微生物学报  2018, Vol. 58 Issue (1): 91-108
http://dx.doi.org/10.13343/j.cnki.wsxb.20170034
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

王迎政, 喻晓蔚, 徐岩. 2018
Yingzheng Wang, Xiaowei Yu, Yan Xu. 2018
巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析
Transcriptome analysis of recombinant Komagataella phaffii with methanol induced expression of phospholipase A2
微生物学报, 58(1): 91-108
Acta Microbiologica Sinica, 58(1): 91-108

文章历史

收稿日期:2017-01-18
修回日期:2017-03-30
网络出版日期:2017-04-05
巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析
王迎政 , 喻晓蔚 , 徐岩     
工业生物技术教育部重点实验室, 江南大学生物工程学院, 江苏 无锡 214122
摘要[目的] 基于转录组学技术研究表达磷脂酶A2的毕赤酵母重组菌在甲醇诱导表达外源蛋白时的基因表达差异,从而解析外源蛋白高效诱导表达机制,为进一步工程菌株的改造提供理论支撑。[方法] 以一株产磷脂酶(PLA2)的毕赤酵母为出发菌株,采用RNA-Seq二代测序方法,研究在甘油培养和甲醇诱导两种条件下,重组毕赤酵母转录组基因表达差异情况。[结果] 重组毕赤酵母中共鉴定到5225个转录本。甘油培养与甲醇诱导相比,共有857个基因发生显著变化。依据代谢途径分类,差异基因集中在核糖体成分、甲醇代谢、磷酸戊糖途径、糖酵解途径、柠檬酸循环、乙醛酸循环以及蛋白质加工过程。[结论] 通过分析甲醇诱导前后的差异表达基因,结果表明碳源改变对胞内代谢会产生全局影响。本研究结果为进一步研究毕赤酵母表达外源蛋白的机制提供了基础。
关键词: 毕赤酵母     转录组     甲醇诱导     甘油培养     代谢机制    
Transcriptome analysis of recombinant Komagataella phaffii with methanol induced expression of phospholipase A2
Yingzheng Wang , Xiaowei Yu , Yan Xu     
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
Received 18 January 2017; Revised 30 March 2017; Published online 5 April 2017
*Corresponding author: Xiaowei Yu, Tel/Fax:+86-510-85918201;E-mail:bioyuxw@aliyun.com
Supported by the National Natural Science Foundation of China (31671799) and by the Six Talent Peaks Project in Jiangsu Province (NY-010)
Abstract: [Objectives] In order to study the mechanism of heterologous protein expression in Komagataella phaffii, transcriptomics technology was used to study the profile of differentially expressed genes in the recombinant K. phaffii during the methanol-induced expression of phospholipase A2. [Methods] We used RNA sequencing (RNA-seq) technology to analyze the differentially expressed genes in the recombinant K. phaffii expressing phospholipase A2 between glycerol and methanol cultivation condition. [Results] In total 5225 transcripts were identified in the recombinant K. phaffii. Compared between glycerol and methanol cultivation condition, 857 genes were significant differentially expressed. According to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, the differentially expressed genes were mostly related to ribosome composition, methanol metabolism, pentose phosphate pathway, citric acid cycle, glyoxylic acid cycle and protein processing process. [Conclusions] A global effect on the metabolism was observed when changing the carbon source from glycerol to methanol for the induction of heterologous protein. Our results provide rich information for further in-depth studies of the mechanism of protein expression in K. phaffii.
Key words: Komagataella phaffii     transcriptome     methanol induce     glycerol culture     metabolic mechanism    

巴斯德毕赤酵母(Komagataella phaffii,原命名为Pichia pastoris[1])是一种高效生产外源蛋白的甲醇营养型酵母,能以甘油或者甲醇作为唯一碳源和能源,其中甲醇可作为诱导剂,诱导醇氧化酶(AOX)启动子表达相关基因,它兼具大肠杆菌表达系统优势,同时克服了酿酒酵母表达系统诸多不足。如:具有强诱导性和强启动性的AOX启动子,适用于外源基因高水平诱导表达;具有真核生物蛋白质翻译后加工折叠和翻译后修饰机制,从而使表达出的真核生物蛋白具有生物活性;胞外蛋白中内源蛋白分泌非常少,有利于表达产物分离纯化;外源基因可整合到酵母染色体上,避免了外源基因的丢失。

毕赤酵母利用甘油和甲醇作为碳源时,发酵过程中胞内代谢状态存在一定差异。重组毕赤酵母在甘油培养和甲醇诱导下,显著性差异基因主要在蛋白酶体、糖基化合成、甲烷代谢、内质网蛋白质加工、不饱和脂肪酸和柠檬酸循环(TCA循环)等[2]。毕赤酵母在不含甘油的甲醇诱导培养基中,甘油代谢相关基因表达量较少[2]。毕赤酵母在发酵过程中,环境条件、胞内代谢系统以及外源基因共同决定了胞内的代谢状态。本实验室前期构建了表达磷脂酶基因的毕赤酵母基因重组菌,实现了其高效分泌表达[3],但是毕赤酵母高水平蛋白表达具体原因和机制还有待进一步深入研究。

RNA-Seq是基于第二代测序技术(Next-generation sequencing,NGS)的转录组学研究方法,揭示基因组中RNA特定时刻的存在和数量的技术[4]。目前已有从蛋白组学和基因组学研究代谢途径,但是利用转录组有针对性研究代谢途径还处于初期阶段[6]。在本研究中,结合RNA-Seq第二代测序技术,在转录层面获得高表达外源蛋白重组菌株中全部基因表达情况,形成表达谱,并构建关键基因的表达通路;通过生物信息学技术分析基因表达差异,研究细胞表型和功能,全面理解蛋白产生机制,进一步为工程菌株的改造提供理论支撑。

1 材料和方法 1.1 材料

1.1.1 菌种: 表达紫红链霉菌磷脂酶A2的毕赤酵母菌株K. phaffii GS115/pPIC9K-PLA2为本实验室构建[3]

1.1.2 YPD平板培养基(g/L): 蛋白胨20.0,无水葡萄糖20.0,琼脂粉20.0,酵母提取物10.0。

1.1.3 种子培养基(g/L): 蛋白胨20.0,YNB 13.4,酵母提取物10.0,生物素4×10-4,甘油10.0,pH 6.0磷酸缓冲液100.0 mmol/L。

