Microbial Biotechnology最新文献

Chlamydomonas reinhardtii, a model green alga for expressing foreign proteins, faces challenges in multigene expression and enhancing protein expression level in the chloroplast. To address these challenges, we compared heterologous promoters, terminators and intercistronic expression elements (IEEs). We transformed Chlamydomonas chloroplast with a biolistic approach to introduce vectors containing the NanoLuc expression unit regulated by Chlamydomonas or tobacco promoters and terminators. We observed that tobacco promoters PrbcL and PpsbA could not effectively regulate protein expression, whereas tobacco terminators TrbcL and Trps16 did not affect the expression of Nluc protein. Further exploration of IEEs specific to Chlamydomonas revealed that Cr-IEE2 had a minor effect on both upstream and downstream protein expression, whereas Cr-IEE5 significantly influenced downstream protein expression. In contrast, tobacco IEE was found to be unsuitable for driving protein expression in Chlamydomonas. Additionally, VOR element and Rep protein derived from beet curly top geminivirus were able to form a minichromosome in Chlamydomonas chloroplast, and this system could enhance protein expression level compared to the traditional method of site-specific integration in the plastome. This study highlights the potential of IEEs and minichromosome in facilitating heterologous protein expression in Chlamydomonas chloroplast.

Teredinibacter turnerae is a cultivable cellulolytic Gammaproteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose and pectin and contribute to wood (lignocellulose) digestion in the shipworm gut. However, the mechanisms by which T. turnerae secretes lignocellulolytic enzymes are incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce membrane vesicles (MVs) that include a variety of proteins identified by liquid chromatography–mass spectrometry (LC–MS/MS) as carbohydrate-active enzymes (CAZymes) with predicted activities against cellulose, hemicellulose and pectin. Reducing sugar assays and zymography confirm that these MVs exhibit cellulolytic activity, as evidenced by the hydrolysis of CMC. Additionally, these MVs were enriched with TonB-dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations indicate a potential role for MVs in lignocellulose utilisation by T. turnerae in the free-living state, suggest possible mechanisms for host–symbiont interaction and may be informative for commercial applications such as enzyme production and lignocellulosic biomass conversion.

The phylum Actinomycetota and genus Streptomyces in particular are the major source for discovery of natural products with diverse chemical structures and a variety of biological activities. Genes encoding biosynthetic pathways for bacterial natural products are grouped together into biosynthetic gene clusters (BGCs). The size of a typical actinobacterial BGC may range from 10 kb to 200 kb, which makes their cloning for heterologous expression a challenging task. Various DNA cloning and assembly methods have been established for capturing BGCs. Among them, the transformation-associated recombination (TAR) in Saccharomyces cerevisiae remains one of the most cost-effective, accessible, customisable and precise approaches. However, the drawback of TAR cloning is a need for intensive screening of clones in order to identify one carrying the BGC. In this study, we report a further development of the TAR cloning approach by introducing the direct selection of colonies with BGC of interest based on the yeast killer phenomenon. For this, a new TAR cloning vector system was constructed and the strategy was validated by successful cloning of chelocardin (35 kb) BGC from Amycolatopsis sulphurea and daptomycin BGC (67 kb) from Streptomyces filamentosus. Both BGCs were functionally expressed in a heterologous host, resulting in the production of the corresponding antibiotics. The proposed approach could be widely applied for precise direct cloning of BGCs from the representatives of phylum Actinomycetota and easily adopted for other bacteria.

