顶头孢霉遗传育种研究进展

顶头孢霉是一类重要的工业微生物,其发酵产物头孢菌素C可用来生产7-ACA,而后者是临床常用抗感染药物头孢类抗生素的重要中间体。头孢菌素C的发酵水平决定了其下游头孢类抗生素的生产水平、产品质量及价格,因此对顶头孢霉的菌种选育工作显得尤其迫切。随着分子生物学的发展,基因工程分子改造在遗传育种领域发挥着越来越重要的作用。文章综述了对头孢菌素C的生物合成以及调控的研究进展,并将国内外对顶头孢霉进行遗传育种的结果进行了归纳总结,提出了可以从提高头孢菌素C发酵水平、延伸代谢途径等不同方面对头孢菌素C生物合成及调控基因,包括外源基因的导入和表达进行改造优化,并对进一步的研究目标进行了展望,认为可以结合比较蛋白质组和基因组改组使遗传育种所获得的工程菌尽快进入产业化。     

菌丝形态分化与头孢菌素C合成的关系*

利用显微图像分析法对顶头孢霉菌的菌丝形态进行了定量研究,并统计分析了头孢菌素C发酵过程中的菌丝形态的变化规律,具体对菌丝长度、菌丝宽度和菌丝生长单位进行了定量分析,分析了菌丝形态分化与头孢菌素C合成的关系。研究表明头孢菌素C的合成是在细长菌丝分化成膨大菌丝片段后才启动的,头孢菌素C可能主要是在膨大菌丝分化成节孢子的过程中被合成。     

大肠杆菌头孢菌素C乙酰酯酶的敲除对头孢菌素C酰化酶应用的影响

7-氨基头孢烷酸(7-ACA)是合成头孢菌素类抗生素的重要中间体,工业上通常采用头孢菌素C酰化酶一步水解头孢菌素C制备,但在该反应产物中存在一个主要杂质3-去乙酰基-7-氨基头孢烷酸(D-7-ACA),该杂质的产生是由大肠杆菌中内源基因aes编码的头孢菌素C乙酰酯酶水解头孢菌素C或7-ACA引起的.为了防止D-7-AC...     

菌丝形态分化与头孢菌素C合成的关系

利用显微图像分析法对顶头孢霉菌的菌丝形态进行了定量研究,并统计分析了头孢菌素C发酵过程中的菌丝形态的变化规律,具体对菌丝长度、菌丝宽度和菌丝生长单位进行了定量分析,分析了菌丝形态分化与头孢菌素C合成的关系。研究表明头孢菌素C的合成是在细长菌丝分化成膨大菌丝片段后才启动的,头孢菌素C可能主要是在膨大菌丝分化成节孢子的过程中被合成。     

RNAi技术降低头孢菌素C工业生产菌株中cefG基因的转录

RNAi是近年来新兴的一种通过抑制基因转录水平来实现特定基因表达沉默的技术。本研究尝试在顶头孢霉工业生产菌株中利用RNAi技术沉默cefG基因的转录,构建了一个含可形成cefG双链RNA转录单元的质粒,并将其导入头孢菌素C工业生产菌株中。结果发现其中两个转化子的cefG基因转录水平在发酵第4天较出发菌株降低了80%以上,而它们的头孢菌素C发酵水平较出发菌株则分别降低了34.6%和28.8%。     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

不同补料控制方式发酵生产头孢菌素C的性能比较

在7 L发酵罐下,对利用顶头孢霉菌(Cephalosporins acremonium)发酵生产头孢菌素C(CPC)过程的最优底物流加工艺进行了研究。提出了一种新式硫铵豆油耦联型的硫铵流加策略。该控制策略可将发酵液中的氨态氮浓度控制在3 6 g/L之间,同时满足了发酵前期细胞生长与CPC合成对氮源和硫源的需求,促进了顶头孢霉菌菌丝分化,为发酵后期的CPC高效生产奠定了前期基础。比较了CPC合成期内间歇、匀速和DO-Stat自动流加3种不同豆油流加方式的发酵性能。研究发现,耦联使用硫铵/后程通富氧空气DO-Stat法进行硫铵和豆油的同时补料和CPC发酵,可将碳源浓度与溶解氧浓度DO同时控制于适中水平,使CPC合成以高浓度和低副产物积累的方式进行,最终CPC浓度和得率分别达到35.77 g/L和13.3%。主代谢副产物脱乙酰氧头孢菌素C(DAOC)的积累量和DAOC/CPC分别仅有0.178 g/L和0.5%。     

