控下,Gal的酶产量随着启动子强度的增加而增加,Gal酶产量变化范围与yEGFP荧光强度的基本一致(达到野生型PPGK1启动子强度的65%–170%);这4个启动子调控表达的Gal酶产量,强启动子PPD是弱启动子PPG的2.6倍;不同启动子调控下,Gal酶产量与lacZ的mRNA水平相关( 图 2)。
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图 2 不同截短启动子构建的重组菌的β-半乳糖苷酶基因表达水平和酶产量的检测 Figure 2 Measurement of β-galactosidase (Gal) production and lacZ mRNA level under the control of the truncated promoters (PPG, PPE and PPD) in the engineered P. pastoris strains. |
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利用2个报告基因对截短启动子进行了系统表征,证实截短启动子PPE、PPG、PPD对不同基因表达调控的普遍适用性。与野生型启动子PPGK1相比,PPG、PPE、PPD长度分别缩短了57%、45%和79%;PPG、PPE、PPD启动子强度约是PPGK1的70%、140%和180%。
2.2 弱启动子和不同终止子组合调控egfp的表达毕赤酵母Mut途径、PPP途径和ROS途径的基因表达量较高,这些基因的终止子可以作为调控靶基因过表达的调控元件[ 14]。我们选取了3个强度的组成型启动子PPG (弱)、PPE (中)和PPD (强)分别与9个终止子TX (TX代表:TAOX1、TADH2、TFLD1、TPGK1、TGAP、TCAT1、TCYC1、TFDH1和TTEF1)进行组合,调控egfp的表达,以研究不同强度启动子与终止子组合调控靶基因表达的能力。对应重组菌(G-TX、E-TX和D-TX)构建过程如 图 3A所示。通过检测报告蛋白yEGFP荧光强度,研究不同启动子-终止子组合调控外源基因表达的能力。
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图 3 弱启动子PPG与不同终止子组合调控egfp基因表达的毕赤酵母重组菌的荧光强度检测 Figure 3 Fluorescence output of Pichia pastoris recombinants expressing egfp gene under the control of the weaker promoter PPG paired with various terminators. A: schematic diagram of the construction of P. pastoris recombinants expressing egfp gene under control of varied promoter paired with 9 terminators; B: box plot diagram of yEGFP fluorescence of G-Tx transformants; C: egfp transcript abundance versus yEGFP fluorescence intensity are plotted. |
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我们构建了弱启动子PPG分别与9个终止子组合调控egfp表达的重组菌G-TX,每个组合分别挑选10个转化子,利用48深孔板培养36 h,检测其荧光强度并进行统计分析。在9个终止子中,启动子PPG与TPGK1、TAOX1组合时,重组菌yEGFP荧光强度最高;其次是与TTEF1组合;与TCAT1、TFDH1的组合调控能力最弱( 图 3B)。
分别选取各组合重组菌中荧光强度最高且egfp基因拷贝数为1的3个转化子用摇瓶培养进行复筛,设3次生物学重复。重组菌G-TX摇瓶培养36 h,测定yEGFP荧光强度,提取RNA检测egfp基因转录水平。PPG为截短的PGK1基因启动子,将PPG与自身基因终止子TPGK1组合(PPG-TPGK1)作为对照,比较各组合调控egfp表达的强度。摇瓶培养后检测结果显示,各组合重组菌G-Tx的荧光强度与48深孔板初筛结果基本一致( 图 3C)。PPG与9个终止子组合调控强度由高到低为:TPGK1、TAOX1、TTEF1、TADH2、TGAP、TCYC1、TFLD1、TCAT1和TFDH1。各组合调控的egfp转录水平与yEGFP荧光强度相关性较好,调控强度是对照组合(PPG-TPGK1)的25%–100% ( 表 3);其中PPG-TPGK1组合的强度是PPG-TFDH1组合的4倍,即PPG-TX组合的调控范围跨度为4倍。
Promoters | Terminators | ||||||||
TAOX1 | TADH2 | TGAP | TCAT1 | TPGK1 | TCYC1 | TFLD1 | TTEF1 | TFDH1 | |
PPG | 93% | 75% | 74% | 31% | 100% | 55% | 34% | 81% | 29% |
PPE | 220% | 142% | 109% | 105% | 100% | 88% | 83% | 55% | 41% |