1.1.4 微量元素PTM1溶液(g/L): CuSO4·5H2O 6.00,Na2MoO4·2H2O 0.20,KI 0.08,ZnSO4·7H2O 42.20,MnSO4·H2O 3.00,FeSO4·7H2O 65.00,Biotin 0.20,H3BO3 0.02,CoCl2·6H2O 0.50,H2SO4 5.00 mL/L。

1.1.5 基础盐培养基(g/L): 甘油40,85% H3PO4 28.70 mL/L,KOH 4.13,K2SO4 18.20,CaSO4 0.93,MgSO4·7H2O 14.90,含有4 mL/L的微量元素PTM1溶液。

1.1.6 补料生长培养液: 甘油500 g/L,含有微量元素PTM1溶液12 mL/L。

1.1.7 诱导培养液: 甲醇溶液,含有12 mL/L的微量元素PTM1溶液。

1.2 样本制备方法

1.2.1 培养方法: 在无菌环境中挑取YPD平板培养基上生长良好的菌落,接种至三角瓶中培养,培养16-20 h,当OD600达到2-6时,接种至发酵罐中进行培养。

配置2.25 L基础盐培养基,加入7 L发酵罐中,121℃灭菌20 min后,室温冷却,控制空气流量为2.5 L/min,自动流加25%氨水将发酵液pH控制在5.5,控制温度30℃。

在甘油生长相中,将种子培养基接种至发酵罐中,接种量为10% (V/V),维持DO值在20%-30%,培养至溶氧DO值急剧升高,此时其中甘油已经完全消耗,维持30 min;在甘油流加相中,流加50%甘油(W/V,含12 mL/L的PTM1),调节流加速率控制DO维持在20%-30%,当细胞生长密度OD600达到110,停止补加甘油,维持饥饿状态,让甘油彻底耗尽;在甲醇诱导相中,流加甲醇(含12 mL/L的PTM1),利用甲醇检测流加控制器(华东理工大学研制)在线控制发酵液中甲醇浓度为0.10%±0.02% (V/V),维持DO在10%-20%。

1.2.2 样品采集: 甘油流加相中当细胞生长密度OD600达到110时取样;在甲醇诱导相中培养48 h时取样。样品处理方法为在无菌条件下收集发酵液样品,在6000 r/min、4 ℃条件下离心15 min,收集菌体沉淀,将菌体沉淀在液氮中速冻5-10 min后-80 ℃保存,用于转录组RNA样品抽提,同时制备2个平行重复样品。

1.3 转录组建库测序

1.3.1 Total RNA样品抽提和检测: Total RNA样品抽提依据TRIzol reagent (Invitrogen,Carlsbad,CA,USA)操作说明提取Total RNA。

Total RNA样品检测:(1)琼脂糖凝胶电泳分析RNA降解程度以及是否有污染;(2) Nanodrop检测RNA的纯度(OD260/280);(3) Qubit对RNA浓度进行精确定量;(4) Agilent 2100精确检测RNA的完整性。

1.3.2 文库构建和库检: 文库构建及测序由诺禾致源生物信息科技有限公司完成。Total RNA检测合格后,通过Epicentre Ribo-ZeroTM试剂盒去除rRNA。随后将RNA打断成150-200 bp短片段,以短片段RNA为模板,用六碱基随机引物合成cDNA,纯化双链cDNA。纯化的双链cDNA再进行末端修复、加A尾并连接测序接头,然后用AMPure XP beads进行片段大小选择。之后降解含有U的cDNA第二链,最后进行PCR富集得到链特异性cDNA文库。文库构建完成后,先使用Qubit 2.0进行初步定量,使用Agilent 2100对文库进行检测,使用QPCR方法对文库的有效浓度进行准确定量,以保证文库质量。

1.3.3 上机测序: 把构建的文库按照有效浓度及目标下机数据量的需求进行Illumina HiSeq 4000测序。

1.4 生物信息分析流程

获得原始测序序列(Sequenced reads)后,在有参考基因组(K. phaffii GS115)的情况下,通过如下方法进行生物信息分析。(1)表达水平分析:对mRNA用cuffdiff软件进行定量分析[6],得到各样本mRNA的FPKM (Reads per kilobase of exon model per million mapped reads)信息;(2)差异表达分析:从统计学意义的角度上考虑,使用cuffdiff软件对mRNA整体进行差异分析;(3)差异表达mRNA所在基因KEGG (Kyoto encyclopedia of genes and genomes)富集分析:基于mRNA的差异分析获得KEGG富集分析的结果和差异表达mRNA KEGG富集通路图,并进行KEGG富集表达聚类的分析。本文中所有代谢路径相关基因皆引自KEGG数据库。

1.5 反转录及荧光定量PCR方法测定mRNA

使用逆转录酶(Avian myeloblastosis virus reverse transcriptase,AMV RT,上海生工),从总RNA合成第一链cDNA的试剂盒,合成第一链cDNA,具体操作步骤见产品说明书。将cDNA样品适当稀释作为模板上机检测。配置荧光定量PCR (QPCR)体系混合液,按照表 1所示反应体系配制反应混合液。按照试剂盒中酶的特性设定荧光定量PCR循环条件,进行荧光定量PCR实验。

表 1. 荧光定量PCR体系 Table 1. Real-time PCR system
Reaction system Volume/μL Final concentration
2xSG Fast qPCR Master Mix (High Rox) 10.0
Primer F (10 μmol/L) 0.5 200 nmol/L
Primer R (10 μmol/L) 0.5 200 nmol/L
ddH2O 7.0 6
Template (cDNA) 2.0 < 20 ng

1.6 总蛋白含量测定

利用考马斯亮蓝法测定发酵液上清中总蛋白浓度[7]

