Metabolomics Study in Methylotrophic Yeast： A Minireview

Methylotrophic yeast has been used as a cost-effective and valuable host for expression of recombinant protein due to its unique methanol utilisation pathway. It has an AOX （alcohol oxidase） protein which has been characterised to be a strong and tightly methanol-inducible dependent promoter. Metabolomics is the systematic study and inclusive analysis of small molecules called metabolites in a biological system. Metabolomics plays an important part in connecting the phenotype and genotype gap because it magnifies the modifications in the proteome and provides a better phenotype representation of an organism. This quantitative study has provided a new perception on the metabolic burden derived from the overexpression of recombinant protein in methylotrophic yeast. In this review, we discuss the fundamental aspect of metabolomics in methylotrophic yeast followed by their latest developments.

Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals,owing to its high degree of reduction,abundance,and low price.Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals.Alternatively,synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms,such as Escherichia coli.Owing to the complex metabolic pathways,limited availability of genetic tools,and methanol/formaldehyde toxicity,the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility.This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms.It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol

Methylotrophic yeasts and bacteria, which can use methanol as carbon and energy source, have beenwildly used as microbial cell factories for biomanufacturing. Due to their robustness in industrial harshconditions, methylotrophic yeasts such as Pichia pastoris have been explored as a cell factory forproduction of proteins and high-value chemicals. Methanol utilization pathway （MUT） is highlyregulated for efficient methanol utilization, and the downstream pathways need extensively constructedand optimized toward target metabolite biosynthesis. Here, we present an overview of methanolmetabolism and regulation in methylotrophic yeasts, among which we focus on the regulation of keygenes involved in methanol metabolism. Besides, the recent progresses in construction and optimizationof downstream biosynthetic pathways for production of high value chemicals, such as polyketides, fattyacids and isoprenoids, are further summarized. Finally, we discuss the current challenges and feasiblestrategies toward constructing efficient methylotrophic cell factories may promote wide applications inthe future.

Engineering the native methylotrophs for the bioconversion of methanol to value-added chemicals:current status and future perspectives

Methanol is becoming an attractive fermentation feedstock for large-scale bioproduction of chemicals,due to its natural abundance and mature production technology.Native methylotrophs,which can utilize methanol as the only source of carbon and energy,are ideal hosts for methanol bioconversion due to their high methanol utili-zation rate and have been extensively employed in the production of value-added chemicals from methanol.Here,we review the natural methanol utilization pathways in native methylotrophs,describing the available synthetic biology tools developed for engineering native methylotrophs,and discuss the strategies for improving their methanol utilization efficiency.Finally,the representative examples of engineering the native methylotrophs to produce value-added products from methanol are summarized.Furthermore,we also discuss the major challenges and possible solutions for the application of native methylotrophs in methanol-based biomanufacturing.

Efficient fatty acid synthesis from methanol in methylotrophic yeast

Methanol is an attractive C1 feedstock with high abundance and low cost in bio-manufacturing.However,the metabolic construction of cell factories to utilize methanol for chemicals production remains a challenge due to the toxic intermediates and complicated metabolic pathways.The group of Zhou rescued methylotrophic yeast from cell death and achieved high-level production of free fatty acids from methanol through a combination of adaptive laboratory evolution,rational metabolic engineering and multi-omics analysis.

Role of methylotrophic bacteria in managing abiotic stresses for enhancing agricultural production

Abiotic stresses are a significant factor that considerably limits plant growth and productivity.Methylotrophs are an essential group of bacteria that utilize volatile carbon compounds,are prolific colonizers of different plant parts,and play a vital role in plant growth promotion(PGP)under stress conditions.Numerous rhizospheric and phyllosphere methylotrophs have been reported to exhibit PGP activities with superior stress-tolerating capacity against drought,heavy metal,salinity,high and low temperatures,solar ultraviolet radiation,and other harsh environmental conditions.Methylotrophs promote plant growth through N2 fixation,phosphate solubilization,production of phytohormones(i.e.,auxins,gibberellins,and cytokinins),1-aminocyclopropane-1-carboxylic acid(ACC)deaminase activity,siderophore,ammonia production,and secondary metabolites.The production of these compounds by methylotrophs protects a plant against adverse environmental conditions and influences its productivity.This review discusses the role methylotrophs play in managing various abiotic stresses,how they help mitigate these stresses,and how they improve agricultural productivity.