PPD | 248% | 180% | 148% | 78% | 100% | 78% | 76% | 46% | 33% |
Fluorescence intensity relative to TPGK1. |
2.3 中强度启动子和不同终止子组合调控egfp的表达
构建中强度启动子PPE与9个终止子组合调控egfp表达的重组菌E-TX,每个组合分别挑选10个转化子,通过48深孔板培养检测其荧光强度。初筛结果显示,在PPE调控下,与终止子TAOX1和TADH1的组合,重组菌yEGFP荧光强度显著高于其他组合重组菌;与TTEF1和TFDH1的组合调控能力最弱( 图 4A)。各组合重组菌E-TX摇瓶复筛结果与48深孔板初筛结果基本一致。PPE与9个终止子组合调控强度由高到低为:TAOX1、TADH2、TGAP、TCAT1、TPGK1、TCYC1、TFLD1、TTEF1和TFDH1。各组合调控的egfp转录水平与yEGFP荧光强度相关性较好,调控强度是对照组合(PPE-TPGK1)的30%–210% ( 表 3);其中PPE-TAOX1组合的强度是PPE-TFDH1组合的7倍,即PPE-TX组合的调控范围跨度为7倍。
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图 4 中强度启动子PPE与不同终止子组合调控egfp表达的毕赤酵母重组菌的荧光强度检测 Figure 4 Fluorescence output of recombinant Pichia pastoris expressing egfp gene under the control of the moderate strength promoter PPG paired with varied terminators. A: box plot diagram of yEGFP fluorescence of E-Tx transformants; B: egfp transcript abundance versus yEGFP fluorescence intensity are plotted. |
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2.4 强启动子和不同终止子组合调控egfp的表达
构建强启动子PPD与9个终止子组合调控egfp表达的重组菌D-
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图 5 强启动子PPD与不同终止子组合调控egfp表达的毕赤酵母重组菌的荧光强度检测 Figure 5 Fluorescence output of recombinant Pichia pastoris expressing egfp gene under the control of the strong promoter PPD paired with varied terminators. A: box plot diagram of yEGFP fluorescence of D-Tx transformants; B: egfp transcript abundance versus yEGFP fluorescence intensity are plotted. |
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2.5 不同启动子和终止子组合调控外源蛋白分泌表达
来源于草酸青霉(Penicillium oxalicum)菌株GXU20的呋喃果糖苷酶(β-fructofuranosidase,β-Ffase)能高效转移果糖生成新低聚果糖(neo-fructooligosaccharides,neo-FOS)[ 16]。Neo-FOS较其他FOS甜度更高,在双歧杆菌增殖方面表现更优异,其市场需求与日俱增。利用毕赤酵
母分泌表达β-Ffase,可为neo-FOS生产奠定基础。因此,以来源于草酸青霉的β-Ffase作为模式蛋白,研究不同启动子-终止子组合对外源基因的调控能力。从上述27个组合中,我们选取调控范围跨度较大的6个组合(PPG-TAOX1、PPE-TAOX1、PPD-TAOX1、PPD-TGAP1、PPD-TPGK1和PPD-TTEF1)进一步验证。这6个组合在调控egfp表达时,yEGFP荧光强度最高的和最低的相比达到10倍。将这6个组合用于调控β-Ffase分泌表达,构建对应重组菌:PG-TA、PE-TA、PD-TA、PD-TG、PD-TP和PD-TT。
各组合重组菌摇瓶培养实验结果表明,不同强度启动子与终止子组合调控β-Ffase基因(PoFF32A)表达,不影响重组菌的生长( 图 6A)。强启动子PPD与不同终止子组合时,随着终止子强度增强,β-Ffase酶产量呈上升趋势;强终止子TAOX1与不同启动子组合时,随着启动子强度增强β-Ffase酶产量也呈上升趋势( 图 6B)。各组合调控的PoFF32A转录水平与β-Ffase酶产量相关性较好,调控范围在对照组合PD-TP (PPD-TPGK1)的30%–180%;其中最强组合PD-TA的胞外β-Ffase酶产量是最弱组合PD-TT的6倍( 图 6C)。
