Microbial Cell Factories最新文献

Metabolic control analysis is used to understand regulation of metabolism and identify bottlenecks to be overcome in metabolic engineering for desired products. Its application has been hampered by the need for either parameterized models or carefully titrated experiments. In this study, we use thermodynamically feasible, sampled parameters to overcome this limitation. We use metabolic control analysis to explore central carbon metabolism of Saccharomyces cerevisiae growing in continuous culture under different nutrient limitations. Furthermore, we demonstrate shifts in flux control patterns in response to the different growth conditions and show how our results for specific reactions agree with the literature. Key advantages of the proposed framework include the incorporation of allosteric effectors, the use of omics data from a single steady-state time point and the computational efficiency; in all cases, 100 feasible models were sampled in less than 20 min on a laptop. The model and framework are freely available for researchers to use on their own data: https://github.com/biosustain/GRASP.git.

Microalgae, a diverse assemblage of oxygen-generating photosynthetic organisms, demonstrate autotrophic, mixotrophic, and heterotrophic metabolic modes, facilitating adaptive regulation of their activity under different culture conditions and providing extensive biotechnological potential. Recently, there has been a growing interest in the cultivation of microalgae as biofilms owing to their potential for efficient biomass generation in confined systems and a wide range of industrial applications. This review offers a comprehensive overview of microalgal biofilm development, emphasizing adhesion mechanisms, variations in biofilm structure across diverse substrates, and the impact of critical physical parameters including cell density, size, morphology, and surface charge. Furthermore, the review examines the incorporation of different methods to attain a comprehensive understanding of biofilm development and molecular behavior while preserving biofilm integrity. The discussion encompasses the present challenges and future prospects of microalgal biofilms as biosensor components, highlighting their applications in environmental monitoring, on-chip biosensing, and biohybrid devices. The intrinsic benefits of microalgae, such as their capacity for self-regeneration, metabolic adaptability, and compatibility with optical and electronic systems, establish them as plausible candidates for sustainable biotechnological progress. By integrating mechanistic understanding, experimental methodologies, and application-focused approaches, this review seeks to establish a comprehensive framework for the advancement of next-generation microalgal biofilm technologies.

Endophytic fungi establish a symbiotic relationship with their host plants, actively engaging in the hosts' physiological and metabolic processes. They can directly or indirectly transform plant metabolites, thereby playing a crucial role in the host's overall health and functioning. In this study, we isolated and identified an endophytic fungus, Candolleomyces candolleanus P9 strain, which produces β-glucosidase from Ural Glycyrrhizae Radix in Altay, Xinjiang Uygur Autonomous Region, China. In addition, the enzyme production conditions of strain P9 were optimized using a wheat bran concentration of 30.6 g/L, beef extract concentration of 11.2 g/L, inoculum size of 2.6%, pH 7.23, at 30 °C, with shaking at 150 rpm, and a fermentation duration of 6 days. Under these conditions, the β-glucosidase activity of strain P9 increased by 13.6-fold compared to the initial level. On this basis, the efficiency of converting diurea-based urea into diurea-based elements was further optimized. The optimized results were as follows: conversion time 12 h, temperature 37℃, liquiritin concentration 0.8 mg/mL, pH value 7.5, and the conversion rate reached 93.09%. In addition, the antibacterial and antioxidant effects of the fermentation broth of the P9 strain after biotransformation were significantly better than those of commercial β-glucosidase and control group. In summary, fermentation with the β-glucosidase-producing Candolleomyces candolleanus P9 strain is a potential method for converting liquiritin into liquiritigenin of Glycyrrhiza uralensis Fisch.

Background: The utilisation of microbes for the production of enzymes and pharmaceutical proteins is an important step towards a sustainable future. However, the energy requirement for agitation, aeration and cooling during industrial fermentation processes is substantial. To address this challenge, we have previously developed a 'breathing' polytetrafluoroethylene fermenter vessel that allows effective gas exchange with ambient air and, thus, does not require sparger aeration. The present study aimed to explore the potential application of the breathing vessel for enzyme production by the Gram-positive bacterial cell factory Bacillus subtilis. Here, we compared production of the secreted α-amylase AmyQ by the genome-reduced multiple protease-deficient B. subtilis strain IIG-Bs-27-31 during parallel culturing in breathing vessels and shake flasks. Enzyme yields and the cellular and extracellular proteome compositions were assessed.

Results: We observed comparable growth characteristics and AmyQ yields per liter in both culture systems. However, proteome analyses indicated statistically significant differences (p < 0.05, log₂fold change>|0.5|) in the utilization of carbon sources and stress responses between cells grown in the breathing vessels versus shake flasks. In particular, bacteria in the breathing vessel presented activation of the Sigma B-dependent general stress response, presumably to sustain bacterial growth and viability. In contrast, bacteria grown in shake flask presented activated cell envelope stress pathways and typical sheer stress symptoms.

Conclusions: While the overall AmyQ yields per liter were similar in both fermentation systems, the total enzyme yields in the breathing fermenter were significantly higher due to the 15-fold increase in culture volume. Our findings imply that breathing fermentation vessels are suitable for B. subtilis enzyme production and, upon further scale-up, they may represent sustainable and cost-effective alternatives to traditional fermentation systems for microbial cell factories.

Background: Single-cell protein (SCP) is gaining attention as a source of food and feed, offering a lower environmental footprint than traditional agriculture. Efficient SCP production requires high cell density culturing (HCDC; >20 g cell dry weight L^- 1), but O₂ supply can become limiting in conventional aerobic systems. To circumvent this bottleneck, we recently proposed an anaerobic strategy using nitrate as an electron acceptor, exploiting denitrification-driven alkalinization in a pH-stat system to regulate substrate provision.

Results: Using the model organisms Paracoccus denitrificans and Paracoccus pantotrophus, we achieved biomass concentrations of up to 60 g dry weight L^- 1 and protein contents up to 75% (75 ± 5%) in a 3 L fed-batch bioreactor. However, growth rates at high cell density were markedly lower than µ_max observed in low-density cultures. In vivo and in silico experiments revealed three interacting constraints: (i) a CO₂-pH lag where CO₂ accumulation delayed alkalization by denitrification and thus substrate injection; (ii) mixing limitations, leading to poor substrate distribution; and (iii) physiological stress from nitrite accumulating during imbalanced denitrification. The CO₂-pH lag emerged as the dominant barrier, resulting in long starvation periods. Lowering the pH setpoint of the pH-stat accelerated CO₂ removal and thus substrate provision, but intensified nitrite toxicity. Insufficient mixing compounded the growth limitations as nitrate was only briefly available to a fraction of the population. Small-batch bioassays ruled out accumulation of inhibiting compounds other than nitrite. However, cells grown at high density in the reactor displayed reduced respiration rates, suggesting chronic stress under these conditions.

Conclusions: Anaerobic HCDC by denitrification is feasible and yields high-quality biomass, but barriers remain to achieving competitive production rates. The CO₂-pH lag appears to be the primary constraint, amplified by incomplete mixing and nitrite toxicity. These factors interact, e.g. mitigating the CO₂-pH lag by lowering the process pH exacerbates nitrite toxicity. Future work should integrate reactor engineering to improve mixing and gas removal and strain selection for tolerance to low pH and nitrite, supported by omics and metabolic modelling to understand denitrifier physiology at high cell density.