1.7 SDS-PAGE分析

发酵上清液样品采用SDS-PAGE分析[8],选用浓缩胶5%,分离胶12%,检测发酵上清液蛋白分子量。

2 结果和分析 2.1 毕赤酵母分批发酵结果

毕赤酵母发酵过程分为3个阶段,分别为甘油生长相、甘油流加相及甲醇诱导相。为了研究甲醇诱导和甘油培养条件下基因表达差异,根据菌株发酵生长曲线,选取具有代表性样品取样点。在甘油生长相结束时即甲醇诱导前取样,样品命名为P;在甲醇诱导相的对数期(48 h)时取样,样品命名为PLM。样品信息如表 2所示。

表 2. 毕赤酵母菌株培养和样本特性 Table 2. Culture of K. phaffii and sample characteristics
Strains Carbon source Description Sample name
GS115/pPIC9K-PLA2 Glycerol Sampling at the end of glycerol fed-batch phase P
GS115/pPIC9K-PLA2 Methanol Sampling at 48 h after methanol induction PLM

在毕赤酵母分批发酵过程中,甘油生长相结束时取样(P),即图 1-A中0 h;甲醇诱导48 h取样(PLM),如图 1-A中48 h。表达磷脂酶A2的毕赤酵母菌体浓度OD600及蛋白浓度变化如图 1-A所示,相比甲醇诱导初期,诱导48 h后总蛋白浓度达1.25 g/L。菌株发酵上清液SDS-PAGE如图 1-B所示,括号所示为表达的磷脂酶A2蛋白,存在3条带,前期实验鉴定表明为不同程度糖基化形式[3]

图 1 毕赤酵母分批发酵过程生物量、胞外总蛋白浓度变化(A)和上清液总蛋白浓度SDS-PAGE图(B) Figure 1 Changes in biomass, total extracellular protein concentration (A) and SDS-PAGE analysis (B) during batch fermentation in K. phaffii. M: molecular weight marker; lane 1: methanol induction for 0 hour; lane 2: methanol induction for 24 hours; lane 3: methanol induction for 40 hours; lane 4: methanol induction for 48 hours

2.2 荧光定量PCR验证转录数据

荧光定量PCR原理是采用荧光信号强度达到预先设定的阈值时(CT)的循环数作为计量的方法。荧光定量PCR利用CT值的变化表示基因在不同表达下差异表达倍数,而RNA-Seq依据基因表达量(FPKM)的比值表示。利用Actin作为内参,计算甲醇诱导与甘油培养条件下基因差异变化倍数(公式1-2)。

公式(1)
公式(2)

利用荧光定量PCR验证RNA-Seq测定的准确性,引物设计如表 3。从表 4中可以看出荧光定量PCR和RNA-Seq结果基本相一致。

表 3. 荧光定量PCR引物列表 Table 3. A list of quantitative PCR primers
Target Primers(5′→3′)
HSP42F AAGCACTCAAGGAGGAAGGC
HSP42R ACTGTGATTCAGGAGCTGGC
PAS_chr2-1_0854F GGGTCAAAGAGAAACAAACGGA
PAS_chr2-1_0854R GGGTCAAAGAGAAACAAACGGA
HSP78F AAAATGACGGGTGTTCCGGT
HSP78R TGACTGCATCGGCAACTGAA
HSP82F TGGGTGTCCATGAAGACAGC
HSP82R TCTGGTGCTCTGGCATTCTG
HSP104F TTCCAGTGTGGTTGGGCAAT
HSP104R TTCCAGTGTGGTTGGGCAAT
ITR2F CGCCAAAGTCATCGGCAAAA
ITR2R TTGATCCTGGGATGATGGCG
RPP1BF GCCAAAGGTTTGGAAGGCAA
RPP1BR GTTCTCCTCTTCGGCCTCCT
RPP1AF GCTTTATCATACGCCGCCCT
RPP1AR TAACGTCAGCCTTGGTGGTC
RPL10F CCAGGTGTTCACTGGCATTT
RPL10R TAAATAGGGGGAAGAAGTTTCA
CPR6F GGTTAACCAGTTACCGGCCT
CPR6R TCACAGCAAACAGCCGATCT

表 4. 不同基因RNA-Seq和荧光定量PCR验证结果 Table 4. Verification of RNA-Seq data by QPCR
Gene name Fold change (QPCR) Fold change (RNA-Seq)
HSP42 9.811679 7.075784
PP7435_CHR3-0854 32.256100 7.247684
HSP84 12.393400 8.619475
HSP82 9.626424 9.308960
HSP104 9.126110 9.954715
ITR2 12.248220 10.769280
RPP1B 0.488524 0.306753
RPP1A 0.302289 0.233974
RPL10 0.063219 0.059403
CPR6 15.215820 15.579320

2.3 毕赤酵母总体代谢途径变化分析

毕赤酵母K. phaffii GS115/pPIC9K-PLA2在甘油和甲醇两种条件下,共鉴定到5225个转录本,有857个基因发生显著变化。图 1展示了每个代谢途径中显著差异基因的数目、比例和显著性,显著变化基因分布在89个KEGG代谢途径中,约占76.7%。

从各KEGG pathway的基因数目可以看出(图 2),发生显著变化基因主要在核糖体、核糖体生物合成、DNA修复、过氧化物酶体、硫代谢和甲醇代谢等代谢过程中。其中,核糖体和核糖体生物合成这两个代谢途径变化最为显著,核糖体中有73个基因发生显著变化,核糖体生物合成中有29个基因发生显著变化。通过RNA-Seq检测转录组结果表明,在甲醇诱导条件下,约65%核糖体组分和42%核糖体生物合成过程相关基因变化显著,说明核糖体对外源蛋白合成起到了重要作用。

