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。     

发酵生产7-ACA基因工程菌的构建

头孢菌素类抗牛素是临床用途最广的抗感染药物,其工业生产的重要中间体7－氨基头孢烷酸（7－ACA）采用顶头孢霉发酵产物头孢菌素C为前体,通过化学合成或两步酶法狭得。介绍了在了解头孢菌素C生物合成的前提下,在建赢了顶头孢霉的遗传改造丛础上,运用合成生物学的知识,在头孢菌素C产生菌顶头孢霉中分别构建了三个头孢菌素C酰化酶的表达框架,通过发酵产物的分析并优选表达框架后,再采用传统发酵工艺的优化获得了一株可以直接发酵7-ACA的高产顶头孢霉工程菌。     

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%。     

藤仓赤霉(Gibberella fujikuroi)分子生物学及发酵工程研究进展

赤霉素是最重要的植物生长调节剂之一,工业化生产是由丝状真菌藤仓赤霉发酵产生.近20年来,随着分子生物学技术的发展,对藤仓赤霉赤霉素生物合成途径中相关基因的分子鉴定和表达调控等研究取得了显著的进展,赤霉素生物合成途径的分子生物学基本研究清楚,使得利用基因工程和代谢工程技术进行赤霉菌改良、提高赤霉素发酵水平成为可能.本文对藤仓赤霉中赤霉素合成机理及其表达调控、关键酶基因功能、外源基因转化系统、发酵技术、利用基因工程技术进行改造等方面的研究进展进行综述.     

高山被孢霉产花生四烯酸及其遗传改造的研究进展

花生四烯酸作为一种重要的多价不饱和脂肪酸,因其具有多种生理功能而被认为是潜在的食品添加剂和药物。近年来,利用高山被孢霉合成花生四烯酸已成为研究热点。前期相关研究主要集中在菌种选育及发酵调控方面。随着研究的不断深入,关于高山被孢霉合成花生四烯酸的代谢途径的研究取得了较大进展。以下简要概述前期工作进展,着重论述花生四烯酸合成途径的关键酶及其高山被孢霉的遗传改造的研究情况,包括生物合成花生四烯酸代谢途径、关键酶及其应用、高山被孢霉的遗传操作系统的构建以及遗传改造的应用,并对其研究前景进行了展望。     

ω-3多不饱和脂肪酸发酵生产中两个调控指标的探讨

为了调控长链ω 3多不饱和脂肪酸的发酵 ,对用于脂肪酸发酵调控的 2个指标 18∶3(α ) /18∶2和∑C18/∑C16进行探讨。对 2个指标从化学反应平衡角度进行阐述 ,进一步证明在合适的条件下 ,头孢霉 18∶3(α ) /18∶2的变化趋势和二十二碳六烯酸变化趋势一致 ;轮梗霉∑C18/∑C16和二十碳五烯酸变化趋势一致。 18∶3(α ) /18∶2和∑C18/∑C16可以作为发酵生产二十二碳六烯酸和二十碳五烯酸的调控指标。     

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。     

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。     

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。     

利用功能基因组学方法研究酿酒酵母乙醇生物合成的调控机理

酿酒酵母在发酵生产乙醇的过程中存在的主要问题是前期高浓度底物葡萄糖的抑制和后期高浓度产物乙醇的抑制。功能基因组学技术的发展为从基因组水平上系统研究酿酒酵母乙醇生物合成的调控机理提供可能。本研究模拟工业发酵的条件,对酿酒酵母实验菌株BY4743为遗传背景的116个单基因缺失菌株进行了乙醇发酵试验,以发现基因和乙醇发酵的关系。结果表明乙醇对菌体得率系数高于平均值30%以上的基因缺失株有20株,其中高于50%以上基因缺失株有5株;低于平均值30%以上的基因缺失株有11株,其中低于45%以上的有5株。本研究为从整个酿酒酵母基因组水平上系统研究乙醇生物合成的调控机理建立了研究方法,提供了可行性验证。     

Solid-state and submerged fermentations show different gene expression profiles in cephalosporin C production by Acremonium chrysogenum

Despite the importance of Acremonium chrysogenum as the only cephalosporin C (CPC) producer, there is still a limited understanding about the molecular mechanisms regulating antibiotic biosynthesis in this fungus. Based on the previously described relationship between environmental pH and antibiotic production in numerous filamentous fungi, we studied the expression of genes related to CPC production in A. chrysogenum. We report for the first time similarities and differences, characterizing CPC production by A. chrysogenum under a variable pH environment, in submerged and solid-state fermentation. This characterization is supported by measurements of parameters, like CPC production, pH, growth, and expression levels of several genes involved, directly or indirectly, in CPC production. Interesting differences in intermediate (Pen N) and certain biosynthetic gene expression levels were observed. Our results point out some relationships between physiological features and gene expression that open important improvement perspectives for both culture systems.     

混合碳源发酵生产头孢菌素C过程优化

本文对头孢菌素C（Cephalosporin C,CPC）发酵过程中碳源补料控制策略进行了优化研究,提出了一种基于DO—Stat的混合碳源流加策略,提高了发酵整体性能。在7L发酵罐上对使用该策略和传统补油策略的头孢菌素发酵性能进行比较,结果表明,采用补加混合碳源（葡萄糖＋豆油）策略时,CPC终浓度最高,达到36．99g／L,CPC得率也从使用传统单纯补油策略时的11．39％提高到22．19％,代谢副产物去乙酰氧头孢菌素C（DAOC）的积累量少,DAOC／CPC只有0．38％,达到生产要求。     

Glutathione metabolism ofAcremonium chrysogenum in relation to cephalosporin C production: Is γ-glutamyltransferase in the center?

Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific gamma-glutamyltransferase (gamma-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum. The accelerated turnover of GSH might be indicative of the existence of a functioning gamma-glutamate cycle, and might supply the CPC biosynthetic machinery with L-cysteine. The gamma-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents. Exogenous cysteine hindered both the uptake of methionine and the induction of gamma-GT, and was not beneficial for CPC production. There was no causal relationship between the redox status of the cells and the observed cell morphology.     

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.     

Analysis of the relationship between growth, cephalosporin C production, and fragmentation in Acremonium chrysogenum.

Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.     

Improvement of Cephalosporin C Production by Recombinant DNA Integration in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Cephalosporins are widely used as anti-infectious β-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.     

Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.     

Transposons in biotechnologically relevant strains of Aspergillus niger and Penicillium chrysogenum

In the past 15 years, many class I and class II transposons were identified in filamentous fungi. However, little is known about the influence of transposons during industrial strain development. The availability of the complete genome sequences of the industrially relevant fungi Aspergillus niger and Penicillium chrysogenum has enabled an analysis of transposons present in these two fungi. Here, a compilation of the transposon-like sequences identified is provided. We investigated a yet undescribed A. niger retrotransposon, ANiTa1, as well as two P. chrysogenum transposons (PeTra1 and PeTra2), which are the first P. chrysogenum transposons ever described, in more detail. Analysis of the genomic distribution of selected transposable elements in five strains of A. niger and seven strains of P. chrysogenum revealed the transposon distribution to be virtually identical. However, one element, Vader-previously published-from A. niger, showed strain-specific differences in transposon distribution, suggesting transposition activity during classical strain improvement programs.     

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.