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图 6 启动子与终止子组合调控β-Ffase在毕赤酵母中的分泌表达 Figure 6 Influence of promoter and terminator combinations on β-Ffase secretory expression in Pichia pastoris. A: growth of recombinant strains expressing PoFF32A gene using under the control of varied promoter paired with TPGK1 or TAOX1 terminator; B: extracellular β-Ffase activity of recombinant strains in shake flask; C: PoFF32A transcript abundance versus β-Ffase activity are plotted; D: yEGFP fluorescence versus β-Ffase activity are plotted. |
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在同一系列启动子-终止子组合调控下,egfp基因表达水平与PoFF32A基因表达水平相关性较好;yEGFP基因表达水平的调控范围达到10倍,而β-Ffase分泌表达水平的调控范围相对变窄,仅达到6倍( 图 6D)。结果表明,这些组合对不同基因具有较一致的调控能力,均能实现对目的基因的较强的表达调控,不管其表达产物是在胞内还是在胞外,可作为代谢工程改造的有力基因调控工具。
3 讨论控制基因表达对于优化代谢途径和合成基因网络至关重要。近几年,研究人员展示了合成生物学组件(主要是启动子和终止子)在酵母表达系统中的潜在适用性和应用前景[ 19]。毕赤酵母可利用的启动子和终止子有限,而代谢工程改造常涉及多个基因的表达调控,这就无法避免相同启动子和终止子序列的多次使用,导致细胞内重复序列发生同源重组,造成遗传的不稳定,影响内源和外源基因表达。构建强度和序列不同的启动子和终止子组合,可作为毕赤酵母代谢途径改造和合成生物应用的有力工具。
本研究构建了毕赤酵母截短的PGK1启动子PPE、PPG和PPD,这些组成型启动子强度是野生型启动子强度的70%–190%,其中强启动子PPD强度是弱启动子PPG的2.7倍。这些启动子的长度较PPGK1缩短了至少45%,便于启动子的改造和外源基因表达框的构建。Arruda等[ 6]报道的截短PGK1启动子(PPD3),其强度与PPGK1相比无差异。相同启动子表征的强度不同,主要原因可能是:两个研究中启动子所调控表达的基因不同,本研究用yEGFP荧光强度和Gal酶产量来表征启动子强度,而Arruda等用胞外α-淀粉酶产量表征;报告基因表达框整合位点也不同,本研究中表达框整合在基因组的His4基因位点,Arruda等将表达框整合在PGK1基因位点。已有研究发现,外源基因整合位点会影响毕赤酵母重组蛋白的产量[ 20]。
我们选取了毕赤酵母的9个终止子,分别与弱(PPG)、中(PPE)和强(PPD)启动子组合调控外源基因的表达,这27个组合可以在较宽广的范围实现基因的连续调控。分别与9个终止子组合时,PPG、PPE和PPD启动子调控egfp基因表达水平最高的和最低的比分别达到4倍、7倍和10倍,强启动子的调控强度变化范围远大于弱、中启动子的。同一终止子分别与3个启动子组合时,大部分终止子与强启动子组合的调控强度高于与弱、中启动子组合的。这说明,当强启动子与不同终止子组合调控时,启动子通过影响基因的转录水平来调控基因表达,且发挥着主导作用。同一启动子与不同终止子组合调控时,重组菌的egfp基因转录水平与yEGFP荧光强度相关性较好,这说明终止子通过影响基因的mRNA量来调控基因的表达,而mRNA量的变化很可能与终止子调控mRNA半衰期有关[ 13]。本研究也表征了一个酿酒酵母的常用终止子TCYC1,在毕赤酵母表达系统中TCYC1强度比大多数内源性终止子弱,尽管其在酿酒酵母中具有较高的强度[ 21]。这很可能是因为,在酿酒酵母和毕赤酵母中,基因表达的调节机制不同。
启动子工程应用于代谢工程改造和合成生物学的相关研究已有较多报道,但对于终止子及其与启动子组合调控的研究较少。我们构建的这些启动子-终止子组合在调控不同基因(egfp和lacZ)表达时都有效,从中选择了6个组合用于调控β-Ffase的分泌表达,以验证其调控基因表达能力。在这6个启动子-终止子组合调控下,胞内表达的yEGFP和Gal酶产量变化范围达到10倍(对照组合的25%–250%),而分泌表达的β-Ffase酶产量变化范围变窄,仅为6倍(对照组合的30%–180%)。这是因为,与胞内表达相比,胞外表达重组酶的酶产量不仅与其编码基因的mRNA量相关,分泌途径中的蛋白质折叠、修饰、降解和转运都影响着其最终酶产量的高低[ 22]。同一系列启动子-终止子组合调控下,胞外的β-Ffase还要经历复杂的分泌途径。在这个过程中部分重组酶β-Ffase很可能来不及折叠而被降解,或转运、分泌速率较低而滞留胞内,最终导致胞外酶产量比实际的总酶产量低。上述结果证明,通过选择合适的启动子-终止子组合,可以达到目的蛋白的较高表达量。因此,我们通过构建和检测不同的启动子-终止子组合,可以实现目的基因的不同水平的表达,获得了毕赤酵母代谢途径优化和靶基因精确调控的有力工具。
[1] | Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microbial Cell Factories, 2009, 8: 29. DOI:10.1186/1475-2859-8-29 |
