鸡碳酸酐酶4基因在毕赤酵母中的表达

[目的]通过毕赤酵母的表达获得鸡碳酸酐酶4(CAⅣ)蛋白。[方法]根据鸡CAⅣ的序列,结合毕赤酵母密码子的偏好性,合成CAⅣ基因。将CAⅣ基因克隆到pPICZαA真核表达载体,获得重组表达质粒pPICZαA-CAⅣ。将其电转毕赤酵母GS115后,获得重组毕赤酵母菌GS115/pPICZαA-CAⅣ。用终浓度为1%的甲醇对重组阳性菌进行诱导表达,通过SDS-PAGE和Westernblot法检测蛋白的表达,并用Ni离子亲和层析法对表达出的蛋白进行纯化。[结果]成功构建了表达载体pPICZαA-CAⅣ,转化重组酵母菌后可分泌出36kDa左右的CAⅣ蛋白,并通过Ni离子亲和层析法获得了单一性的目的蛋白CAⅣ。[结论]获得分子量约36kDa的鸡CAⅣ蛋白。     

中华绒螯蟹Δ9-脂肪酸去饱和酶基因克隆和真核表达系统的构建

Δ9-脂肪酸去饱和酶是多不饱和脂肪酸合成途径的关键酶之一,可催化脂肪酸链上特定位置形成双键。本研究在已克隆到中华绒螯蟹(Eriocheir sinensis)的Δ9-脂肪酸去饱和酶(Δ9 fatty acyl-Co A desaturase,Δ9-FAD)基因基础上,为进一步了解其功能,根据中华绒螯蟹Δ9-FAD基因c DNA序列(accession number:JQ693685)以及真核表达载体p PIC3.5K,设计引物并在其上下游分别加入Bam HⅠ和Eco RⅠ酶切位点;利用反转录PCR(RT-PCR)技术,克隆其开放阅读框(open reading frame,ORF)片段,构建重组质粒p PIC3.5k-Δ9-FAD;利用电穿孔仪将经限制性内切酶SacⅠ线性化后的重组质粒p PIC3.5k-Δ9-FAD转入毕赤酵母(Pichia pastoris)GS115菌株中。经筛选与PCR验证,得到含有重组质粒p PIC3.5k-Δ9-FAD的毕赤酵母转化株。本实验成功构建了中华绒螯蟹Δ9-脂肪酸去饱和酶的毕赤酵母表达系统,为深入研究Δ9-脂肪酸去饱和酶的功能做了基础性的工作。     

玉米赤霉烯酮降解酶毕赤酵母表达载体的构建及其表达

目的构建毕赤酵母表达载体pPIC9-ZEN-jjm,筛选高效分泌表达活性目的蛋白的菌株。方法克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切连接至pPIC9中,电转化至毕赤酵母GS115。利用RDB培养基和甲醇诱导表达进行筛选。HPLC检测表达蛋白降解玉米赤霉烯酮的活性。结果测序表明ZEN-jjm成功插入pPIC9中,SDS-PAGE表明获得1株高效表达目的蛋白的重组酵母,其分子量约29 kDa。HPLC表明其能有效地降解玉米赤霉烯酮。结论玉米赤霉烯酮降解酶在毕赤酵母中获得了高效分泌表达。     

共表达伴侣蛋白对重组毕赤酵母产米根霉(Rhizopus oryzae)脂肪酶发酵过程的影响

[目的]通过共表达伴侣蛋白Erolp和PDI获得米根霉(Rhizopus oryzae)脂肪酶在毕赤酵母中的高效表达.[方法]利用毕赤酵母基因重组菌高密度发酵的方法,在7L发酵罐水平上分析共表达伴侣蛋白菌株(BH128)与非共表达伴侣蛋白菌株(H238)对脂肪酶表达的差异.[结果]在相同条件下,BH128发酵产酶能力高于H238,最高酶活可达到2 338.7U/mL,最大比生长速率达到0.02 h-1,最大产物比形成速率达到944.5 U/(gDCW·h),最大底物比消耗速率也能达到0.15 gmethanol/(gDCW·h),分别是H238的1.7、0.5、4.1和1.3倍,且发酵周期缩短了20h.[结论]毕赤酵母基因重组菌BH128通过共表达伴侣蛋白Ero1p和PDI,提高了米根霉脂肪酶的产量,而且缩短了发酵周期,为工业化生产奠定了基础.     