头孢菌素C产生菌的诱变育种及培养基优化

通过对顶头孢霉（Cephalosporium acremonium）FC-01进行诱变选育及特定种子培养基的优化,提高了头孢菌素C的发酵产量。分别采用紫外-氯化锂和钴-60（60Co）γ射线对FC-01进行诱变选育,筛选到高产菌株FC-1-4和FC-4-2,产量较出发菌株分别提高了26%和54.5%。运用Plackett-Burman设计方法和响应面法对种子培养基进行优化,头孢菌素C发酵效价较对照分别提高了34.7%和13.2%,优化后的种子培养基主要成分为玉米浆3.70%、葡萄糖2.62%和硫酸镁0.15%,得到的菌株及相应的种子培养条件已成功应用在160M³工业发酵罐生产中,具有重要的工业生产能力。     

顶头孢霉中己糖激酶编码基因Achka的功能研究

己糖激酶在真菌中广泛存在,参与葡萄糖磷酸化等生理过程。本文从头孢菌素产生菌顶头孢霉中克隆并鉴定了一个己糖激酶编码基因,命名为Achka。通过RT‐PCR证明Achka含有4个内含子,其推测的编码蛋白含有484个氨基酸,分子量为53.7k Da。在顶头孢霉中敲除Achka后发现,突变株在以果糖、蔗糖或甘露糖为唯一碳源的培养基上生长受到严重限制。我们在大肠杆菌中异源表达了Achka,并对表达的重组蛋白AcHKA进行了分离纯化。酶学动力学分析表明,AcHKA对果糖的最大反应速率要大于葡萄糖,但是却对葡萄糖有更高的亲和力。上述结果进一步证明AcHKA为己糖激酶,在顶头孢霉体内主要负责果糖、蔗糖和甘露糖等的代谢。     

头孢菌素酰化酶基因在大肠杆菌中的表达规律

假单孢菌sp .130头孢菌素酰化酶催化戊二酰 7 氨基头孢烷酸的水解反应 ,生成 7 氨基头孢烷酸。 7 氨基头孢烷酸是医药工业合成大多数头孢菌素衍生物的起始原料。在 6种大肠杆菌表达质粒上构建了表达该酶的不同载体 ,得到了不同表达结果的大肠杆菌转化子。这些质粒有各自的特点 ,适用于不同的场合。     

Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum

By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.     

Recombinant microorganisms for industrial production of antibiotics

The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7-aminocephalosporanic acid (7-ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) and adipyl-7-ACA, compounds that can be transformed into the economically relevant 7-ADCA and 7-ACA intermediates. Escherichia coli expression of the genes encoding D-amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7-ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 216-226, 1997.     

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.     

Coordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporin-producing mutant of Acremonium chrysogenum and simultaneous loss of the three enzymes in a non-producing mutant

Abstract An early blocked mutant in cephalosporin biosynthesis ( Acremonium chrysogenum N₂) had simultaneously lost 3 enzymes of the cephalosporin biosynthetic pathway (isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase) and accumulated the tripeptide α-aminoadipyl-cysteinyl-valine. An overproducing mutant ( A. chrysogenum C-10) showed a 2-fold increase in the same 3 enzymes throughout fermentation, with respect to the low-producing strain A. chrysogenum CW-19. These results suggest that expression of the genes coding for cephalosporin biosynthetic enzymes is altered in a coordinate form in these mutants.     

Correlation of cephalosporin C synthesis and proteolytic enzymes in a differentiating culture of Acremonium chrysogenum (Cephalosporum acremonium) mutants

The effect of the growth conditions and composition of the nutrient medium on the synthesis of cephalosporin C, alkaline exoproteases and cell differentiation was studied in various strains of Acremonium chrysogenum. It was observed that the changes in the above processes occurred simultaneously and depended on the aeration rate, temperature, illumination level and concentrations of methionine and soybean meal. Close correlation between the synthesis of alkaline exoproteases, cephalosporin C and formation of secondary structures in the strains of A. chrysogenum was shown.