Influence of the Crc global regulator on substrate uptake rates and the distribution of metabolic fluxes in Pseudomonas putida KT2440 growing in a complete medium

Abstract

When the soil bacterium Pseudomonas putida grows in a complete medium, it prioritizes the assimilation of preferred carbon sources, optimizing its metabolism and growth. This regulatory process is orchestrated by the Crc and Hfq proteins. The present work examines the changes that occur in metabolic fluxes when the crc gene is inactivated and cells grow exponentially in LB complete medium. Analyses were performed at three different moments during exponential growth, examining the assimilation rates for the compounds present in LB, changes in the proteome, and the changes in metabolic fluxes predicted by the iJN1411 metabolic model for P. putida KT2440. During the early exponential phase, consumption rates for sugars, many organic acids and most amino acids were higher in a Crc‐null strain than in the wild type, leading to an overflow of the metabolic pathways and the leakage of pyruvate and acetate. These accelerated consumption rates decreased during the mid‐exponential phase, when cells mostly used sugars and alanine. At later times, pyruvate was recovered from the medium and utilized. The higher consumption rates of the Crc‐null strain reduced the growth rate. The lack of the Crc/Hfq regulatory system thus led to unbalanced metabolism with poorly optimized metabolic fluxes.

Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida

“… 105 Blocking Crc-mediated regulation has been shown to alter the consumption of available carbon compounds in a complex medium, resulting in metabolite overflow and, consequently, inefficient growth. 106 In particular, glucose consumption was enhanced during early exponential growth, significantly decreasing in the late exponential phase. These effects are also relevant under bioconversion conditions, whereby a non-native substrate (3-FBz) is supplied along sugars, and provide an explanation for the low glucose consumption of strain PMP1053d pre-grown in a rich medium.…”

Specific and Global RNA Regulators in Pseudomonas aeruginosa

“…In Pseudomonas, the hierarchical assimilation of carbon sources is governed by the RNA chaperone Hfq [29] and the catabolite repression control protein Crc [146,[170][171][172][189][190][191][192][193][194][195][196] (Figure 3a). Under conditions of carbon catabolite repression (CCR), Hfq and Crc form a repressive complex on target mRNAs, the encoded proteins of which are involved in uptake and/or utilization of carbon and nitrogen sources other than the preferred one [29,147,197].…”

Optimizing a High Performing Multiplex-CRISPRi P. putida Strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

“…The next step to employ 13 C-MFA was to determine the extracellular metabolites secretion rates to constrain the MFA model. P. putida has been previously reported to secrete gluconate and 2ketogluconate (2KG) during growth on glucose minimal medium (del Castillo et al, 2007;Kohlstedt and Wittmann, 2019;Nikel et al, 2021Nikel et al, , 2015 or complex LB medium (Molina et al, 2019a(Molina et al, , 2019b). An analysis of the extracellular medium revealed both compounds were secreted by the WT strain, reaching of 1.4 ± 0.1 mM of gluconate and 3.9 ± 0.3 mM of 2KG (~1.0 ± 0.1 g/L total secretion) towards the end of the growth phase (Fig.…”