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%。     

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。     

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10

Aims: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid‐state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. Methods and Results: Solid‐state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7·8, but also under 6·4. Using initial pH lower than 6·8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 μg gdm^?1 (g^?1 of dry matter). Conclusion: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. Significance and Impact of the Study: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.     

Production of cephalosporin C,and its intermediates,by raised-titre strains of Acremonium chrysogenum

Three different strains of Acremonium chrysogenum have been grown under identical fermentation conditions and their profiles with respect to cephalosporin C and its intermediates were compared. Clear differences were found between the strains; one notably accumulated a large pool of penicillin N, showing a reduced ability to convert this antibiotic to the later intermediates in the pathway, deacetoxycephalosporin C, deacetylcephalosporin C and cephalosporin C.     

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed. Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999     

Specific cephalosporin C production of Acremonium chrysogenum is independent of the culture density

Specific cephalosporin C production of Acremonium chrysogenum grown on a glucose-based minimal medium using conventional batch and dialysis membrane reactor systems was independent of the cell density in the range of 0.4 to 40 g biomass l^–1.     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H⁺-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyP5 with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H⁺-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.     

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。     

Undefined xylose media extracted from biorefinery waste for enhanced and eco-friendly production of cephalosporin C by Acremonium chrysogenum M35

Xylose-rich undefined broth, extracted from the dilute acid pretreatment wastes of barley straw, serves as resourceful media for Acremonium chrysogenum M35 culture and production of cephalosporin C (CPC). Concentrating the extract with proper reprocessing enables to prepare various concentrations of xylose broth (2%–8%). The undefined xylose media were prepared for CPC production from A. chrysogenum M35 by the addition of other nutrients. Cell growth and CPC production were the most effective at 6% xylose and additional 2% glycerol, with maximum CPC production of 9.07 g/L after 6 days, which is higher production than that in defined media prepared with laboratory-level nutrients and reagents. Investigation of autotrophic and reverse trans-sulfuration pathways for cysteine synthesis, a limited element of three precursors for CPC synthesis, supports the enhanced CPC production in undefined media. Abundance of xylose ensures a maintained NADPH concentration required for sulfate reduction and synthesis of amino sulfide such as cysteine. Cystathionine-γ-lyase activity profiling indicated more efficient biosynthesis in undefined media than in other cultures use glycerol and glucose, and the biosynthesis pathway of CPC production by the cephalosporin gene cluster (i.e. pcbC and cefG genes) was investigated. The process using undefined xylose media was designed, and process simulation program confirmed our results.     

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.     

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.     

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。     

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。     

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.     

The last step in cephalosporin C formation revealed: crystal structures of deacetylcephalosporin C acetyltransferase from Acremonium chrysogenum in complexes with reaction intermediates

Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum β-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the α/β hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl-enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.     

Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k _frag) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.     

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.