Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing

Abstract

Clavulanic acid (CA) is an irreversible β-lactamase enzyme inhibitor with a weak antibacterial activity produced by Streptomyces clavuligerus (S. clavuligerus). CA is typically co-formulated with broad-spectrum β‑lactam antibiotics such as amoxicillin, conferring them high potential to treat diseases caused by bacteria that possess β‑lactam resistance. The clinical importance of CA and the complexity of the production process motivate improvements from an interdisciplinary standpoint by integrating metabolic engineering strategies and knowledge on metabolic and regulatory events through systems biology and multi-omics approaches. In the large-scale bioprocessing, optimization of culture conditions, bioreactor design, agitation regime, as well as advances in CA separation and purification are required to improve the cost structure associated to CA production. This review presents the recent insights in CA production by S. clavuligerus, emphasizing on systems biology approaches, strain engineering, and downstream processing.

Robustifying Experimental Tracer Design for13C-Metabolic Flux Analysis

“…In this work, we take the industrially relevant non-model organism S. clavuligerus as an example. The Gram-positive bacterium is a natural producer of CA, a potent inhibitor of β-lactamase enzymes secreted by bacteria as a defense mechanism against β-lactam antibiotics (Brown et al, 1976;Ramirez-Malule, 2018;Ramirez-Malule et al, 2018;López-Agudelo et al, 2021). S. clavuligerus grows on GLYC as main carbon source, while amino acid supplementation, e.g., ARG, improves CA titer.…”

“…The combination of CA and amoxicillin is a well-established broad-spectrum antibacterial treatment (World Health Organization, 2019a,b), which was prescribed in 2018 almost 8 million times in the US alone (Kane, 2021). Although bioprocess development and genetic engineering successfully improved CA yields (Li and Townsend, 2006;Ünsaldı et al, 2017;Ramirez-Malule et al, 2018;Gómez-Ríos et al, 2019;López-Agudelo et al, 2021), rational metabolic engineering based on the precise knowledge of the fluxome of S. clavuligerus is expected to push the limits further. The knowledge about in vivo fluxes is, however, still very limited (Medema et al, 2010;Ramirez-Malule et al, 2018;Gómez-Ríos et al, 2020), which makes this system an ideal showcase for the proposed R-ED methodology.…”

Environmental Factors Modulate the Role of orf21 Sigma Factor in Clavulanic Acid Production in Streptomyces Clavuligerus ATCC27064

“…CA biosynthesis begins at the L-arginine and glyceraldehyde-3-phosphate condensation, catalyzed by carboxyethylarginine synthase (CeaS); the next five consecutive enzymatic reactions, generally referred to as early steps, lead to (3S,5S)-clavaminic acid formation ( Figure 1 ) [ 2 ]. The formation of clavaminic acid is an important branch point in the CA pathway since it can either be converted to (3R,5R)-clavulanic acid, or to 3S,5S clavams, by the late reactions of the pathway [ 7 ]. For CA biosynthesis, different compounds result from the intermediate steps involved in the transition of the clavaminic acid into CA.…”

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus

“…In this sense, many important achievements have been made regarding nutrients, environmental and operational conditions on CA production as previously reviewed by Ser et al [ 5 ]. Although recent studies have contributed to a deeper understanding of S. clavuligerus metabolism [ 6 , 7 , 8 ] as a basis for strain engineering, system biology, and downstream processing (see recent review by López-Agudelo et al [ 9 ]), the interaction of byproduct accumulation and product synthesis is not understood yet. Nonetheless, several S. clavuligerus strains have been engineered, yielding up to 6.7 g L −1 of CA in the supernatant [ 10 , 11 ].…”

“…Five GEMs have been published and used as tools for assessing the complexity of S. clavuligerus metabolism in silico, especially in connection with antibiotics production (CA and cephamycin C) [ 20 , 21 , 27 , 28 , 29 ]. The previous stoichiometric metabolic models for S. clavuligerus were not reconstructed de novo from the organism-specific reference sequences; instead, rather new biochemical information was added to the first GEM without changing its initial structure [ 9 , 21 ]. Only recently, an automated “bottom-up” reconstruction of the S. clavuligerus metabolism based on a reference genome sequence for the strain (refseq GCA_001693675.1) was used as a model draft.…”