Search Results (125)

Ascidians are a group of marine invertebrates, most of which are sessile and soft-bodied. Their lack of an adaptive immune system makes them rely on innate immune responses to detect and eliminate invading microbes. Antimicrobial peptides (AMPs) play an essential part in this process. In this paper, we present the isolation, structure elucidation, and bioactivities of two new cysteine-rich peptides (CRPs) from the Arctic marine ascidian Synoicum turgens. The sequences and structures of the peptides were determined by Edman degradation sequencing, mass spectrometry, and NMR analysis. This revealed two novel 2 kDa peptides, St-CRP-1 and St-CRP-2, with neutral net charge and C-terminal amidation. St-CRP-1 consisted of 18 amino acids and displayed selective and moderate growth inhibition of two Gram-positive bacterial strains (Bacillus subtilis and Corynebacterium glutamicum) at 24.6 µM, whereas St-CRP-2 consisted of 19 amino acids and inhibited the growth of B. subtilis at 49.2 µM. St-CRP-1 had no effect on two mammalian cell lines or the brine shrimp Artemia salina at the highest concentration tested. Structural analysis of the St-CRPs indicated a Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 disulfide connectivity, which is also found in alpha-defensins. The results from this study show that Arctic marine ascidians are a rich source of novel bioactive peptides. Full article

The MarR (multiple antibiotic resistance regulator) family regulators, which are widely conserved across various organisms, play pivotal roles in metabolism, stress response mechanisms, and virulence factor production. However, the regulatory functions of these factors in the degradation of aromatic compounds within Corynebacterium glutamicum remain largely uncharacterized. In this study, we identified a MarR-type regulator, designated AcsR (encoded by ncgl2425), which directly represses the expression of the catechol 2,3-dioxygenase gene ncgl2007 (c23o) and the heavy metal (nickel) transport system permease gene ncgl2351, while activating the expression of ncgl2258 encoding an ABC-type C4-dicarboxylate-binding periplasmic protein. AcsR binds specifically as a dimer to a 6 bp inverted repeat sequence, and this binding is disrupted by catechol in vitro. Correspondingly, catechol induces the expression of c23o in vivo. Phenotypic analysis revealed that the ΔacsR mutant exhibited enhanced resistance to multiple aromatic compounds but increased sensitivity to antibiotics, heavy metals, and oxidants. Collectively, these findings demonstrate that AcsR is an important regulator of stress adaptation in C. glutamicum and provide new insights into the regulatory mechanisms of aromatic compound degradation in this industrially important bacterium. Full article

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor, yet its industrial supply still relies largely on multi-step chemical synthesis. Here, using the industrial chassis Corynebacterium glutamicum ATCC 13032, we proposed and validated a strategy based on a minimal heterologous entry coupled to endogenous pathway continuation, resulting in a distinct PLP-producing route. Three engineered strains were constructed and compared: S1 expressing ecepd from Escherichia coli; S2 co-expressing ecepd plus ecpdxB from Escherichia coli (a minimal two-gene module); and S3 carrying an additional ecpdxA from Escherichia coli and smpdxJ from Sinorhizobium meliloti to form a four-gene module as a benchmark for heterologous reconstruction. The wild-type (WT) strain produced a basal PLP level of 10.6 mg/L. Overexpressing ecepd alone increased the titer to 40.4 mg/L (3.8-fold vs WT), whereas the minimal two-gene module in S2 yielded the highest PLP titer of 95.5 mg/L (9.0-fold vs WT; 136.0% higher than S1). Notably, the four-gene module (S3) reached 70.0 mg/L, which was 36.3% lower than S2 under matched conditions. These results indicated that introducing only a minimal two-gene entry could cooperate with the endogenous metabolic network of Corynebacterium glutamicum to establish a new and highly effective PLP biosynthetic route, with production performance exceeding that of a multi-gene heterologous reconstruction in the tested window. This work provides a low-burden and scalable framework for sustainable PLP biomanufacturing and motivates further optimization targeting the endogenous continuation steps and regulatory constraints. Full article

