氧化葡萄糖酸菌转化制备米格列醇关键中间体

米格列醇作为一种新型α－葡糖苷酶抑制剂,能够有效治疗Ⅱ型糖尿病,并已迅速成为首选药物。葡萄糖酸菌细胞膜上含有多种脱氢酶,能够催化一系列重要产物的微生物转化,米格列醇就是其中之一。本文将详细说明不同的微生物转化过程,并进行比较。     

氧化葡糖杆菌sndh-sdh基因簇的克隆表达

目的:从氧化葡糖杆菌H763中克隆sndh-sdh基因簇,在大肠杆菌和氧化葡糖杆菌621H中分别表达山梨酮脱氢酶-山梨糖脱氢酶(SNDH-SDH),并检测其活性。方法与结果:以氧化葡糖杆菌H763基因组DNA为模板,PCR扩增包括启动子、结构基因及终止序列在内的sndh-sdh基因簇,回收3533 bp的扩增产物,连入pMD18T载体,转化至大肠杆菌DH5α中表达;以山梨糖或木糖为底物,DCIP法检测菌体裂解液,DCIP检测液颜色由蓝绿色变为黄色,表明大肠杆菌表达产物具有脱氢酶活性。构建pBBR1MCS2-sndh-sdh载体,通过接合转移导入氧化葡糖杆菌621H,重组葡糖杆菌在以山梨醇或山梨糖为底物的培养基中培养,采用薄层层析检测法检测其培养上清中的代谢产物,层析板上显示了2-酮基-L-古龙酸斑点。结论:重组大肠杆菌DH5α和氧化葡糖杆菌621H中均表达了有脱氢酶活性的SNDH-SDH。     

利用山梨糖脱氢酶活性筛选氧化葡糖杆菌PQQ合成基因簇

【目的】利用山梨糖脱氢酶醌酶活性从氧化葡糖杆菌H24中分离PQQ生物合成基因簇。【方法】利用ptsG位点整合sdh基因的大肠杆菌JM109作为宿主菌构建了氧化葡糖杆菌H24的基因组DNA文库。通过山梨糖脱氢酶活性检测,从文库中筛选具有PQQ合成能力的单菌落并进行亚克隆。【结果】从氧化葡糖杆菌H24的基因组文库中筛选得到一株具有山梨糖脱氢酶活性的单菌落,亚克隆后序列分析显示插入片段全长5400bp,对应5个编码框(pqqABCDE),与其他细菌PQQ生物合成基因簇有很高的序列同源性。【结论】利用山梨糖脱氢酶醌酶活性成功从氧化葡糖杆菌H24中分离克隆得到了PQQ生物合成基因簇pqqABCDE。     

高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响

【目的】研究高浓度的2-KLG对其生产菌株氧化葡萄糖酸杆菌生产过程中关键的脱氢酶合成基因、辅因子合成基因及其转运蛋白编码基因的影响。【方法】测定高浓度梯度2-KLG下氧化葡萄糖酸杆菌的生长情况,确定合适的添加浓度对氧化葡萄糖酸杆菌进行胁迫。使用实时定量PCR技术检测2-KLG合成中关键山梨醇脱氢酶基因sld AB、关键辅因子PQQ合成基因pqq ABCDE及5个潜在转运蛋白合成基因的变化。【结果】根据氧化葡萄糖酸杆菌在2-KLG高浓度梯度下生长测定实验结果,选定40、80和120 g/L 2-KLG作为添加浓度。实时定量PCR结果显示,在高浓度的2-KLG压力下,PQQ合成基因pqq ABCDE未受到显著影响,山梨醇脱氢酶基因sld AB以及部分PQQ潜在转运蛋白编码基因的表达均显著下调。【结论】高浓度2-KLG会抑制氧化葡萄糖酸杆菌中山梨醇脱氢酶基因的表达,有可能会影响辅酶PQQ的转运,但不会显著影响辅酶PQQ的合成。     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Use of population genetics to derive nonrecombinant Saccharomyces cerevisiae strains that grow using xylose as a sole carbon source

According to scientific dogma, Saccharomyces cerevisiae cannot grow utilizing xylose as a sole carbon source. Although recombinant DNA technology has overcome this deficiency to some degree, efficient utilization of xylose appears to require complex global changes in gene expression. This complexity provides a significant challenge to the development of yeasts suitable for the utilization of xylose-rich lignocellulosic substrates. In contrast to the dogma, we have found that native strains of S. cerevisiae can grow on xylose as a sole carbon source, albeit very slowly. This observation provided the basis for a new approach using natural selection to develop strains of S. cerevisiae with improved ability to utilize xylose. By applying natural selection and breeding over an extended period, we have developed S. cerevisiae strains that can double in less than 6 h using xylose as a sole carbon source. Strains with improved growth rate possessed increased xylose reductase and xylitol dehydrogenase activities, with the latter showing the greater improvement. This unique, completely nonrecombinant approach to developing xylose-utilizing strains of S. cerevisiae opens an alternative route to the development of yeast that can fully utilize lignocellulosic substrates.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Solution properties of an exopolysaccharide from a Pseudomonas strain obtained using glycerol as sole carbon source

We report the solution properties of a new exopolysaccharide (EPS) obtained from a Pseudomonas strain fed with glycerol as the sole source of carbon. This high molecular mass (3 × 10⁶ g mol⁻¹) biopolymer is essentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolyte character. The Smidsrod parameter B computed from the ionic strength dependence of the intrinsic viscosity indicates that the EPS backbone is rather flexible. In salt free aqueous solutions, the zero shear viscosity scaling with concentration follows a typical polyelectrolyte behavior in bad solvent, whereas at high ionic strength the rheological response is reminiscent from neutral polymers. Light scattering data indicate that the EPS adopts a globular conformation as a result of hydrophobic interactions. EPS solutions are stable within 4 days as particle sizing does not indicate EPS aggregation. Both globular conformation and stability against precipitation from solution are attributed to the low charge density of the polyelectrolyte.     

A Rapid Procedure for the Large Scale Purification of Elastase and Cathepsin G from Human Sputum

A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatography on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum a re identical to the same enzymes isolated directly from the leukocytes of human blood.     

Acetate as sole carbon and energy source for growth of methanosarcina strain 227

[This corrects the article on p. 995 in vol. 39.][This corrects the article on p. 996 in vol. 39.].     

para-Aminobenzoic acid as a sole source of carbon and energy for Pseudomonas desmoliticum]

A strain of Psuedomonas desmoliticum has been isolated from soil. It utilizes, as a source of carbon and energy, p-aminobenozic (PABA), p-fluorobenzoic acids, and some other aromatic compounds. The strain has been isolated by inoculating soil suspensions onto Petri plates with a solid mineral medium containing 0.1% PABA as a carbon source. The preparatory metabolism of PABA was studied in this work; p-hydroxybenzoic and protocatechuic acids were found to be its intermediate products. Enzyme systems catalysing oxidation of aromatic compounds and glucose are inducible.