图 2 毕赤酵母在甘油培养和甲醇诱导条件下KEGG pathway热图及相关KEGG pathway基因数目和富集因子 Figure 2 Heat map of KEGG pathway with gene number and enrichment factor in K. phaffii in the comparison of glycerol and methanol conditions. Gene number: The number of genes enriched in the pathway; Rich factor: The ratio of the number of genes enriched in the pathway to the number of all the annotation genes of the pathway. 1: Ribosome; 2: Ribosome biogenesis in eukaryotes; 3: DNA replication; 4: Peroxisome; 5: Sulfur metabolism; 6: Methane metabolism; 7: Base excision repair; 8: Valine, leucine and isoleucine biosynthesis; 9: Pyrimidine metabolism; 10: Purine metabolism; 11: Pantothenate and CoA biosynthesis; 12: Riboflavin metabolism; 13: 2-Oxocarboxylic acid metabolism; 14: Tyrosine metabolism; 15: Sphingolipid metabolism; 16: Thiamine metabolism; 17: Cysteine and methionine metabolism; 18: Carbon metabolism; 19: Phenylalanine metabolism; 20: Biosynthesis of amino acids; 21: Fatty acid elongation; 22: Pentose and glucuronate interconversions; 23: Fatty acid degradation; 24: Tryptophan metabolism; 25: Butanoate metabolism; 26: Vitamin B6 metabolism; 27: Selenocompound metabolism; 28: Histidine metabolism; 29: Glutathione metabolism; 30: Glyoxylate and dicarboxylate metabolism; 31: RNA polymerase; 32: beta-Alanine metabolism; 33: Ascorbate and aldarate metabolism; 34: Valine, leucine and isoleucine degradation; 35: Galactose metabolism; 36: Arginine and proline metabolism; 37: Biosynthesis of unsaturated fatty acids; 38: ABC transporters; 39: Phenylalanine, tyrosine and tryptophan biosynthesis; 40: Glycerolipid metabolism; 41: Mismatch repair; 42: Glycolysis/Gluconeogenesis; 43: Porphyrin and chlorophyll metabolism; 44: Fatty acid metabolism; 45: Fructose and mannose metabolism; 46: Alanine, aspartate and glutamate metabolism; 47: Non-homologous end-joining; 48: One carbon pool by folate; 49: Nucleotide excision repair; 50: Pyruvate metabolism; 51: Homologous recombination; 52: Steroid biosynthesis; 53: Meiosis-yeast; 54: Taurine and hypotaurine metabolism; 55: Degradation of aromatic compounds; 56: Glycerophospholipid metabolism; 57: Propanoate metabolism; 58: Lysine biosynthesis; 59: Nitrogen metabolism; 60: Amino sugar and nucleotide sugar metabolism; 61: Cell cycle-yeast; 62: Inositol phosphate metabolism; 63: Cyanoamino acid metabolism; 64: Ubiquinone and other terpenoid-quinone biosynthesis; 65: Fatty acid biosynthesis; 66: Lysine degradation; 67: Glycine, serine and threonine metabolism; 68: Pentose phosphate pathway; 69: Phosphatidylinositol signaling system; 70: Starch and sucrose metabolism; 71: Terpenoid backbone biosynthesis; 72: Sulfur relay system; 73: Aminoacyl-tRNA biosynthesis; 74: Protein export; 75: Phagosome; 76: Nicotinate and nicotinamide metabolism; 77: Regulation of autophagy; 78: mRNA surveillance pathway; 79: Citrate cycle (TCA cycle); 80: N-Glycan biosynthesis; 81: Oxidative phosphorylation; 82: RNA degradation; 83: RNA transport; 84: Endocytosis; 85: Basal transcription factors; 86: Protein processing in endoplasmic reticulum; 87: Ubiquitin mediated proteolysis; 88: Spliceosome

文献报道利用RNA-Seq测序技术研究米黑根毛霉脂肪酶在毕赤酵母中甲醇诱导表达时,多聚核糖体的比例在总核糖体(40S,60S,80S/monosomes和polysomes)中提高了约2倍[9-10]。甲醇提供碳源的同时,也诱导外源蛋白大量表达,此时不仅需要合成大量氨基酸,而且需要更多核糖体参与蛋白质合成[9]。因此,通过提高胞内核糖体数量和核糖体加工过程相关基因的表达量,可以作为提高外源蛋白表达量的一种策略。

2.4 毕赤酵母中心碳代谢基因差异表达

毕赤酵母细胞碳代谢途径主要包括甲醇代谢、糖酵解、TCA循环、磷酸戊糖途径、糖醛酸途径、糖原合成、糖原分解、糖异生以及其他己糖代谢等。尤其是甲醇代谢途径、糖酵解途径、TCA循环和磷酸戊糖途径等代谢过程会产生大量中间代谢产物,为其他代谢过程提供底物。甘油生长期,胞内代谢途径主要集中在氧化磷酸化、糖酵解、TCA循环以及电子呼吸链;甲醇诱导期,胞内的代谢途径主要集中在甲醇代谢途径[11]。甘油和甲醇分别经同化吸收后,进入以TCA循环为中心的碳代谢过程。有研究者研究了甲醇代谢途径(MUT pathway)和其相关代谢路径[12-14]。结合毕赤酵母代谢路径和本全转录组数据绘制了甲醇代谢以及磷酸戊糖途径代谢过程路径图(图 3),主要包含5部分:细胞吸收甲醇在过氧化物酶体中经过甲醇代谢(图 3-Part 1),代谢产生的H2O2在ROS (图 3-Part 2)途径[13]的作用下降低氧化力生成O2,一部分甲醇氧化成甲醛经过异化过程生成CO2(图 3-Part 3),另一部分甲醛进入磷酸戊糖途径,最终1分子甲醇生成1/3分子甘油三磷酸(GAP),用于生物质合成(图 3-Part 4,图 3-Part 5)。

图 3 毕赤酵母甲醇代谢以及磷酸戊糖途径代谢过程路径图及差异表达基因 Figure 3 Differentially expressed genes of MUT pathway and pentose phosphate pathway in K. phaffii. Part 1:Canonical MUT pathway; Part 2: ROS defense; Part 3: Dissimilative branch of the MUT pathway; Part 4: Pentose phosphate pathway; Part 5: Assimilative branch of the MUT pathway. Red: Significant up-regulated gene; Green: Significant down-regulated gene; Black: No significant change gene. Fold change: FPKM (Methanol)/FPKM (Glycerol); P-value: Enrichment analysis of P value (< 0.05)