The selection of an optimal antifoam is critical for efficient fermentation, as industrial agents often have detrimental side effects like growth inhibition, while some can enhance productivity. We studied the efficacy of novel silicone–polyol antifoam emulsions for use in fermentation as defoamers. Except for agent 3L10, all antifoams tested did not show inhibition on six bacterial and one fungal culture. Interestingly, agent 3L10 strongly inhibited Gram-positive bacteria (especially Corynebacterium glutamicum) but not Gram-negative strains. A comprehensive evaluation protocol—combining chemical design, cytotoxicity screening across diverse microorganisms, the determination of minimum effective concentrations (MECs), and validation in model bioreactor fermentations—was established. Through this process, 6T80 was identified as a promising antifoam agent for fermentation. It exhibited a low MEC, high emulsion stability, and no cytotoxicity and did not impair growth or recombinant protein production in Bacillus subtilis or Pseudomonas putida fermentations. This study concludes that agent 6T80 is suitable for further application in processes involving Gram-negative and certain Gram-positive hosts. The developed methodology enables the targeted selection of highly efficient and biocompatible antifoams for specific biotechnological processes. Full article

Background: Adopting novel feed ingredients and aligning feeding strategies with these formulations are key to improving aquaculture sustainability. This study assessed the combined effects of alternative protein and lipid sources and feeding regime on growth, nutrient utilization, and body composition of European sea bass (Dicentrarchus labrax) juveniles. Methods: Two isoenergetic and identical digestible protein diets (39%) were formulated: a control (conventional fishmeal/fish oil (FM/FO) and plant proteins, containing 20% FM and 6% FO) and an alternative diet replacing 50% of FM and 25% of vegetable proteins with a blend of poultry by-products, insect meal, and single-cell protein (Corynebacterium glutamicum) and totally replacing fish oil with alternative lipid sources (microalgae and by-product oils). Fish (28 g of initial body weight) were fed for 210 days either to apparent satiety (AS) or under moderate restriction (85% and 65% of AS). The number of fish used was 65 fish per 500 L tank (triplicate for each experimental group). Growth performance, feed conversion, nutrient efficiency ratios, protein retention, and proximate and fatty acid composition were measured. Results: The alternative diet significantly improved growth, feed and nutrient efficiency, and protein retention compared with the control. Whole-body fatty acid profiles of fish fed the alternative diet showed higher contents of nutritionally important fatty acids, including DHA. Restricted feeding at 65% of AS enhanced nutrient efficiency ratios and protein retention relative to 85% and AS, but reduced growth. Feeding to AS produced the highest feed intake and growth but poorer feed conversion and nutrient efficiency. No significant interaction between diet and feeding strategy was observed. Conclusions: Incorporating novel protein and lipid sources can improve sea bass performance and product nutritional value while supporting sustainability. Feeding at ~85% of AS may offer a practical compromise between growth and efficient nutrient utilization. Full article

1,5-Pentanediamine (PDA) is an important monomer for the synthesis of nylon materials. However, its microbial production from glucose is severely limited by product cytotoxicity, which slows the metabolism of both precursor lysine and glucose uptake. To overcome this limitation, a PDA-responsive dynamic regulatory switch (PDRS) was constructed using the transcriptional repressor CgmR and the P_cgmA promoter. By replacing promoters and ribosome-binding sites, the response window of the PDRS was optimized to a PDA concentration range of 38.9–87 g/L. Based on this system, the PDRS was employed to enhance lysine biosynthesis and glucose uptake. Following fermentation optimization, the optimal strain Corynebacterium glutamicum YY3.6 produced 105.5 g/L PDA within 36 h, achieving a PDA productivity of 2.93 g/L/h and a yield of 0.36 g/g glucose. Collectively, these results provide an effective strategy for the microbial production of PDA from glucose. Full article

Corynebacterium glutamicum is a versatile microbial cell factory, but efficient recovery of intracellular macromolecules remains a major challenge. In this study, we engineered environmentally controllable lysis systems to enable programmable product release. A glucose-responsive module, combining the cg3195 promoter with phage-derived holin–endolysin genes (clg51-50), triggered lysis when extracellular glucose dropped below 0.19–0.36 g/L. A separate temperature-inducible system employing the cI857-CJ1OX2 module activated lysis at 42 °C. These modules were further integrated into a dual-input AND-gate circuit, enhancing regulatory precision and suppressing premature lysis, with additional operator copies allowing temporal tuning of induction. Functional validation using fluorescence, cell density measurements, and scanning electron microscopy confirmed robust, tunable responses under defined environmental cues. Collectively, these programmable lysis systems demonstrate that stimulus-responsive genetic circuits can be rationally designed to control cell disruption, providing a promising approach to streamline downstream processing and reduce extraction costs in industrial fermentation of Corynebacterium glutamicum. Full article

