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。     

头孢菌素C生物合成调控研究进展

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。     

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。     

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%。     

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。     

脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF的基因克隆及序列分析

利用氯化苄分别从真菌顶头孢（Cephalosporium acremonium)和产黄头孢（Acremonium chrysogenum)中提取总DNA,通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF,结果只能从黄头孢DNA趸扩增出cefEF基因。测序结果表明,其与已报道的基因序列只有3个碱基的差异,推断的氨基酸序列只有2个氨基酸有差异,并未涉及活性中心。同时表明,国外指所指的与该酶有关的顶头孢（Cephalosporium acremonium或Acremonium chryso-geum)对应的是国内的产黄头孢（Acremonium chrysogenum)。     

不同补料发酵方式对发夫酵母产虾青素的影响

对发夫酵母的不同补料发酵方式进行了研究,期望提高发酵产率,以便对工业化生产提供一定的指导作用。实验结果表明：采用恒pH葡萄糖-氨水流加培养,色素量和生物量均具有最大值,分别为54.3μg/ml和49.5μg/ml;其次是指数流加培养,色素量和生物量分别为49.9μg/ml和47.4mg/ml;恒pO2流加方式下色素量和生物量分别为34.04ug/ml和35.4mg/ml;恒pH葡萄糖流加方式所得的色素量和生物量最小,分别为32.3μg/ml和31.5mg/ml。不同的补料发酵方式对发夫酵母生长和色素形成的影响很大。     

脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF的克隆及序列分析

利用氯化苄分别从真菌顶头孢(Cephalosporium acremonium)和产黄头孢(Acremonium chrysogenum)中提取总DNA,通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF,结果只能从产黄头孢DNA中扩增出cefEF基因。测序结果表明,其与已报道的基因序列只有3个碱基的差异,推断的氨基酸序列只有2个氨基酸有差异,并未涉及活性中心。同时表明,国外所指的与该酶有关的顶头孢(Cephalosporium acremonium或Acremonium chrysogenum)对应的是国内的产黄头孢(Acremonium chrysogenum)。     

补料发酵工艺的应用及其研究进展

综述了补料工艺在发酵工业中应用和研究。介绍了补料发酵工艺及其优点,着重讨论了补料发酵动力学和控制理论研究,以期为补料发酵的应用提供充分的参考依据。     

聚-ε-赖氨酸补料发酵的初步研究

在5L自动发酵罐中,通过分批发酵和补料分批发酵,对白色链霉菌(Strepomyces albulus)生物合成聚-ε-赖氨酸(ε-PL)进行了初步研究。结果表明,在分批发酵中,利用pH控制策略,ε-PL产量从0.6 g/L提高到2.4 g/L。在分批补料发酵中,采用pH控制策略,当糖浓度在10g/L以下,补加葡萄糖和硫酸铵,则菌体大量生长,ε-PL产量提高到16.81 g/L,为分批培养的27倍。     

Performance improvement of cephalosporin C fermentation by Acremonium chrysogenum with DO-Stat based strategy of co-feeding soybean oil and glucose

Cephalosporin C (CPC) fermentation by Acremonium chrysogenum featured with two major problems: (1) high raw materials cost (low CPC yield from soybean oil) and (2) low oxygen transfer rate between gaseous/aqueous phases leading to low CPC productivity and quality instability of CPC fermentation product due to the accumulation of deacetoxycephalosporin C (DAOC). To solve the problems, in this study, we proposed a novel DO-Stat based co-substrates feeding strategy by simultaneously supplementing soybean oil and glucose, and testified the effectiveness of the strategy in a 7 L bioreactor. The CPC fermentation performance were significantly improved when co-feeding soybean oil and glucose at a weight ratio of 1:0.7, as compared with those when feeding pure soybean oil: (1) final CPC concentration and yield reached higher levels of 37 g/L and 23.5%, the increments were 46% and 82%, respectively; (2) oxygen transfer rate was largely improved, oil consumption rate and CPC productivity were enhanced by 31% and 136%, respectively; and (3) DO could be controlled at adequately high levels so that DAOC accumulation could be minimized and the quality of CPC fermentation product be ensured. The proposed strategy showed application potential in improving the economics of industrial CPC productions.     