2.4.1 毕赤酵母甘油代谢和甲醇代谢差异表达: 毕赤酵母细胞以甘油和甲醇分别作为唯一碳源时,细胞生长状态不尽相同,胞内代谢系统也相应做出部分调整。毕赤酵母以甘油作为唯一碳源培养时,主要经过甘油代谢同化外界碳源。首先,甘油在甘油激酶不可逆催化下形成磷酸甘油,再经磷酸甘油脱氢酶和磷酸丙糖异构酶(TPI1)分别催化形成磷酸二氢丙酮和3-磷酸甘油醛,此后,即可进入糖酵解生成丙酮酸,进而生成乙酰CoA,进入三羧酸循环。甘油促进细胞生长,此时外源蛋白几乎不表达[15]。在甲醇作为唯一碳源时,甲醇促进细胞生长同时诱导外源蛋白表达,甲醇氧化场所在过氧化物酶体中[13]。从KEGG数据库获取甲醇代谢相关基因,毕赤酵母以甲醇为唯一碳源进行新陈代谢和甘油培养时,基因差异表达倍数如表 5所示。

表 5. 毕赤酵母甲醇诱导与甘油培养甲醇代谢基因差异倍数 Table 5. The foldchange of the significant differentially expressed genes involved in methanol metabolism genes in the comparison of methanol and glycerol cultivation in K. phaffii
Gene ID Gene name Description Fold change P value
PAS_chr3_0834 DAS2 Transketolase, similar to Tkl2p 200.85 2.89E-15
PAS_chr3_0832 DAS1 Transketolase, similar to Tkl2p 131.60 6.12E-12
PAS_chr4_0821 AOX1 Alcohol oxidase 1 24.59 0.000124
PAS_chr2-2_0177 SHB17 Putative protein of unknown function 22.32 2.55E-06
PAS_chr3_0932 FDH1 NAD-dependent formate dehydrogenase, may protect cells from exogenous formate 21.71 0.000236
PAS_chr3_0867 FGH1 Non-essential intracellular esterase that can function as an S-formylglutathione hydrolase 11.55 0.001231
PAS_chr3_1028 FLD1 S-(hydroxymethyl)glutathione dehydrogenase 9.32 0.007054
PAS_chr3_0403 ACS2 acetate-CoA ligase 8.11 2.97E-06
PAS_chr4_0416 AGX1 Alanine:glyoxylate aminotransferase (AGT), catalyzes the synthesis of glycine from glyoxylate 5.06 0.011088
PAS_chr3_0841 DAK2 Dihydroxyacetone kinase, required for detoxification of dihydroxyacetone (DHA) 4.06 0.126775
PAS_chr3_0868 FBP1 Fructose-1, 6-bisphosphatase, key regulatory enzyme in the gluconeogenesis pathway 3.56 0.095237
PAS_chr2-1_0657 SER3 3-phosphoglycerate dehydrogenase, catalyzes the first step in serine and glycine biosynthesis 3.25 0.005038
PAS_chr4_0152 AOX2 Alcohol oxidase 2 2.03 0.256310
PAS_chr4_0415 SHM2 Cytosolic serine hydroxymethyltransferase 1.38 0.552604
PAS_chr4_0285 SER2 Phosphoserine phosphatase of the phosphoglycerate pathway, involved in serine and glycine biosynthes 1.21 0.637429
PAS_chr1-1_0427 YOR283W Hypothetical protein 1.19 0.661960
PAS_chr3_0566 SER1 3-phosphoserine aminotransferase -1.23 0.615148
PAS_chr4_0587 SHM1 Mitochondrial serine hydroxymethyltransferase -1.40 0.584437
PAS_chr1-4_0047 PFK2 Beta subunit of heterooctameric phosphofructokinase involved in glycolysis -1.64 0.460753
PAS_chr2-1_0402 PFK1 Alpha subunit of heterooctameric phosphofructokinase involved in glycolysis -1.74 0.415801
PAS_chr3_0693 GPM3 Tetrameric phosphoglycerate mutase -2.14 0.056185
PAS_chr3_0826 GPM1 Tetrameric phosphoglycerate mutase -2.45 0.143465
PAS_chr1-1_0072 FBA1-1 Fructose 1, 6-bisphosphate aldolase, required for glycolysis and gluconeogenesis -2.55 0.252666
PAS_chr3_0082 ENO1 Enolase I, a phosphopyruvate hydratase that catalyzes the conversion of 2-phosphoglycerate to phosph -3.89 0.109996
PAS_chr2-1_0767 ACS1 Acetate-CoA ligase -5.58 0.060806
Fold change: FPKM (PLM)/FPKM (P).

表 5中展示了甲醇代谢途径中相关基因差异表达情况,其中DAS2DAS1SHB17ACS2AOX1FDH1FGH1SER3FLDAGX1均显著上调。在甲醇诱导条件下,甘油代谢相关基因STL1(Glycerol proton symporter of the plasma membrane)和GUT1 (Glycerol kinase)分别下调了3倍和1倍,这和实验结果相符合。Liang等发现甲醇诱导毕赤酵母后,甲醇代谢相关基因转录水平也发生了显著上调[16]。甲醇代谢途径的中间代谢产物GAP和二羟基丙酮(DHAP)为TCA循环、磷酸戊糖途径以及糖酵解途径中生物质合成提供前体物质及能量。另外,过氧化物酶体功能基因PMP20PEX12PEX13PEX2分别上调了156.9倍、2.8倍、6.1倍和4.4倍(表 6)。过氧化物酶体中甲醇代谢相关基因显著上调(如表 5所示),以及过氧化物酶体结构组分表达量也提高,促进了对碳源吸收,为生物质合成提供碳源和能量,磷脂酶被大量合成,胞外蛋白总浓度达1.25 g/L(图 1)。上述研究结果表明,在甲醇诱导条件下细胞做出精细的系统调控,维持细胞内环境稳定。