[2] | Zhu TC, Sun HB, Wang MY, Li Y. Pichia pastoris as a versatile cell factory for the production of industrial enzymes and chemicals: current status and future perspectives. Biotechnology Journal, 2019, 14(6): 1800694. DOI:10.1002/biot.201800694 |
[3] | Virdi V, Palaci J, Laukens B, Ryckaert S, Cox E, Vanderbeke E, Depicker A, Callewaert N. Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model. Nature Biotechnology, 2019, 37(5): 527-530. DOI:10.1038/s41587-019-0070-x |
[4] | Patra P, Das M, Kundu P, Ghosh A. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnology Advances, 2021, 47: 107695. DOI:10.1016/j.biotechadv.2021.107695 |
[5] | Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D. Metabolic engineering of Pichia pastoris. Metabolic Engineering, 2018, 50: 2-15. DOI:10.1016/j.ymben.2018.04.017 |
[6] | Arruda A, Reis VCB, Batista VDF, Daher BS, Piva LC, De Marco JL, De Moraes LMP, Torres FAG. A constitutive expression system for Pichia pastoris based on the PGK1 promoter. Biotechnology Letters, 2016, 38(3): 509-517. DOI:10.1007/s10529-015-2002-2 |
[7] | Nong LY, Zhang YM, Duan YH, Hu SL, Lin Y, Liang SL. Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris. Biotechnology Letters, 2020, 42(12): 2703-2709. DOI:10.1007/s10529-020-02979-x |
[8] | Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, Köhler EM, Baumschlager A, Fischer JE, Hyden P, Wagner M, Baumann M, Borth N, Geier M, Ajikumar PK, Glieder A. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nature Communications, 2018, 9: 3589. DOI:10.1038/s41467-018-05915-w |
[9] | Curran KA, Karim AS, Gupta A, Alper HS. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic Engineering, 2013, 19: 88-97. DOI:10.1016/j.ymben.2013.07.001 |
[10] | Ito Y, Terai G, Ishigami M, Hashiba N, Nakamura Y, Bamba T, Kumokita R, Hasunuma T, Asai K, Ishii J, Kondo A. Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression. Nucleic Acids Research, 2020, 48(22): 13000-13012. DOI:10.1093/nar/gkaa1066 |
[11] | Rajkumar AS, Varela JA, Juergens H, Daran JMG, Morrissey JP. Biological parts for Kluyveromyces marxianus synthetic biology. Frontiers in Bioengineering and Biotechnology, 2019, 7: 97. DOI:10.3389/fbioe.2019.00097 |
[12] | Ito Y, Kitagawa T, Yamanishi M, Katahira S, Izawa S, Irie K, Furutani-Seiki M, Matsuyama T. Enhancement of protein production via the strong DIT1 Terminator and two RNA-binding proteins in Saccharomyces cerevisiae. Scientific Reports, 2016, 6: 36997. DOI:10.1038/srep36997 |
