New reactions in clavulanic acid biosynthesis

Clavulanic acid is only a modestly effective antibiotic against bacterial infections in humans, but a potent inhibitor/inactivator of beta-lactamase enzymes that confer bacterial resistance. The biosynthetic pathway to clavulanic acid is considerably more complex than that to the structurally related penicillins and cephalosporins and has revealed several interesting reactions.     

克拉维酸产生菌的优化培养

为提高克拉维酸产量,通过摇瓶实验考查了多种氮,碳源及无机盐对克拉维酸产量的影响,并对产生菌的部分性能进行了考查,试验表明实验中所用棒状链霉菌（Strepto-myces elavuligerus）合成克拉维酸的最佳氮,碳源及无机盐是黄豆粉,甘油,KH2PO4,MgSO4,其最佳培养基配比（%）分别为2.8、1.8、0.04、0.05。为保证克拉维酸高产,稳产,产生菌斜面冷藏时间不宜超过1个月,斜面传代次数不宜超过4代,沙土,20%甘油溶液,冷冻干燥管均可用于产生菌的孢子保藏。     

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。     

Kinetics of beta-lactamase inhibition by clavulanic acid]

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.     

影响克拉维酸生物合成的氨基酸

发酵液的氨基酸分析显示,谷氨酸,精氨酸,天门冬氨酸,丙氨酸易被棒状链霉菌利用,发酵培养基中添加上述氨基酸后,谷氨酸,精氨酸有利于克拉维酸的生物合成,适时添加谷氨酸,精氨酸可分别提高克拉维酸的产量约25%和12%;而蛋氨酸,半胱氨酸含S氨基酸对克拉维酸生物合成不利,不同来源的黄豆粉作发酵培养基氮源,因其组成中某些氨基酸含量的差异。可使克拉维酸的产量相差百分之十几。     

The inhibition of staphylococcal beta-lactamase by clavulanic acid.

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.     

Effects of preculture variability on clavulanic acid fermentation.

The production profile of clavulanic acid by Streptomyces clavuligerus was shown to be strongly dependent on inoculum activity. Two sets of fermentations (A and B) were investigated at industrial pilot-plant scale using complex media. Type A fermentations were inoculated using late exponential growth phase mycelia. Type B fermentations were inoculated using mycelia harvested at stationary phase. Productivities throughout type A fermentations were consistently higher than type B, reaching a maximum at about 70 h and then decaying to the same final productivities at 140 h of type B runs. Several scheduling alternatives, based on combinations of the two inocula types and different fermentation lengths, were compared in terms of the overall process economics (fermentation and downstream). An increase of ca. 22% on the overall process profit is predicted using late exponential growth phase inocula and a fermentation duration of only 96 h. A new operating strategy was thus proposed for inoculum production based on the control of preculture activity using off-gas analysis. This method ensures higher productivity and better batch-to-batch reproducibility of clavulanic acid fermentations than traditional methods based on constant age inocula.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Application of lat gene disruption to increase the clavulanic acid production of Streptomyces clavuligerus

A 1.7 kb fragment of lat was obtained from Streptomyces clavuligerus NRRL 3585, and recombinant plasmid pKC1139-lat, which was used to disrupt the lat gene was constructed. pKC1139-lat was introduced into S. clavuligerus by bi-parental conjugation from Escherichia coli ET12567 to S. clavuligerus. The apramcin-resistant transformants were obtained and through homogeneous single-crossover between recombinant plasmid pKC1139-lat and the S. clavuligerus chromosome lat disrupted mutant strains were obtained. The genome of S. clavuligerus NRRL 3585 and the lat disrupted mutants were analyzed by PCR technique, the bioactivity of cephamycin C in the two kinds of strains were also tested. Both results proved that lat was disrupted by the insertion of pKC1139 in the lat disrupted mutants. And the production of clavulanic acid of these two kinds of strains were analyzed by HPLC with different incubation time interval (96 and 120 h), and the yield in the lat mutants was approximately 2.6 fold higher at their highest production point.     

Kinetic studies of clavulanic acid recovery by ion exchange chromatography

Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus. Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid–liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 °C and 20 °C during adsorption and 30 °C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations

 Clavulanic acid (CA) was produced by Streptomyces clavuligerus in medium containing glycerol and soy meal or soy meal extract. With regard to growth and CA productivity, the microorganism showed significant differences if solid soy meal as such or its extract were applied as the major nitrogen source. If the extract is used, growth and CA production take place simultaneously and in the stationary phase the CA concentration is stagnant or reduces. If soy meal is used, growth is threefold faster and CA is only generated in the stationary phase. In the case of using the soy meal extract, the decrease of the CA concentration is mainly due to decomposition or re-metabolisation of CA in the presence of the microorganism. This conclusion is supported by in vivo and in vitro data on CA decomposition. Received: 17 July 1995 / Received revision: 7 September 1995 / Accepted: 13 September 1995     

Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus

Summary Production of cephamycin and clavulanic acid by Streptomyces clavuligerus is controlled by the phosphate concentration. Phosphate represses the biosynthesis of cephamycin synthetase, expandase and clavulanic acid synthetase. In the presence of 2 mM phosphate, the specific activities of expandase, cephamycin synthetase and clavulanic acid synthetase were higher than in the presence of 75 mM phosphate. The specific activity of cephamycin synthetase is maximal with an initial phosphate concentration of 10 mM, whereas the specific activity of expandase is maximal with 1 mM phosphate. A correlation between cephamycin synthetase specific activity and expandase specific activity was established at phosphate concentrations higher than 10 mM. This shows that the expandase is an important enzyme in the mechanism by which the phosphate concentration affects the biosynthesis of cephamycin.     

Expansion of the clavulanic acid gene cluster: identification and in vivo functional analysis of three new genes required for biosynthesis of clavulanic acid by Streptomyces clavuligerus

Clavulanic acid is a potent inhibitor of beta-lactamase enzymes and is of demonstrated value in the treatment of infections by beta-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219-236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.     

The antimicrobial activity of Augmentin (amoxicillin/clavulanic acid) compared to other antibacterial agents]

Sensitivity of clinical strains of gram-positive and gram-negative bacteria to Augmentin was studied in comparison to other antibiotics. Augmentin was shown to be advantageous in the level and spectrum of its antibacterial activity over ampicillin and other broad-spectrum antibiotics.     

Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerus

AIMS: To improve the production of clavulanic acid through the development of strains, the selection of a production medium and a pH shift strategy in a bioreactor. METHODS AND RESULTS: Streptomyces clavuligerus mutant 15 was selected by antibacterial activities. As a result of pH control in a 2.5 l bioreactor, the highest productivity (3.37 microg x ml(-1) x h(-1)) was obtained at a controlled pH of 7.0. CONCLUSIONS: The highest level of production obtained was an increase of about 36% compared with a non-controlled pH. When the production of clavulanic acid reached the maximum level, the pH was shifted from 7.0 to 6.0 for reduction in decomposition rate. The maximum concentration of clavulanic acid was maintained for 24 h as a result of the pH shift control, and a significant reduction in the decomposition of clavulanic acid was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: Clavulanic acid decomposition was considerably reduced as a result of the pH shift control. The results of this study can be applied for the efficient production of beta-lactamase inhibitory antibiotics.     

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.