表 6. 毕赤酵母甲醇诱导与甘油培养过氧化物酶体显著差异基因差异倍数 Table 6. The foldchange of the significant differentially expressed genes involved in peroxisome in the comparison of methanol and glycerol cultivation in K. phaffii
Gene ID Gene name Description Fold change P value
PAS_chr1-4_0547 PMP20 Peroxiredoxin 156.94 1.14442E-12
PAS_chr3_0099 PMP47 Peroxisome membrane protein 47 17.60 7.84274E-05
PAS_chr2-2_0207 PEX13 Integral peroxisomal membrane protein required for translocation of peroxisomal matrix proteins 6.16 0.00308638
PAS_chr4_0416 PAS_chr4_0416 Alanine: glyoxylate aminotransferase (AGT) 5.06 0.01107180
PAS_chr3_0043 PEX2 RING-finger peroxin and E3 ubiquitin ligase 4.35 0.00815035
PAS_chr2-1_0230 MRP1 Mitochondrial ribosomal protein of the small subunit 3.52 0.00246982
PAS_chr4_0788 RSM26 Mitochondrial ribosomal protein of the small subunit 2.96 0.01130610
PAS_chr4_0759 PEX12 C3HC4-type RING-finger peroxin and E3 ubiquitin ligase 2.84 0.03105350
PAS_chr2-1_0580 IDP1 Cytosolic NADP-specific isocitrate dehydrogenase -3.02 0.02428140
PAS_chr2-2_0272 PXA2 Subunit of a heterodimeric peroxisomal ATP-binding cassette transporter complex (Pxa1p-Pxa2p) -3.28 0.02075670
PAS_chr1-4_0071 SOD2 Mitochondrial manganese superoxide dismutase -3.85 0.02331130
PAS_chr3_0975 SPS19 Peroxisomal 2, 4-dienoyl-CoA reductase, auxiliary enzyme of fatty acid beta-oxidation -4.87 0.00745497
PAS_chr4_0352 FAA2 Medium chain fatty acyl-CoA synthetase, activates imported fatty acids -5.60 0.02847880
PAS_chr2-1_0502 PAS_chr2-1_0502 Thiol-specific peroxiredoxin -7.58 0.00236159
PAS_chr3_0069 CAT2 Carnitine acetyl-CoA transferase present in both mitochondria and peroxisomes -10.85 0.00112011
PAS_chr1-4_0074 YAT1 Outer mitochondrial carnitine acetyltransferase -18.24 0.00042053
Fold change: FPKM (PLM)/FPKM (P).

2.4.2 毕赤酵母内磷酸戊糖途径基因差异表达: 磷酸戊糖途径不但为微生物提供能量,还可为微生物提供大量前体物质,是重要的代谢途径。有学者研究发现甲醇诱导外源蛋白表达时,菌株细胞生物质产生降低了25%,但是磷酸戊糖途径代谢增强了重组蛋白的表达量[17-18]

磷酸戊糖途径(图 3-Part 4)分为氧化阶段和非氧化阶段[19]。氧化阶段是细胞产生还原力(NADPH)的主要途径,氧化阶段涉及的酶有葡萄糖-6-磷酸脱氢酶(ZWF1)、内酯酶(SOL3)和6-磷酸葡萄糖酸脱氢酶(PDG1),其中ZWF1是糖酵解途径的限速酶基因,催化不可逆反应。非氧化阶段是指全部磷酸戊糖途径除上述3个酶催化反应外,都是非氧化阶段。非氧化阶段是细胞内结构分子的重要来源,并为各种单糖相互转变提供前提条件。非氧化阶段的酶有异构酶、差向异构酶、转酮酶和转醛酶,催化不同单糖之间的转换[19]。从图 3中我们看出磷酸戊糖途径氧化阶段相关基因(ZWF1SOL3PGD1)未发生显著变化。有研究者采用过表达磷酸戊糖途径氧化阶段基因(ZWF1ZWF1/SOL3SOL3)显著提高了蛋白表达量[17-18]。因此氧化阶段有待进一步研究。

转录测序结果显示非氧化阶段发生显著变化,如核酮糖-5-磷酸差向异构酶(RPE2),核糖-5-磷酸酮醇异构酶(RKI1)和转醛醇酶(TAL1-2)分别显著上调3.86倍、14.46倍和52.52倍(表 7)。磷酸戊糖途径为微生物代谢提供丰富的中间产物,因此我们认为在表达磷脂酶时,细胞为了缓解胞内代谢压力,仅调整非氧化阶段相关基因表达量,非氧化阶段对生物质的合成起到重要作用,促进蛋白表达量的提高。

表 7. 在甘油和甲醇两种条件下毕赤酵母磷酸戊糖途径基因差异倍数 Table 7. The foldchange of the significant differentially expressed genes involved in pentose phosphate pathway in the comparison of methanol and glycerol cultivation in K. phaffii
Gene ID Gene name Description Fold change P value
PAS_chr2-2_0338 TAL1-2 Transaldolase 52.52 1.96476E-11
PAS_chr4_0213 RKI1 Ribose-5-phosphate ketol-isomerase 14.46 8.71245E-06
PAS_chr3_0441 RPE2 D-ribulose-5-phosphate 3-epimerase 3.86 0.0105208
PAS_chr3_0868 FBP1 Fructose-1, 6-bisphosphatase 3.55 0.0951730
PAS_chr2-1_0771 PGM3 Phosphoribomutase 3.18 0.0221160
PAS_chr4_0212 RKI1-2 Ribose-5-phosphate ketol-isomerase 1.75 0.3453440
PAS_chr1-1_0006 PRS1 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetase 1.56 0.2902980
PAS_chr1-4_0669 YDR248C Putative gluconokinase 1.37 0.5827380
PAS_chr3_0062 PRS3 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetase 1.08 0.8577890
PAS_chr2-1_0308 ZWF1 Glucose-6-phosphate dehydrogenase (G6PD) -1.11 0.8872530
PAS_chr2-2_0174 PRS5 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetase -1.13 0.7540340
PAS_chr1-1_0436 PRS4 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetaseheteromultimeric complexes -1.19 0.6699240
PAS_chr3_0604 RBK1 Putative ribokinase -1.22 0.6286320
PAS_chr3_1126 SOL3 6-phosphogluconolactonase -1.30 0.5544950
PAS_chr1-1_0277 SOL1 Protein with a possible role in tRNA export -1.31 0.5186960
PAS_chr1-4_0047 PFK2 Beta subunit of heterooctameric phosphofructokinase -1.64 0.4606660
PAS_chr2-1_0402 PFK1 Alpha subunit of heterooctameric phosphofructokinase -1.74 0.4157120
PAS_chr3_0277 PDG1 6-phosphogluconate dehydrogenase (decarboxylating) -1.96 0.4571960
PAS_chr1-4_0264 PGM2 Phosphoglucomutase -2.42 0.2047380
PAS_chr1-1_0072 FBA1-1 Fructose 1, 6-bisphosphate aldolase -2.54 0.2525790
PAS_chr2-2_0337 TAL1-1 Transaldolase -2.69 0.1738280
PAS_chr3_0456 PGI1 Glycolytic enzyme phosphoglucose isomerase -2.94 0.1027780
PAS_chr1-4_0150 TKL1 Transketolase -3.40 0.0888672
Fold change: FPKM (PLM)/FPKM (P).

