Microbial Biotechnology最新文献

Commercial probiotics are often formulated as multi-strain cocktails, but the effects of social interactions, particularly between strains of a species, are often neglected, despite their potential to contribute to higher-order interactions where these interactions could affect those with a third party. In this study, we investigated the probiotic potential of a collection of Bacillus subtilis strains against Salmonella Typhimurium in single-strain and mixed cultures. The results indicate a promising probiotic potential of B. subtilis as 38 out of 39 strains significantly inhibited the growth of S. Typhimurium. Next, we tested the effect of mixing B. subtilis strains that differ in their inhibitory potency against S. Typhimurium. The results show that strong inhibition by one strain can be significantly reduced by mixing with a less effective strain. Moreover, mixing similarly effective strains mostly resulted in a decreased growth inhibition of the pathogen. Additionally, we found a group of highly aggressive strains, which completely eliminated other B. subtilis strains in the two-strain mixtures. Overall, this work shows that intraspecies interactions between B. subtilis strains can significantly alter the probiotic effect against S. Typhimurium, which is of great importance for future research on the development of multi-strain probiotics.

Listeria monocytogenes is a pathogenic bacterium that can form biofilms in food processing plants, allowing the bacteria to survive despite the control measures applied. As the surface of the bacteria is covered with versatile polysaccharides and proteins, these influence the interactions of the bacterium with any surface. The unique properties and high stability of fungal proteins make them good candidates for the control of bacteria by targeting surface structures. We screened a group of fungal lectins and protease inhibitors from different fungal species, protein folds and known targets for their antibacterial and antibiofilm activity against model strains of Listeria innocua and Listeria monocytogenes. Several of them significantly decreased the viability of biofilm bacteria, but had no effect on bacterial growth parameters at 37°C and thus had no antibacterial activity. Fungal lectins significantly impaired biofilm development even at room temperature, which was attributed to exposure to lectins during adhesion. The tested fungal proteins also reduced biofilm development on biological model surfaces. The observed antibiofilm activity of fungal proteins suggests that they have the potential to modulate interactions between bacteria and/or between bacteria and surfaces, which could be used in the future to reduce surface contamination by Listeria.

In Pseudomonas putida KT2440, a prime chassis for biotechnology, the clustered distribution of glucose catabolism genes and four related transcription factors (TFs) may facilitate the tight regulation of glucose catabolism. However, the genes under the direct control of these TFs remain unidentified, leaving their regulatory roles elusive. Furthermore, the carbon source gluconate was metabolised similarly to glucose in KT2440, but the responses of these catabolic and TF genes to gluconate were unclear. Here, these mysteries were unravelled through multi-omics analysis integrated with physiological studies. First, we found that the expression of these catabolic and TF genes were significantly induced by both glucose and gluconate in KT2440. The independent responses of these genes to glucose and gluconate were differentiated in the gcd deletion mutant. We then defined the regulon of GnuR, one of the four related TFs, and discovered that GnuR directly repressed the expression of catabolic genes involved in the Entner–Doudoroff and the peripheral glucose and gluconate metabolism pathways. These results were further confirmed by physiological studies. Finally, a regulatory mode of an incoherent feedforward loop involving GnuR is proposed.

Microbial biosurfactants have garnered significant interest from industry due to their lower toxicity, biodegradability, activity at lower concentrations and higher resistance compared to synthetic surfactants. The deep-sea Rhodococcus sp. I2R has been identified as a producer of glycolipid biosurfactants, specifically succinoyl trehalolipids, which exhibit antiviral activity. However, genome mining of this bacterium has revealed a still unexplored repertoire of biosurfactants. The microbial genome was found to host five non-ribosomal peptide synthetase (NRPS) gene clusters containing starter condensation domains that direct lipopeptide biosynthesis. Genomics and mass spectrometry (MS)-based metabolomics enabled the linking of two NRPS gene clusters to the corresponding lipopeptide families, leading to the identification of 20 new cyclolipopeptides, designated as rhodoheptins, and 33 new glycolipopeptides, designated as rhodamides. An integrated in silico gene cluster and high-resolution MS/MS data analysis allowed us to elucidate the planar structure, inference of stereochemistry and reconstruction of the biosynthesis of rhodoheptins and rhodamides. Rhodoheptins are cyclic heptapeptides where the N-terminus is bonded to a β-hydroxy fatty acid forming a macrolactone ring with the C-terminal amino acid residue. Rhodamides are linear 14-mer glycolipopeptides with a serine- and alanine-rich peptide backbone, featuring a distinctive pattern of acetylation, glycosylation and succinylation. These molecules exhibited biosurfactant activity in the oil-spreading assay and showed moderate antiproliferative effects against human A375 melanoma cells.

Live biotherapeutic products (LBPs), including engineered bacteria, are rapidly emerging as potential therapeutic interventions. These innovative therapies can serve as live in situ drug delivery platforms for the direct deposition of therapeutic payloads, including complex biologics, at sites of disease. This approach offers a platform likely to enhance therapeutic efficacy and decrease off-target side effects. LBPs also can likely be distributed at a relatively low price point, as their production can be economically scaled up. LBPs represent an exciting new means for ensuring healthy lives and promoting well-being for all ages, aligning with the World Health Organization's sustainable development goal 3.