[13] | Yamanishi M, Ito Y, Kintaka R, Imamura C, Katahira S, Ikeuchi A, Moriya H, Matsuyama T. A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a terminatome toolbox. ACS Synthetic Biology, 2013, 2(6): 337-347. DOI:10.1021/sb300116y |
[14] | Vogl T, Sturmberger L, Kickenweiz T, Wasmayer R, Schmid C, Hatzl AM, Gerstmann MA, Pitzer J, Wagner M, Thallinger GG, Geier M, Glieder A. A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synthetic Biology, 2016, 5(2): 172-186. DOI:10.1021/acssynbio.5b00199 |
[15] | Qin XL, Qian JC, Yao GF, Zhuang YP, Zhang SL, Chu J. GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Applied and Environmental Microbiology, 2011, 77(11): 3600-3608. DOI:10.1128/AEM.02843-10 |
[16] | Xu QS, Zheng XQ, Huang MP, Wu M, Yan YS, Pan JM, Yang Q, Duan CJ, Liu JL, Feng JX. Purification and biochemical characterization of a novel β-fructofuranosidase from Penicillium oxalicum with transfructosylating activity producing neokestose. Process Biochemistry, 2015, 50(8): 1237-1246. DOI:10.1016/j.procbio.2015.04.020 |
[17] | Qin X, Qian J, Xiao C, Zhuang Y, Zhang S, Chu J. Reliable high-throughput approach for screening of engineered constitutive promoters in the yeast Pichia pastoris. Letters in Applied Microbiology, 2011, 52(6): 634-641. DOI:10.1111/j.1472-765X.2011.03051.x |
[18] | De Almeida JRM, De Moraes LMP, Torres FAG. Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast, 2005, 22(9): 725-737. DOI:10.1002/yea.1243 |
[19] |
Zhao Y, Zhao YK, Liu SQ, Li J, Li SL, Xiao DG, Yu AQ. Advances in molecular genetics and synthetic biology tools in unconventional yeasts. Acta Microbiologica Sinica, 2020, 60(8): 1574-1591.
(in Chinese) 赵禹, 赵雅坤, 刘士琦, 李建, 李圣龙, 肖冬光, 于爱群. 非常规酵母的分子遗传学及合成生物学研究进展. 微生物学报, 2020, 60(8): 1574-1591. |
[20] | Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K. Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories, 2016, 15: 84. DOI:10.1186/s12934-016-0486-7 |
[21] | Curran KA, Morse NJ, Markham KA, Wagman AM, Gupta A, Alper HS. Short synthetic terminators for improved heterologous gene expression in yeast. ACS Synthetic Biology, 2015, 4(7): 824-832. DOI:10.1021/sb5003357 |
[22] | Sun ZH, Brodsky JL. Protein quality control in the secretory pathway. The Journal of Cell Biology, 2019, 218(10): 3171-3187. DOI:10.1083/jcb.201906047 |