2.4.3 毕赤酵母糖酵解途径和TCA循环基因差异表达: 微生物细胞碳代谢主要包括糖酵解途径、磷酸戊糖途径和TCA循环。图 4展示了碳代谢过程酵母细胞吸收的甲醇在甲醇代谢作用下生成GAP,随后进入糖酵解途径,部分碳源经过代谢作用生成果糖-6-磷酸,进入磷酸戊糖途径。另一部分碳源在糖酵解作用下胞内丙酮酸(Pyruate)分两个代谢路径:一部分是进入线粒体中进行TCA循环,另一部分参与乙醛酸循环。TCA循环和乙醛酸循环均为微生物提供能量和前体物质[20-21]

图 4 毕赤酵母TCA循环和乙醛酸循环代谢图及差异表达基因 Figure 4 Differentially expressed genes of citric acid cycle and glyoxylate in K. phaffii. Red: Significant up-regulated gene; Green: Significant down-regulated gene; Black: No significant change gene. Fold change: FPKM (PLM)/FPKM (P); P value: Enrichment analysis of P value (< 0.05)

甘油培养和甲醇诱导两种条件相较,糖酵解途径第一阶段基因如TPI1、甘油醛-3-磷酸脱氢酶(TDH1)、果糖-2, 6-二磷酸酶(FBP1)和PFK1表达量均无明显变化(图 4),且表达量较低,与文献报道一致[22]。说明毕赤酵母在甘油和甲醇培养两种条件下,糖酵解第一阶段基因处于本底表达状态。过氧化物酶体中甲醇代谢产物为GAP和DHAP,经过氧化物酶体膜转运至胞质进入糖酵解途径第二阶段(图 4)。果糖1, 6-二磷酸醛缩酶(FBA1-2)可逆催化果糖1, 6-二磷酸(FDP)转化为GAP和DHAP。甲醇代谢途径产生的GAP促使FBA1-2表达量增加,形成大量代谢产物(FDP)进入磷酸戊糖途径(图 4)。测序结果显示FBA1-2基因上调84.45倍。上述结果证明甲醇代谢产物GAP部分进入磷酸戊糖途径,并为微生物提供还原力和前体物质。

在糖酵解过程磷酸甘油酸激酶(PGK1)催化GAP为磷酸烯醇式丙酮酸(PEP)中,结果显示PGK1下调约5.17倍(图 4),导致糖酵解能力下降。此外甲醇诱导阶段糖异生途径中磷酸烯醇丙酮酸羧化激酶(PCK1)表达量较低,糖异生途径形成较少GAP,而胞内GAP的主要来源是甲醇代谢途径[10]TPI1催化GAP和DHAP之间可逆转换[18],提供还原力[23],但在本次实验中表达水平较低。综上结果表明细胞内甲醇代谢形成大量GAP转化为FDP,主要通过糖酵解途径第一阶段,进入磷酸戊糖途径,继续进行下一步代谢。

Almeida等[24]认为毕赤酵母基因组中PGK1以单拷贝形式存在,通过Northern blot分析不同碳源对PGK1表达影响时,葡萄糖培养条件下PGK1表达水平是甘油的2倍,说明在不同碳源中PGK1基因的启动子活力不同。在甲醇生长条件下,PGK1基因表达显著降低,这成为了糖酵解能力下降的主要原因。

TCA循环作为糖酵解下游代谢途径,本次实验中TCA循环关键基因(如苹果酸合酶基因、琥珀酰-CoA基因)并未发生明显变化,仅线粒体中催化柠檬酸形成异柠檬酸的乌头酸酶基因(ACO2)显著上调3.86倍。在甘油培养和甲醇诱导两种条件下,毕赤酵母线粒体中异柠檬酸脱氢酶基因(IDP1)下调3.03倍。

有研究者[25]比较酵母葡萄糖和甲醇作为碳源培养时的生长状况,得出甲醇生长条件下TCA循环代谢流降低3倍,同时TCA循环中没有转录水平或者蛋白水平发生显著改变,仅柠檬酸产量增加。此外,依据酵母代谢组和转录组的结果分析,假丝酵母甲醇培养条件下TCA循环中物质和基因表达量普遍下降[26]

上述结果表明在甲醇诱导条件下,胞内TCA循环过程的电子链氧化磷酸化供能需求降低,TCA循环代谢流降低,TCA循环为其他代谢途径提供前体物质能力减弱。

2.4.4 毕赤酵母乙醛酸循环基因差异表达: 乙醛酸循环中有异柠檬酸裂合酶(ICL1)和苹果酸合酶(MLS)两个关键酶,催化异柠檬酸和乙酰辅酶A转化成苹果酸和琥珀酸(图 4)。在甲醇诱导条件下,ICLMLS表达量分别比甘油培养条件下降低了约13.55倍和6.54倍。乙醛酸循环中间代谢产物富马酸(Succinate)由线粒体琥珀酸富马酸转运子(SFC1,Mitochondrial succinate-fumarate transporter)转运至线粒体TCA循环中,结果表明在甲醇诱导表达外源蛋白时SFC1下调8.51倍,这与文献报道[27]一致。SFC1下调导致乙醛酸循环进入TCA循环的代谢流进一步降低。以上研究结果表明,以甲醇作为碳源,乙醛酸代谢途径和TCA循环部分受到抑制,而甲醇代谢途径及磷酸戊糖途径得到增强,重新建立代谢平衡。

研究发现毕赤酵母表达外源蛋白时ICL1基因表达量下调[27]。但是,也有研究报道提高酿酒酵母中乙醛酸循环相关基因(如ADH2MLSCIT2ICL1等)的表达有利于提高糖基化外源蛋白表达量[28]。在本研究中甲醇诱导外源蛋白表达过程中发现ICLMLS表达量显著下调,其机制还有待进一步验证。

