产HSA—CP融合蛋白毕赤酵母的发酵条件优化

为提高重组毕赤酵母生产人血清白蛋白-C肽融合蛋白（HSA—CP）的产量和生产强度,在摇瓶条件下考察了甲醇诱导时间和浓度对目的蛋白产量的影响。结果表明,质量浓度10g/L的甲醇诱导72h最适于产物表达。通过对7L发酵罐中各因素的优化,得到最佳条件为：初始甘油质量浓度10g/L,30℃培养,菌体生长期和诱导期的pH及溶氧分别控制在pH5．0、30％溶解O2或pH6．0、15％的溶解O2。10g/L的甲醇诱导72h,最终使干细胞质量浓度达到56．43g/L,目的蛋白产量达368．45mg／L。生产强度为3．920mg/（L·h）,目标蛋白的比生产速率为5．12mg/（L·h）。     

重组毕赤酵母发酵牛肉风味肽的中试研究

研究分泌表达16拷贝牛肉风味肽（beefy meaty peptide, BMP）毕赤酵母工程菌株(Pichia pastoris GS115-16B2)的500L发酵罐中试发酵工艺。在500L发酵罐中采用三步发酵法进行了3批次中试发酵实验,设定菌体培养阶段温度为30℃,pH5.0,诱导表达阶段温度为28℃,pH3.0。在发酵程中通过调节搅拌速度、通气量和罐压等措施来使DO维持在20%~35%左右,总共发酵126h（诱导时间为96h）,当诱导88h后（发酵118h）,600nm波长下的光密度值(OD600)达到184.5,菌体湿重达到318.7g/L,目的产物BMP的表达量达到最高为172 mg/L,实现了BMP的高效表达。通过中试发酵初步建立了工程菌P. pastoris GS115-16B2的中试发酵工艺,为BMP的进一步研究开发奠定了良好基础。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度,在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明,以下条件：初始甘油浓度40g／L、初始甲醇浓度3.1g甲醇／gDCW、每24h添加0.51g甲醇／gDCW、诱导表达周期72h、250mL三角瓶诱导培养基装液量30mL、初始pH6,0,最适于菌体生长与产物表达。在此基础上,7L罐上通过恒速流加甘油进一步提高细胞密度,诱导阶段甲醇采取前期恒速流加和后期DO-stat,发酵结束菌体干重达80g／L,酶活为217U／mL,比摇瓶结果提高了66.2％。     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

发酵条件对毕赤酵母表达重组人干扰素ω糖基化的影响

发酵条件是影响毕赤酵母 (P .pastoris)表达外源重组糖蛋白时糖基化的重要因素。通过菌体浓度、起始pH值、甲醇诱导浓度和周期、装液量等摇瓶发酵实验 ,研究不同发酵条件对毕赤酵母表达分泌型重组人干扰素ω(rhIFNω)过程中糖基化的影响 ;同时 ,在连续培养过程中考察pH值变化对rhIFNω糖基化的影响和分批发酵过程中rhIFNω糖基化的变化。结果表明 ,控制菌体密度 250g L(WCW)、起始pH值 6 0、装液量小于 30mL、甲醇诱导浓度 15g L、甲醇诱导 3次 (每 24h诱导一次 )等发酵条件 ,有利于摇瓶发酵过程中rhIFNω的糖基化 ;控制pH值 70～75可促进rhIFNω的糖基化 ;分批发酵过程中 ,糖基化与非糖基化rhIFNω的含量有同比变化趋势 ,但糖基化rhIFNω所占比例明显低于摇瓶发酵实验的结果 ,其原因有待进一步研究。     