甲醇营养型酵母高密度培养过程中甲醇和乙醇的GC快速检测

采用气相色谱法 (GC)快速检测甲醇营养型酵母发酵液中的甲醇、乙醇含量 ,具有样品处理简便 ,测定时间较短 ,结果重现性好的特点。在 1～ 10mg/mL范围内具有很好的线性关系。在毕赤酵母高密度高表达发酵过程中应用此法对甲醇和乙醇进行实时监控 ,细胞终密度超过 30 0g/L(干重 )。本方法为甲醇营养型酵母工程菌的发酵中试工艺研究提供了重要的发酵生化参数。

甲基营养菌WB-1甲胺磷降解酶的产生、部分纯化及性质

甲基营养菌WB 1菌株在以甲胺磷作碳氮源的无机盐培养基上生长时 ,产生甲胺磷降解酶情况较好 ,培养 2 0h为细胞收获的适宜时期。所得细胞经过超声波破碎、吐温 2 0抽提、热处理 ( 60℃ ,9min)、DEAE 纤维素 32和CM 纤维素 32柱层析等步骤 ,得到的酶制品比活力为 1 8 0U/mg ,收率 78 8% ,纯化倍数 2 2 8。纯化的酶制品经连续聚丙烯酰胺凝胶电泳 ,呈现一条主带 ,酶活力染色则呈现一条与之对应的活性谱带 ;该酶催化反应的适宜 pH为9 0 ,底物专一性不强 ,Hg2 +、Mn2 +、Cu2 +对酶有强烈的抑制作用。部分纯化酶制品在 - 2 0℃( 0 .1mol/L的磷酸盐溶液中 )存放 5d ,酶活力丧失约 60 %。

基因工程菌Pichia pastoris高密度培养条件研究

基因工程菌pichiapastoris最佳种子培养基为添加 4mL/LPTMl的BMGY培养基 ;全合成高密度摇瓶培养基是甘油 4% ,(NH4) 2 SO41 0g/L ,CaSO40 93g/L ,K2 SO41 8 2g/L ,MgSO4·7H2 O 1 4 9g/L ,0 1mol/L磷酸缓冲液 (pH =6 0 )配制 ,培养 2 6h后细胞密度OD60 0 可达到 65。经SDS PAGE电泳图谱分析 ,甲醇诱导培养 72h结果 :1 2h有重组人血清白蛋白表达 ,2 4h达到最大。此全合成摇瓶培养基与批补料发酵培养基相类似 ,有利于指导发酵罐上发酵培养。

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 。

影响甲醇酵母中外源蛋白表达的因素

甲醇酵母系统由于其在表达蛋白方面无可比拟的优越性 ,已越来越得到广泛的应用。但不同的外源蛋白表达量有很大差异。介绍了影响甲醇酵母中外源蛋白表达的因素 ,这将有助于揭示甲醇酵母中外源蛋白表达的内在机制。

基因工程菌Pichia pastoris连续培养的生长及抑制动力学

在同一稀释率μ(μ=0 .1 4h-1)下用不同浓度的甘油流加进行连续培养 ,研究甘油浓度对基因工程菌毕赤酵母 ( Pichia pastori)生长的影响。结果表明甘油浓度对细胞生物量 ( DCW和WCW)、底物得率系数 ( YX/ S)、底物比消耗速率 ( qs)、呼吸熵 ( RQ)与二氧化碳释放率 ( CER)都有影响 ,在低甘油残留浓度 ( <63.3g/L)下 ,甘油是激活剂 ,菌体生长符合 Monod方程 ,Ks=1 9.62 g/L,甘油激活常数 Ka=1 9.45 g/L;而在高甘油残留浓度 ( >63.3g/L )下甘油是抑制剂 ,菌体生长特征符合 Haldance方程 ,Ks=0 .0 1 4g/L,KI=1 5 6.67g/L。毕赤酵母生长的甘油抑制浓度为 5 5 .42

微生物酶催化甲胺磷降解机理初探

甲胺磷 (MAP)农药经微生物酶 -甲胺脱氢酶催化后 ,采用聚酰胺薄膜层析、钼酸铵试剂和 HPL C等分法 ,分别可检测出氨基、磷酸根和甲醇等化合物 .将去除氨基的 MAP溶液作小鼠灌胃试验 ,发现该农药的毒性明显降低 .文中还对 MAP在自然界的降解过程等问题进行了讨论 .

毕赤氏酵母工程菌高密度发酵表达恶性疟原虫融合抗原的研究

The effects of cultural conditions,which were fermentation period when methanol was as sole carbon source,methanol concentration,range of pH,on the expression of the chimeric protein of Plasmodium falciparum in genetically engineered methylotrophic Pichia pastoris were investigated by shake flask experiments in this paper.The results showed:(1)fermentation period with methanol inducement is about 96 hours;(2)the optimum methanol concentration as sole carbon source is 10g/L;(3)the range of pH is from 6.0 to 7.0. On the base of above experimental results the cell high-density fermentation had been done on the FMG-5L fermentor with multi-sensors.The results showed that the cell optical density (OD 600) can reached 550 and the ma-ximum expression level of target proteins was 780mg/L which was 4 times higher or more than the shake flask culture’s.