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。     

The effect of thiols and the Ca2+ ionophore A23817 on growth and antibiotic production in Cephalosporium acremonium

Abstract Yeast-like blastosporic growth, lowered levels of isopenicillin N synthetase and deacetoxycephalosporin C synthetase/hydroxylase, and lowered antibiotic production were induced in Cephalosporium acremonium strain C.O. 728 by inclusion of cysteine or glutathione in the growth medium. These effects were similar to those observed under normal conditions with strain M 8650/4, which had higher levels of glutathione reductase and protein disulphide reductase than strain C.O. 728. The addition of cytochalasins A and B slightly increased yeast-like growth in C.O. 728, although levels of biosynthetic enzyme activity and antibiotic secretion were not significantly affected. The Ca²⁺ ionophore A23187 inhibited C.O. 728 growth, slowed penicillin secretion into the medium, and totally inhibited cephalosporin secretion. These results show that maintenance of Ca²⁺ levels and the absence of thiols are important for antibiotic production in Cephalosporium acremonium .     

Immobilized Enzyme Three-Phase Reactors for Oxidation of Cephalosporin C

The enzymatic oxidation of Cephalosporin C (CEPHC) was catalyzed by D-aminoacid oxidase, from the red yeast Trigonopsis variabilis, immobilized on Duolite A365. The study was performed in two different three phase bioreactors, gas-liquid-solid (immobilized enzyme): the fluidized-bed batch reactor, fed continuously with oxygen and discontinuously with CEPHC, and the UF-membrane reactor continuously fed with both substrates. Only the first reactor allowed significant product yield (>70%) while the second was a very useful tool for laboratory investigation of both bioconversion kinetics and enzyme stability.

Optimum reaction temperature was 15d`C for the control of CEPHC spontaneous degradation (roughly 15% in 30 h), and enzyme deactivation (half-life greater than 30 h). Immobilization improved (one order of magnitude longer half-life) enzyme resistance to mechanical stresses induced by liquid stirring and gas bubbling. Roughly 0.04g of CEPHC was adsorbed per gram of enzyme carrier. The limiting step in oxygen transfer was the gas to liquid transport. In order to attain kinetic control of the bioconversion the mildest conditions were atmospheric gas pressure and oxygen flow rate equal to 2 × 10 ²NmL/s per mL of liquid phase.     

Production enhancement of Rhizopus arrhizus lipase by feeding oleic acid

A strategy for Rhizopus arrhizus lipase production enhancement by feeding oleic acid was developed. The oleic acid was proved to have strong inducing effect on lipase production, but high concentration oleic acid could repress lipase production. The decrease rate of oleic acid concentration using peanut oil as initial carbon source was figured out according to the change of oleic acid concentration in the fermentation broth. Our feeding strategy designed based on the decrease rate of oleic acid could avoid the repression of lipase production that is caused by high concentration of oleic acid in the fermenting liquor, and this strategy worked as a new feeding method showing excellent performance. The maximum lipase activity was gained by feeding dilute oleic acid every 12 h starting at 60 h, which maintained the oleic acid concentration around 18 mg/L, and the lipase activity was 31% higher than that of no feeding.     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant K_m values of EP-PEI-CCA and EP-EDA-CCA were 22?mM and 30?mM, respectively, which were lower than that of the free enzyme (43?mM), suggesting that the support’s zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.     

Repression and inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate

Cephalosporin production by growing cells of Streptomyces clavuligerus was reduced by 100 mM inorganic phosphate. Resting cell production was repressed by prior growth in high phosphate and inhibited by phosphate. The cell-free activity of desacetoxycephalosporin C synthetase (ring expansion activity) was repressed by prior growth in high phosphate and inhibited by phosphate. Isopenicillin N synthetase (cyclase) was inhibited but not repressed. Penicillin epimerase was neither inhibited nor repressed by phosphate.Abbreviations DCW dry cell weight - MOPS 3-(N-morpholino) propane-sulfonic acid