酵母表达肌肉生长抑制素前肽发酵工艺的优化

突变型肌肉生长抑制素前肽（MMP）在治疗肌肉萎缩症和培育多肌肉牲畜上有着广泛的应用前景。以重组表达MMP的毕赤酵母工程菌为模式,对该工程菌在30L发酵灌中培养与诱导备件进行优化,建立该表达系统大规模发酵的最佳生产条件,以期荻得最短的发酵时间和最低的生产损耗。毕赤酵母发酵过程通常分为3个阶段：分批培养（基础培养）阶段、分批补料培养阶段、诱导阶段。对发酵过程的第2与第3阶段进行了优化。通过分步提高甘油流加速度：以20mL／L·h^-1的速度流加5h、以30mL／L·h^-1的速度流加5h、以50mL／L·h^-1速度流加14h,并通入纯氧气,维持60％的溶氧度,使甘油流加阶段的时间从常规的48h缩短至24h,即只需24h即可达到常规方法48h才能达到的菌体密度。在诱导表达阶段,通过甘油与甲醇的交替流加,同时对发酵液中甲醇含量的实时监测,保持甲醇的浓度不超过0．5％的高限,使诱导的时间从常规的72h缩短至36h,而且表达量提高了约1倍。优化后,整个发酵周期从120～148h缩短至72～80h,显著提高了MMP蛋白的生产效率。     

出芽短梗霉发酵过程溶氧控制的研究

在搅拌罐式生物反应器中,通过控制DO（溶氧浓度）的变化,对出芽短梗霉（Aureobasidium pullulans）发酵过程的控制进行了研究。以100g/L玉米粉水解液做碳源,比较了不同溶氧控制条件下发酵参数的变化及其对出芽短梗霉发酵结果的影响。结果表明,过低的DO对菌体生长和多糖生产都不利,过高的DO使培养液中糖大部分消耗在菌体的生长上,也不利于多糖的生产,通过控制搅拌速度和通气量能将DO维持在较合适的水平。     

甲醇／山梨醇共混诱导改善重组毕赤酵母表达猪a干扰素发酵过程和NADH再生效率

在10L发酵罐中利用重组毕赤酵母诱导表达猪a干扰素（pIFN-a）,考察甲醇／山梨醇共混诱导策略对pIFN-a表达水平提高和能量（NADH）再生效率的影响。结果表明：在诱导稳定期,甲醇／山梨醇共混诱导可弱化细胞的甲醇代谢,有利于缓解毒副中间产物（过氧化氢、甲醛等）的生成积累;以0．785g／（L·h）的速率缓慢共混流加山梨醇时,pIFN-a抗病毒活性最大,最高活性可达1．8×107IU／mL,与30℃常温甲醇单独诱导（最高活性1．0X10。IU／mL）和20℃低温甲醇单独诱导（最高活性1．4×lO6IU／mL）相比,活性均大幅提高,且胞外pIFN-a的降解减缓;发酵体系的抗高甲醇浓度冲击能力有效提高,发酵生产的稳定性增强;能量利用效率大幅提高,NADH的再生利用效率提高了29％-84％。     

酵母菌表达倍菲隆发酵工艺研究

目的 :为了探索用酵母菌表达乙型干扰素 (倍菲隆 )的大批生产的发酵工艺流程及优化的重要参数。选用 1 5L不锈钢发酵罐作为乙型干扰素发酵工艺研究系统。方法 :探索了种细胞密度对初始发酵期 (甘油利用期 )的影响 ,启动甘油补充期的溶解氧浓度 ,诱导目的蛋白表达时添加甲醇的速率条件控制 ,pH对酵母菌生长与蛋白表达的影响以及发酵时间对乙型干扰素表达量影响等重要发酵工艺参数。结果 :接种高密度的种菌 (OD60 0为 9)时酵母菌增殖快 ,湿细胞重量于1 8h即达到 1 5 7g L。当DO恢复至 67%时即开始补充甘油可使酵母菌在 5h内增殖达到 2 1 9g L。添加甲醇速率从 2 %(甲醇终浓度为 0 0 3%)开始 ,逐步增加 ,在 2 4h内达到 33%的添加速率 (甲醇终浓度为 0 5 %)。甲醇诱导前pH为 6时最有利于酵母菌生长 ,在蛋白表达时期 ,控制发酵罐内pH值在 3 0 ,可得最高蛋白表达量。发酵 70h乙型干扰素表达量达到高峰。结论 :接种高密度种菌可促进工程菌生长 ,当DO恢复至 67%时开始补充甘油可加快酵母菌生长 ,在诱导期中缓慢添加甲醇并维持发酵罐pH为 3 0 ,发酵 70h可获得最佳蛋白生产量。     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.