In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

Background: Clavulanic acid (CA), a β-lactamase inhibitor, is industrially produced by the fermentation of Streptomyces clavuligerus. The efficiency of CA production is associated with media composition, culture conditions and physiological and genetic strain characteristics. However, the molecular pathways that govern CA regulation in S. clavuligerus remain unknown. Methods and Results: Here we used RNA-seq to perform a comparative transcriptome analysis of S. clavuligerus ATCC 27064 wild-type strain grown in both a favorable soybean-based medium and in limited media conditions to further contribute to the understanding of S. clavuligerus metabolism and its regulation. A total of 350 genes were found to be differentially expressed between conditions; 245 genes were up-regulated in favorable conditions compared to unfavorable. Conclusion: The up-regulated expression of many regulatory and biosynthetic CA genes was positively associated with the favorable complex media condition along with pleiotropic regulators, including proteases and some genes whose biological function have not been previously reported. Knowledge from differences between transcriptomes from complex/defined media represents an advance in the understanding of regulatory paths involved in S. clavuligerus’ metabolic response, enabling the rational design of future experiments.

Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus.

The clinical and industrial importance of clavulanic acid (CA) promotes the search for novel strategies to boost productivity at lower costs. In this contribution, the production of CA was evaluated in two reference strains of Streptomyces clavuligerus (Sc), ATCC27064 and DSM41826; the strains were tested in different culture media, using product yields and culture media costs as selection criteria. On average, ScATCC reached a concentration of CA 1.6-fold higher than ScDSM, using the isolated soybean protein (ISP) culture medium. A central composite experimental design was used to optimize the medium, by studying the influence of ISP and K2HPO4 concentration on the production of CA. The maximum CA concentration obtained in this optimized medium was 56 mg·L -1 , close to the theoretical optimum. Our results confirm that a culture medium containing a rich carbon source such as glycerol and/or starch provides the best conditions for CA production.Keywords: clavulanic acid; culture media design; strain selection; Streptomyces clavuligerus; antibiotics.Selección de una cepa de Streptomyces clavuligerus para la biosíntesis de ácido clavulánico: un estudio basado en los efectos de la composición del medio de cultivo y análisis estadístico Resumen La importancia clínica e industrial del ácido clavulánico (AC) promueve la búsqueda de nuevas estrategias para aumentar su productividad a un menor costo. En esta contribución, se evaluó la producción de AC en dos cepas de referencia de Streptomyces clavuligerus (Sc), ATCC27064 y DSM41826; éstas se probaron en diferentes medios de cultivo; los rendimientos de producto y costo de los medios se usaron como criterios de selección. En promedio, ScATCC alcanzó una concentración de AC 1.6-veces mayor que ScDSM, en el medio de proteína de soya (ISP). Se optimizó el medio con un diseño experimental central compuesto, estudiando la influencia del ISP y K2HPO4 sobre la producción de AC. La concentración máxima de AC obtenida en el medio optimizado (56mg·L -1 ) estuvo cerca del óptimo teórico. Nuestros resultados confirman que medios de cultivo con fuentes de carbono como glicerol y/o almidón proporcionan las mejores condiciones para la producción de AC.Palabras clave: ácido clavulánico; diseño de medios de cultivo; selección de cepa; Streptomyces clavuligerus; antibióticos.

The role of fundamental parameters in the conduction of penicillin semicontinuous fermentations is analyzed. Biomass concentration, penicillin production, and main nutrient consumption are particularly studied. Furthermore, the conduction of the operation is simulated with regard to conditions of constant specific rate of growth and of constant oxygen concentration in the broth. An intermediate condition is also considered.