Postbiotics are metabolic by-products from microorganisms that provide health benefits to the host. Their secretion can be influenced by various conditions affecting bacterial metabolism. This study presents a novel approach for producing potential postbiotics, specifically extracellular products (ECPs), from the probiotic strain Shewanella putrefaciens SpPdp11, grown under different culture conditions. These conditions include aquafeed media, with partial or total microalgae/cyanobacteria replacement as the microbial substrate, as well as variations in temperature and growth phase. The use of microalgae/cyanobacteria as substrates may represent a valuable strategy for generating novel postbiotics with unique properties. The ECPs assessed were evaluated for their in vitro cytotoxic, hydrolytic and antimicrobial activities. Three conditions (ECPs derived from aquafeed media with partial (FM2324 and FM1548) or total (M2324) microalgae/cyanobacteria replacement) were non-cytotoxic to various fish cell lines and hydrolysed key nutritional compounds (casein, lipids, amylase and gelatin). Proteomic analysis of these ECP conditions revealed common structural and regulatory DNA-associated proteins, while differentially expressed proteins were associated with amino acid metabolism and antioxidant system (FM2324 and FM1548) and chemotaxis system (M2324). The results highlight the potential of the selected postbiotics as feed additives for future in vivo studies, aligning with sustainable development for aquaculture.

Pseudomonas putida is widely used in industrial applications, including the recombinant proteins production, because of its natural advantageous properties. In this study, the gene encoding FleQ, the primary regulator of flagellar synthesis, was deleted to construct a new non-motile P. putida KT2440-derived strain (ΔfleQ). The non-motile cells showed reduced biofilm formation and enhanced expression of a heterologous gene in nutrient-rich media compared with the wild-type (WT) strain, attributed to the reallocation of cellular resources from flagellar synthesis and cellular motility. Additionally, the ΔfleQ strain exhibited enhanced tolerance to chloramphenicol, indicating higher ribosome production, confirmed by a higher RNA/protein ratio relative to the WT. While the WT strain showed decreased growth and a three-fold increase in reporter gene activity in minimal media, the ΔfleQ strain maintained consistent reporter gene expression and exhibited a relatively higher growth rate. This suggests that the FleQ is involved in modulating proteome allocation based on nutrient quality. The removal of FleQ allows for more flexible resource allocation, creating a chassis strain with nutrient quality-independent gene expression capacity, which could be valuable in industrial applications where consistent output is essential.

Yeast extract (YE) is a complex nutritional source associated with high performance on microbial production processes. However, its inherent compositional variability challenges its scalability. While prior efforts have focused on growth-associated products, the dynamics of growth-uncoupled production, which leads to higher production rates and conversion yields, still need to be explored. This production scenario is common in large-scale applications. This study presents a systematic approach to replace YE for the production of the terpene amorpha-4,11-diene in Escherichia coli. Sequential processing was successfully applied to identify glutamic acid, alanine, leucine, valine, isoleucine and glycine as the key amino acids (AAs) under slow-growth conditions. Thoroughly applying biomass retention as part of sequential processing increased production capacity by 45% using these AAs instead of YE. Further studies, including flux balance analyses, targeted pyruvate as the common AA precursor. The optimized fed-batch process feeding pyruvate with 0.09 g_Pyr h⁻¹ enhanced amorpha-4,11-diene production by 37%, although adding only 1% carbon via pyruvate. Flux balance analysis revealed the criteria for optimum pyruvate feeding, for example, to prevent succinate secretion and maintain the NADH/NAD⁺ balance. These findings illustrate the interplay between media composition and metabolic activity and provide a successful guideline for identifying lean, best-performing media for industrial applications.

Escherichia coli, a common microbial host for industrial bioproduction, experiences a highly dynamic environment in industrial-scale bioreactors due to significant glucose and oxygen gradients. In this study, we mimic the combined gradients of glucose and oxygen in high-throughput bioreactors to study the transcriptional response of E. coli to industrial-scale conditions. Under oscillating oxygen conditions, E. coli formed less biomass and accumulated the anaerobic by-product acetate. With respect to oxygen-responsive genes, we found that genes of the TCA cycle and of different electron transport chain complexes were differentially expressed. A global analysis of the expression data revealed that oxygen oscillations had caused a transition towards a catabolite-repressed state and upregulation of several stress-related regulatory programs. Interestingly, the transcriptional changes persisted after oxygen limitation stopped. In contrast, the changes we observed due to glucose starvation, such as induction of the stringent response, were primarily transient. Most importantly, we found that effects of combined oxygen and glucose oscillations were distinct from the ones of oxygen and substrate oscillations alone, suggesting an important interplay between the different metabolic regimes in industrial-scale bioreactors.

Despite the great potential of DNA vaccines for a broad range of applications, ranging from prevention of infections, over treatment of autoimmune and allergic diseases to cancer immunotherapies, the implementation of such therapies for clinical treatment is far behind the expectations up to now. The main reason is the poor immunogenicity of DNA vaccines in humans. Consequently, the improvement of the performance of DNA vaccines in vivo is required. This mini-review provides an overview of the current state of DNA vaccines and the various strategies to enhance the immunogenic potential of DNA vaccines, including (i) the optimization of the DNA construct itself regarding size, nuclear transfer and transcriptional regulation; (ii) the use of appropriate adjuvants; and (iii) improved delivery, for example, by careful choice of the administration route, physical methods such as electroporation and nanomaterials that may allow cell type-specific targeting. Moreover, combining nanoformulated DNA vaccines with other immunotherapies and prime-boost strategies may help to enhance success of treatment.