Better understanding of the fitness and flexibility of microbial platform organisms is central to biotechnological process development. Live-cell experiments uncover the phenotypic heterogeneity of living cells, emerging even within isogenic cell populations. However, how this observed heterogeneity in growth relates to the variability of intracellular processes that drive cell growth and division is less understood. We here approach the question, how the observed phenotypic variability in single-cell growth rates links to metabolic processes, specifically intracellular reaction rates (fluxes). To approach this question, we employ the Maximum Entropy (MaxEnt) principle that allows us to bring together the phenotypic solution space, derived from metabolic network models, to single-cell growth rates observed in live-cell experiments. We apply the computational machinery to first-of-its-kind data of the microorganism Corynebacterium glutamicum, grown on different substrates under continuous medium supply. We compare the MaxEnt-based estimates of metabolic fluxes with estimates obtained by assuming that the average cell operates at its maximum growth rate, which is the current predominant practice in biotechnology. Full article

In this study, we constructed a recombinant Corynebacterium glutamicum strain for γ-aminobutyric acid (GABA) biosynthesis via the heterologous expression of glutamate decarboxylase (GAD) derived from Lactiplantibacillus plantarum. We systematically analyzed the fermentation strategy, the balance between cell growth and GAD expression, and the intracellular and extracellular glutamate and GABA levels during fermentation in recombinant C. glutamicum. The results demonstrated that a fermentation strategy combining variable-rate feeding with two-stage pH control at an initial glucose concentration of 50 g/L effectively enhanced cell proliferation, facilitated continuous glutamate synthesis and improved the catalytic efficiency of GAD. The intracellular and extracellular GABA synthesis improved up to 3.231 ± 0.024 g/L (a six-fold increase compared to the uncontrolled supplementation conditions). Furthermore, we fitted empirical equations relating cell growth, glucose consumption, GAD activity, and GABA synthesis during the fermentation. The maximum specific growth rate, glucose consumption rate, and GABA synthesis rate of recombinant C. glutamicum were 0.316 h⁻¹, 1.407 g/(g∙h), and 0.0697 g/L/h, respectively. The fermentation regulation strategy and the dynamic analysis of the fermentation process in this study provide support for future metabolic regulation strategies. Full article

Dehydroshikimate (DHS) dehydratase (DSD) catalyzes the conversion of DHS into 3,4-dihydroxybenzoic acid (3,4-DHBA), a compound with promising applications across various industries. The DSD from Podospora anserina (DSD_Pa) was characterized and its catalytic properties were compared with those of previously investigated enzymes, AsbF (Bacillus thuringiensis), Qa-4 (Neurospora crassa), and QsuB (Corynebacterium glutamicum), both in vitro and in vivo using tube fermentation. Escherichia coli and C. glutamicum were used as platforms to construct model 3,4-DHBA producers. To increase DHS availability in both hosts, shikimate dehydrogenase AroE was inactivated, and the plasmid pVS7-aroG4, encoding 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (E. coli), was introduced. In E. coli, heterologous 3,4-DHBA synthesis was achieved through chromosomal integration of dsd genes. The fungal genes were codon-optimized for this bacterium. The same genes were cloned into the pVK9 vector and introduced into C. glutamicum, where 3,4-DHBA degradation was disrupted (ΔpcaHG). AsbF (k_cat ~ 1 s⁻¹) showed poor 3,4-DHBA accumulation in both hosts (1–1.5 g/L). The enzymes with better catalytic characteristics, QsuB (k_cat ~ 60 s⁻¹), DSD_Pa (k_cat ~ 125 s⁻¹), and Qa-4 (k_cat ~ 220 s⁻¹), provided 5 g/L 3,4-DHBA in E. coli and 3 g/L 3,4-DHBA in C. glutamicum, except for Qa-4. The low production (~1.5 g/L) observed for Qa-4 in C. glutamicum might be attributed to a non-optimal nucleotide sequence rich in codons rare for C. glutamicum. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Efficient co-utilization of glucose and xylose from lignocellulosic biomass remains a critical bottleneck limiting the viability of sustainable biorefineries. While Corynebacterium glutamicum has emerged as a promising industrial host due to its robustness, further improvements in mixed-sugar co-utilization are needed. Here, we demonstrate how a single amino acid substitution can dramatically transform cellular sugar transport capacity. By combining rational strain engineering with continuous adaptive laboratory evolution, we evolved a ptsG-deficient C. glutamicum strain in glucose–xylose mixtures for 600 h under consistent selection pressure. Whole-genome sequencing revealed a remarkable finding: a single point mutation; exchanging proline for alanine in the myo-inositol/proton symporter IolT1 was sufficient to boost glucose uptake by 83% and xylose uptake by 20%, while increasing the overall growth rate by 35%. This mutation, located in a highly conserved domain, likely disrupts an alpha helical structure, thus enhancing transport function. Reverse engineering confirmed that this single change alone reproduces the evolved phenotype, representing the first report of an engineered IolT1 variant in PTS-independent C. glutamicum that features significantly enhanced substrate uptake. These results both provide an immediately applicable engineering target for biorefinery applications and demonstrate the power of evolutionary approaches to identify non-intuitive solutions to complex metabolic engineering challenges. Full article