玉米赤霉烯酮降解酶多拷贝毕赤酵母菌株的构建及高效表达

通过密码子优化、体外多拷贝构建实现玉米赤霉烯酮(Zearalenone,ZEN)降解酶基因(zlhy-6)在毕赤酵母GS115菌株中的高效表达。按酵母密码子偏好性优化zlhy-6基因的密码子,与α因子信号肽编码序列一起合成,插入到pAO815质粒中,通过酶切酶连构建含1–6个表达盒的表达质粒,将其转入毕赤酵母GS115菌中,获得ZEN降解酶重组菌株。重组蛋白分子量为28.9 kDa,与预期一致。重组菌用甲醇诱导3 d,蛋白浓度达最高,之后下降;在pH 5.0、4.5条件下诱导培养,表达量最高;每天添加0.8%的甲醇、接种量10%表达水平最高;4拷贝的转化子表达水平最高,三角瓶发酵3 d,酶活性达到10 U/mL。在1 g玉米渣中添加0.1–0.5 mL发酵上清液,水解24 h,玉米渣中ZEN的降解率为44.08%–75.51%。研究结果为ZEN降解酶工业生产及在食品饲料中的应用奠定了基础。     

灰葡萄孢菌激发子pebC1在毕赤酵母中的分泌表达及其生物活性分析

为了实现激发子PebC1编码基因在毕赤酵母中的分泌表达,采用PCR方法从灰葡萄孢菌BC-4-2-2-1菌株中扩增获得激发子PebC1的编码序列,将其亚克隆至酵母分泌型表达载体pPIC9K中,以此片段构建了pPIC9K-pebC1重组表达质粒。重组表达质粒经Bgl Ⅱ线性化处理,电击转化至毕赤酵母宿主菌GS115,经MD、G418-YPD平板和PCR法筛选,获得了重组毕赤酵母菌GS115/pPIC9K-pebC1。用甲醇诱导重组酵母菌表达目标蛋白,发酵液经SDS-PAGE电泳分析,在约39 kDa处出现特异目标条带。Western blotting检测结果说明,重组表达产物具有良好的抗原性。生物活性检测表明,酵母重组表达蛋白PebC1能够诱导拟南芥和黄瓜幼苗对灰霉病的抗性。     

猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

为了研制高活性的重组猪β干扰素,对PoIFN-β成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαA-PIB。pPICZαA-PIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFN-β,其中B1株酵母的PoIFN-β产量最高,约为2.5×105U/mL,其表达量约为60μg/mL,比活为4.17×106U/mg。将发酵上清液用PEG20000浓缩后进行SDS-PAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFN-β阳性抗血清发生特异反应。表达产物比PoIFN-β理论推导分子量(约20.8kDa)大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFN-β对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFN-β对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。     

SARS冠状病毒M基因的克隆及其表达

从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RT-PCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZαB,电穿孔法将重组体整合入酵母菌P.pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS-PAGE检测其表达产物.Mut+酵母转化菌经甲醇诱导可分泌表达约65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性.     

SARS冠状病毒M基因的克隆及其表达

从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RTPCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZaB,电穿孔法将重组体整合人酵母菌P．pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut^-菌用甲醇诱导表达,SDS-PAGE检测其表达产物。Mut^-酵母转化菌经甲醇诱导可分泌表达约65kDa和42kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

Production and purification of a novel thermostable phytase by Pichia pastoris FPHY34

A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L⁻¹ and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (Y_X/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL⁻¹ at a methanol feed rate of 3.0 mL L⁻¹ h⁻¹ after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg⁻¹ and 3281 U mg⁻¹ in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production.     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114–174, 206–257, and 258–276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114–276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris

The scene of the protein micro-heterogeneity of recombinant hirudin-II (HV2) expressed in Pichia pastoris was investigated. It was shown that three derivatives of HV2 were present in the fermentation broth of P. pastoris, which were intact HV2 and its two derivatives truncated the C-terminal amino acid residue Gln and Leu-Gln, respectively. To purify the minor degradation derivatives of HV2, a simple, biocompatible and scale-up-feasible purification process with two-step ion-exchange chromatography was established instead of usual reverse phase chromatography. The purities of end products were over 96% and the residual endotoxin less than 0.5 EU/ml.