Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus

Among four different commercially available nitrogen sources containing soybean derivatives, a protein extract of soybean gave the highest yield for clavulanic acid production by Streptomyces clavuligerus. A statistical method based on factorial design of experiments was applied to optimise the medium. An empirical model was obtained by applying response surface statistical analysis. The analysis of variance showed that concentrations of protein extract of soybean and glycerol and the interaction between these two variables were significant at 95% level of confidence. The maximum clavulanic acid concentration obtained in 72 h was 1.2 g l^–1.     

Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding

Glycerol at 10–20 g l^–1 increased clavulanic acid production by Streptomyces clavuligerus in shake-flask culture. The biosynthesis of clavulanic acid continued for longer by feeding glycerol and production increased to 250 mg l^–1 compared with 115 mg l^–1 without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased further to about 280 mg l^–1 but also remained stable up to 130 h.     

脲对棒状链霉菌棒酸合成的影响

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。     

Improvement of clavulanic acid production by Streptomyces clavuligerus in medium containing soybean derivatives

The effect of the nitrogen source in the production medium on the level of clavulanic acid production by Streptomyces clavuligerus has been investigated. Batch cultures using two types of synthetic culture medium and two types of complex culture medium containing soybean derivatives were employed. To allow comparison of the various media, all of them were formulated with 4.0 g total nitrogen/l. It was observed that the production of clavulanic acid using synthetic medium reached values slightly greater than those usually found in the literature. However, in trials with complex media, it was found that when Samprosoy 90NB (protein extract of soybean) was utilized, production of clavulanic acid went up to 920 mg/l, twice as high as when soy meal was used, and notably higher than values reported in the literature (300–500 mg/l) for complex medium.     

Production of clavulanic acid and cephamycin C by Streptomyces clavuligerus in palm-oil medium

Palm and palm-kernel oils and their olein and stearin fractions were suitable as the main carbon sources for growth and production of clavulanic acid by Streptomyces clavuligerus. However, oleic and lauric acids were not utilized for growth. A spontaneous mutant, which was selected for higher cephamycin C production, also produced more clavulanic acid with these oils in the medium.     

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

Streptomyces clavuligerus produces a large array of natural compounds with antibiotic, antitumor, beta-lactamase inhibition or inmunomodulating activities. The production of cephamycin C, clavulanic acid and other compounds with a clavam structure has been studied for many years. A network of regulatory mechanisms is present in S. clavuligerus to control the formation of different compounds by pathway-specific regulators or pleiotropic regulators. The possible existence of a gamma-butyrolactone signaling system in this streptomycete is emerging. In addition, S. clavuligerus possesses a stringent control mechanism somehow different from those previously reported in other Streptomyces species.     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

The biosynthetic genes for clavulanic acid and cephamycin production occur as a 'super-cluster' in three Streptomyces

Abstract The cosmid cloning vector pHC79 has been used to clone fragments of chromosomal DNA from the Streptomyces: S. clavuligerus, S. jumonjinensis and S. katsurahamanus . These strains all produce both the β-lactam antibiotic, cephamycin and the β-lactamase inhibitor, clavulanic acid. Although structurally related these two β-lactams are known to be derived from different biosynthetic precursors. Hybridisation studies and restriction mapping have shown that the gene clusters encoding the two biosynthetic pathways are chromosomally adjacent in these strains, thus creating a 'super-cluster' of genes involved in both the production and enhancement of activity of a β-lactam antibiotic.     

棒状链霉菌lat基因的中断对棒酸产量的影响

从棒状链霉菌中克隆1.8kb的lat基因片段,构建了基因置换质粒pXAL1和pXAL2。运用接合转移方法把中断载体导入棒状链霉菌中进行lat的中断,得到1株接合转移子AmrThios,命名为XAL863。通过Southern杂交分析及赖氨酸转氨酶活性测定,证明此菌株的lat基因被中断。通过发酵培养,HPLC方法检测棒酸含量,发现棒酸产量明显提高,约为原产量的1.8倍。     

Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for β-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M _r of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free l-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium.     

Flux balance analysis in the production of clavulanic acid by Streptomyces clavuligerus

In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α‐ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1226–1236, 2015     

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     

Characterization of pbpA and pbp2 Encoding Penicillin-binding Proteins Located on the Downstream of Clavulanic Acid Gene Cluster in Streptomyces clavuligerus

Two genes, pbpA (orf18) and pbp2 (orf19) located on the downstream of clavulanic acid (CA) gene cluster of Streptomyces clavuligerus were cloned into pET-28a(+), and confirmed to encode a family of high molecular-weight penicillin-binding proteins (PBPs). Both genes were amplified from genomic DNA by PCR and expressed in E. coli BL21 (DE3). Hydropathy plots of the proteins revealed a single stretch of hydrophobic amino acids indicating them to be transmembrane proteins. Pbp2 had lower affinity to penicillin G compared to PbpA, and was essential to the cell growth in contrast to PbpA. Revisions requested 3 November 2005; Revisions received 13 December 2005     

Morphology and clavulanic acid production of Streptomyces clavuligerus: Effect of stirrer speed in batch fermentations

Streptomyces clavuligerus ATCC 20764 was grown from spore-inocuia on a glycerol, malt extract, bacteriological peptone medium in 5-L batch fermentations at 490, 990, and 1300 rpm. Dry cell weights, clavulanic acid production, and the morphological parameters main hyphal length, total hyphal length, number of tips, and hyphal growth unit were measured. Growth and productivity were hardly dependent on stirrer speed. After early growth fragmentation of long, highly branched mycelia to shorter, less branched fragments occurred. This was followed by regrowth and, at 1300 rpm, a second fragmentation phase. The effect of increasing stirrer speed was to accelerate the initial fragmentation phase. It was clearly possible to obtain the same biomass concentration and clavulanic acid liter, with different morphologies depending on stirrer speed. This shows that for this fermentation at least there is no direct link between morphology and productivity and, hence, that it might be possible to manipulate them independently to improve fermentor performance.     

Utilization of vegetable oil in the production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>ATCC 27064

Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min^–1) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l^–1) was utilized to further investigate CA production. Medium containing 23 g l^–1 led to the highest CA productivity (722 mg l^–1 in 120 h) and that one containing 30 g l^–1 gave the highest CA titre (753 mg l^–1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism.     

Characterisation of percentage viability of Streptomyces clavuligerus using image analysis

A staining procedure has been developed to identify the different physiological states of Streptomyces clavuligerus using the fluorescent, bacterial viability stain BacLight. An image analysis method was used to quantify the extent of the differently stained regions, and therefore to measure the percentage viability. In a batch fermentation, the viability of the mycelia decreased to 64% during the rapid growth phase. Following a period of fragmentation, the viability was approximately constant at 75% until late in the fermentation, when it increased to 93%.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Effect of aeration on antibiotic production byStreptomyces clavuligerus

Summary During the rapid growth phase ofStreptomyces clavuligerus in a 10 litre fermentor, the level of dissolved oxygen (DO) was found to drop to almost zero for a period of approximately 10 h, delaying the appearance of and lowering the production of the antibiotic cephamycin C. Controlling the DO at either 50% or 100% throughout the fermentation did not significantly alter the specific growth rate of the culture, but did elevate final antibiotic levels two- and three-fold respectively. The improved oxygen availability affected antibiotic production both by increasing the rate of specific cephamycin C bisosynthesis and by maintaining this higher rate throughout the production period. These results demonstrate that controlling dissolved oxygen levels close to saturation during periods of rapid growth markedly improves the efficiency and duration of cephamycin C biosynthesis inS. clavuligerus.