甲醇酵母基因表达系统的研究进展

在大肠杆菌这一传统表达系统被频繁用作研究各种基因表达时，一种新型且有效的基因表达系统——甲醇酵母正逐渐引起人们的注意。此系统不仅具有高表达、高稳定、高分泌的特点，而且其宿主甲醇酵母——巴斯德毕赤酵母（Ｐｉｃｈｉａｐａｓｔｏｒｉｓ）具有高密度生长的特性。因此近年来此表达系统的研究得到迅速发展，在其中表达了多种具有商业价值的外源蛋白。本文对甲醇酵母基因表达系统的特点及研究进展作一简要综述。

多形汉逊酵母外源基因表达系统

多形汉逊酵母是一种有很大潜力的外源基因表达系统 ,已在科研和工业化生产上广泛应用。用它生产来源于真核生物的外源基因有许多优点 ,如重组菌减数分裂稳定、能进行正确的翻译后加工和修饰、表达量高等。许多有商业价值的蛋白质在这一系统中得到成功表达 ,有的已投入市场。本文综述多形汉逊酵母宿主菌的生物学特性、基因工程操作技术。

甲基营养型酵母表达载体系统研究进展

Methylotrophic yeast expression system is one of the eukaryotic expression systems which was widely applied and devoloped fast in rencent years. Expression of the methanol inducible genes, expression vector, fermentation, purification and character of secretion proteins, factors affecting heterologous gene expression, comparision of methylotrophic yeast and other yeasts are reviewed in this paper. Ref

改变毕赤酵母信号肽对人三叶因子3表达的影响

为了表达具有天然N 末端的人三叶因子 3,构建了含两种信号肽序列的毕赤酵母表达载体 ,其中一种表达载体在分泌信号肽序列α factor后保留STE13信号肽切割序列 ,另一种表达载体去除了该序列。将 2种表达载体分别转化到毕赤酵母GS115中进行表达。经甲醇诱导后目的蛋白分泌到发酵液上清中 ,SDS PAGE和Western印迹证明 2种重组蛋白均以二体的形式分泌表达 ,分子质量约为 13ku ,都能被TFF3抗体所识别。N 端氨基酸测序证明其中一个重组蛋白具有天然人TFF3的N端 ,质谱检测分子质量与天然蛋白一致 ;另一重组蛋白N 端则带有 2个未切割完全的信号肽序列GluAla ,质谱检测该蛋白中还含有部分切割完全的蛋白。

蚯蚓纤溶酶7#组分基因在甲醇酵母中的克隆与表达

为了使蚯蚓纤溶酶(EFE)最佳活性组分EFE_7#基因在甲醇酵母(Pichiapastoris)中获得表达,构建了表达载体pPIC9K_EFE,通过对电击条件的优化,转化率达到5×103个/μgDNA.为了便于筛选阳性克隆,以该基因的原核表达产物为抗原免疫动物,获得了与天然EFE_7#组分具有相同免疫特异性的抗体.用该抗体为探针对上述转化子进行筛选,得到了上百株阳性重组子.RT_PCR和westernblotting证明,EFE_7#基因在这些重组子中获得了表达,表达产物相对分子质量高于天然产物.

含短C肽人胰岛素原类似物DesB^30在毕赤酵母中的表达及纯化

合成含短C肽AAK,并去掉B链第30位苏氨酸(T)的人胰岛素原类似物:HMPIDesB30(human mini-proinsulin des B30)的cDNA,将其插入大肠杆菌和酵母菌的穿梭质粒pPIC9K.用电转移的方法将重组质粒HMPIDesB30/pPIC9K转入甲醇酵母GS115.用含不同G418浓度的YPD平板筛选高拷贝重组子.经优化条件下的高密度发酵,发酵液用SIPI-40大孔树脂吸附,大孔吸附树脂洗脱液经SP柱进一步纯化后,再用10～20mmol/L Zn2+沉淀,能得到纯度为95%的HMPIDesB30.通过16.5%Tricine SDS-PAGE、HPLC和质谱分析,表达产物分子质量与理论分子质量相符,经高密度发酵,表达量可达1.0g/L.纯化回收率可达60%.说明人胰岛素原类似物HMPIDesB30能在甲醇酵母中高效分泌表达,并能通过经济有效的方法纯化.