The textile industry generates millions of tons of waste annually, posing significant environmental challenges. Addressing this issue, our study explores a sustainable biotechnological approach to convert cotton textile waste into valuable bioproducts. We evaluated the potential of Corynebacterium glutamicum ATCC 21492 for the production of L-lysine and other amino acids using glucose derived from cotton textile waste. Two experimental strategies were implemented: Sequential Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). In SHF, optimization of initial glucose concentration, temperature, and inoculum size led to the highest L-lysine concentration of 2.38 g/L under conditions of 45 g/L glucose, 35 °C, and 2% inoculum. The production of L-lysine, along with varying proportions of other amino acids such as alanine, threonine, methionine, and leucine, was significantly influenced by these parameters. In SSF, the highest L-lysine yield of 3.10 mg/g untreated cotton was achieved at 14% cotton loading, 7% enzyme dose, 35 °C, and 10% inoculum concentration, corresponding to an L-lysine concentration of 0.5 g/L. This reduced concentration, compared to SHF, is primarily attributed to limitations in cotton hydrolysis under the studied conditions. Nevertheless, C. glutamicum utilized alternative carbon sources present in the culture medium, leading to a diversified amino acid profile in the final product. These findings support the feasibility of textile waste bioconversion using C. glutamicum and highlight its potential as a sustainable platform for amino acid production, aligning with circular economy principles and contributing to the reduction of the textile industry’s environmental impact. Full article

To accurately detect internal and environmental cues, bacteria have evolved signal transduction pathways such as two-component systems (TCSs) to reprogram appropriate genetic and physiological functions for adaptation and survival. The MprAB TCS is commonly found in actinobacteria and has been associated with important processes such as mycobacterial virulence, nutrient starvation, and environmental stress, particularly cell envelope stress. However, a comprehensive investigation of the function and response network of the MprAB TCS in corynebacteria remains to be carried out. In this study, we report that the MprAB TCS (previously named CgtSR2) plays a critical role in regulating genes involved in cell envelope remodeling in C. glutamicum. The results indicated that the MprAB TCS directly controls a broad regulon, including cell wall biosynthesis proteins, alternative sigma factors, secreted proteins of unknown function, and the mprAB gene locus itself. Among these, the HtrA-like serine protease confers vancomycin and penicillin resistance. Furthermore, we found that the function of the cell envelope was disrupted during overexpression of mprA, resulting in elongated cell morphology and increased cell membrane permeability, as well as enhanced excretion of L-alanine. In conclusion, our findings provide novel insights into how the conserved MprAB TCS controls cell envelope homeostasis in distant actinobacteria. Full article

The regulation of intracellular NADPH levels is currently a hotspot for research into bacterial modification and fermentation process optimization, and Corynebacterium glutamicum, an important industrial microorganism, achieves enhanced L-lysine production by regulating intracellular NADPH levels. In previous studies, transcriptome analysis was performed on C. glutamicum with different intracellular NADPH levels. The results showed that the expression level of transcription factor AtrN changed significantly. Moreover, experiments showed that transcription factor AtrN can sense high intracellular levels of NADPH and negatively regulate its synthesis. In this study, we integrated the pntAB gene of Escherichia coli into the genome of C. glutamicum XQ-5, successfully constructing a chassis cell with a high intracellular NADPH level. It was named TQ-1. On this basis, we knocked out and complemented the AtrN in strain TQ-1, resulting in strains TQ-2 and TQ-3, respectively. Then, the changes in cell growth, intracellular redox substances and cell membrane among these three strains were investigated. We found that the growth of TQ-2 was inhibited in the early growth stage and the cell survival rate was decreased because of the high increase in the intracellular NADPH level. In addition, the deletion of the AtrN gene also led to a decrease in the fluidity and an increase in the permeability of the cell membrane. Compared with TQ-1, TQ-3 showed slow growth only in the late growth stage, and the fluidity of its cell membrane was also enhanced. This indicates that AtrN guides the cells to make some adaptive changes to maintain cell growth when facing excessive intracellular reductive stress. This will facilitate future research on how potential upstream regulatory genes regulate AtrN and how AtrN regulates downstream genes to cope with cellular reductive stress. It also provides theoretical guidance for the specific modification of high-yield lysine-producing strains. Full article