2.4.5 毕赤酵母外源蛋白质在内质网加工过程中的差异表达: 蛋白质分选、折叠和二硫键形成是蛋白表达的限速步骤[29]。外源蛋白合成过程中,内质网参与蛋白质折叠、分选以及降解等相关功能。成熟mRNA在核糖体中翻译成多肽,信号识别颗粒(SRP)牵引多肽一端至内质网膜上,SRP受体与SRP结合,在结合蛋白(KAR2)的参与下,多肽由细胞质转移到内质网腔内进行加工和修饰。

Kar2p是热激蛋白70家族重要的伴侣蛋白,促使蛋白正确折叠,参与蛋白质易位、ER相关的降解(ERAD)、以及未折叠蛋白的调控[30]。另外KAR2激活Ire1形成二聚体,调节HAC1合成,降低内质网压力,促进蛋白质合成[31]。伴侣蛋白基因KAR2作为胞内UPR效应[29]的标志,在内质网中积聚错误折叠蛋白,引发保护机制UPR效应[32-33]。甲醇诱导条件下,毕赤酵母内质网中KAR2表达量提高5.90倍(图 5)。上述测序结果表明,在甲醇诱导条件下,KAR2表达量上升,进入内质网中的多肽增多,促使蛋白大量表达,磷脂酶表达过程中产生未正确折叠的状态,引发了毕赤酵母内质网中UPR效应。因此,提高UPR效应相关基因表达量有利于提高外源蛋白表达量。

图 5 毕赤酵母中外源蛋白质在内质网中加工过程示意图及差异表达基因 Figure 5 Differentially expressed genes involved in protein processing in endoplasmic reticulum in K. phaffii. Red: Significant up-regulated gene; Green: Significant down-regulated gene; Black: No significant change gene. Fold change: FPKM (PLM)/FPKM (P); P value: Enrichment analysis of P value (< 0.05)

胞内蛋白质未正确折叠,稳定性降低,不仅会激活UPR效应,还会引发内质网相关联的蛋白降解途径(ERAD)。有研究者发现ERAD效应可通过标签基因(HRD3DER1SEC61)[34]或者解聚酶基因(HSP104)和AAA ATP酶基因(CDC48)[35]表达量检测得到。本实验中HRD3DER1SEC61分别非显著上调1.1倍、1.2倍和1.9倍,同时HSP104CDC48未有明显变化。说明本研究过程中,磷脂酶基因在毕赤酵母中表达未引起ERAD效应,表明毕赤酵母中磷脂酶的表达量依然有上升的空间。

蛋白质二硫键异构酶(PDI1)催化膜表面蛋白和分泌蛋白形成二硫键,二硫键的形成是蛋白质合成主要的限速步骤[36]。本研究中,外源蛋白磷脂酶中存在3对二硫键。相对于甘油培养条件,在甲醇诱导条件下PDI1转录水平提高了2.62倍。研究者发现毕赤酵母中共表达PDI1可增强外源蛋白分泌作用,蛋白表达量显著提高[30, 37-39]。在微生物代谢过程中,蛋白折叠和内质网压力影响胞内的氧化平衡,PDI1可维持胞内氧化还原的平衡[40]。因此,在毕赤酵母中可通过共表达PDI1调节细胞内的氧化还原平衡,很可能进一步提高磷脂酶A2的表达量。

3 讨论

利用第二代测序技术可加深对微生物认识深度,为探究微生物代谢机制寻求突破。本实验室前期利用毕赤酵母表达磷脂酶A2已经取得较好表达效果[3],本文进一步探讨毕赤酵母在甲醇诱导条件下高效表达外源蛋白的机制。当环境条件变化时,微生物胞内的代谢会做适当调整。毕赤酵母由甘油培养转变为甲醇诱导时,代谢差异基因主要集中在核糖体、核糖体生物合成、DNA修复、过氧化物酶体、硫代谢和甲醇代谢等途径。尤其是核糖体含量提高,增强核糖体和核糖体生物合成途径,能够提高蛋白加工速度和加工准确性,从而提高蛋白表达量。

毕赤酵母以甲醇为唯一碳源进行新陈代谢时,甲醇代谢相关基因显著上调,甲醇代谢途径中间代谢产物(GAP和DHAP)为TCA循环、磷酸戊糖途径以及糖酵解途径中生物质合成提供前体物质及能量,提高了外源蛋白的表达量。但研究发现毕赤酵母在磷酸戊糖途径氧化阶段相关基因(ZWF1SOL3PGD1)无明显变化,非氧化阶段的TAL1-2RPE2分别发生了显著上调14.46倍和52.52倍,我们推测细胞为了缓解胞内的代谢压力,仅调整非氧化阶段相关基因的表达量,非氧化阶段在表达磷脂酶基因过程中对提高外源蛋白表达起到了重要作用。另一方面,细胞质中乙醛酸循环的关键酶基因(ICLMLS)表达水平显著下调,说明乙醛酸循环流量降低。位于线粒体TCA循环中的IDP1下调约3倍,胞内TCA循环过程的电子链氧化磷酸化供能需求降低,TCA循环代谢流降低。综上所述,甲醇诱导和甘油培养相较,胞内碳代谢表现为甲醇代谢上调,磷酸戊糖途径部分上调,乙醛酸循环下调以及TCA循环下调。说明碳代谢中心(TCA循环)供能的作用减弱,代谢主要集中在甲醇代谢和磷酸戊糖途径。

内质网参与蛋白加工过程,是研究蛋白质合成的重要场所,也是微生物定向改造的重要目标之一。内质网蛋白识别因子如KAR2LHS1SCJ1JEM1在甲醇诱导时上调,对于蛋白识别、牵引蛋白进入内质网有重要作用。研究报道表明,为了促进内质网中外源蛋白的折叠与加工,共表达内质网中伴侣蛋白及UPR相关基因,如KAR2PDI1,以促进蛋白质正确折叠和修饰[30]。微生物细胞内质网合成大量未正确折叠外源蛋白,在内质网中积累,引发ERAD效应[30]。本实验中检测得到ERAD效应标签基因表达量较低,说明磷脂酶基因在毕赤酵母中表达过程中未引起ERAD效应。因此,毕赤酵母中磷脂酶表达量依然有上升的空间。

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