Pyrolysis of lignocellulosic biomass commonly produces syngas, a mixture of gases such as CO, CO₂ and H₂, as well as an aqueous solution generally rich in organic acids such as acetate. In this study, we evaluated the impact of increasing acetate shock loads during syngas co-fermentation with anaerobic microbiomes at different pH levels (6.7 and 5.5) and temperatures (37°C and 55°C) by assessing substrates consumption, metabolites production and microbial community composition. The anaerobic microbiomes revealed to be remarkably resilient and were capable of converting syngas even at high acetate concentrations of up to 64 g/L and pH 5.5. Modifying process parameters and acetate loads resulted in a shift of the product spectrum and microbiota composition. Specifically, a pH of 6.7 promoted methanogens such as Methanosarcina, whereas lowering the pH to 5.5 with lower acetate loads promoted the enrichment of syntrophic acetate oxidisers such as Syntrophaceticus, alongside hydrogenotrophic methanogens. Increasing acetate loads intensified the toxicity of undissociated acetic acid, thereby inhibiting methanogenic activity. Under non-methanogenic conditions, high acetate concentrations suppressed acetogenesis in favour of hydrogenogenesis and the production of various carboxylates, including valerate, with product profiles and production rates being contingent upon temperature. A possible candidate for valerate production was identified in Oscillibacter. Across all tested conditions, acetate supplementation provided additional carbon and energy to the mixed cultures and consistently increased carboxydotrophic conversion rates up to about 20-fold observed at pH 5.5, 55°C and 48 g/L acetate compared to control experiments. Species of Methanobacterium, Methanosarcina and Methanothermobacter may have been involved in CO biomethanation. Under non-methanogenic conditions, the bacterial species responsible for CO conversion remain unclear. These results offer promise for integrating process streams, such as syngas and wastewater, as substrates for mixed culture fermentation allowing for enhanced resource circularity, mitigation of environmental impacts and decreased dependence on fossil fuels.

The highly pathogenic avian influenza virus (HPAIV) H5N1, first isolated in 1996 in China, spread rapidly across Eurasia and caused major epizootics in wild and domesticated birds, as well as spillover infections in humans characterised by high mortality. Avian influenza viruses are therefore candidate viruses for a human pandemic. Surprisingly, HPAIV was not isolated in North America until 2014. With the help of intensive biological sampling and viral genome sequencing, the intrusion of HPAIV into North America could be retraced to two separate events. First, migratory birds carried HPAIV from East Siberia via Beringia and dispersed the virus along the Pacific flyway. After reassortment with genes of local low pathogenic avian influenza viruses, HPAIV H5 caused 2015 a major epizootic on poultry farms in the US Mid-West. After costly containment, HPAIV dropped below the detection limit. In 2021, Eurasian HPAIV H5 viruses arrived a second time in North America, carried by migratory birds to Canada via the Atlantic flyway, using Iceland as a stop. The H5 virus then spread with water birds along the East Coast of the United States and dispersed across the United States. In contrast to the 2015 poultry outbreak, spillover infections into diverse species of mammals were now observed. The events culminated in the 2024 HPAIV H5 epizootic in dairy cows affecting 300 dairy herds in 14 US states. The cattle epizootic was spread mainly by milking machinery and animal transport. On affected farms infected cats developed fatal neurological diseases. Retail milk across the United States frequently contains viral RNA, but so far only a few milk farm workers have developed mild symptoms. The tracing of HPAIV with viral genome sequencing complicated the taxonomical naming of influenza viruses raising fundamental problems in how to mirror biological complexity in written plain language, rendering communication with the lay public difficult.

A major factor limiting the biodegradation of organofluorine compounds has been highlighted as fluoride anion toxicity produced by defluorinating enzymes. Here, two highly active defluorinases with different activities were constitutively expressed in Pseudomonas putida ATCC 12633 to examine adaption to fluoride stress. Each strain was grown on α-fluorophenylacetic acid as the sole carbon source via defluorination to mandelic acid, and each showed immediate fluoride release and delayed growth. Adaptive evolution was performed for each recombinant strain by serial transfer. Both strains adapted to show a much shorter lag and a higher growth yield. The observed adaptation occurred rapidly and reproducibly, within 50 generations each time. After adaption, growth with 50-70 mM α-fluorophenylacetic acid was significantly faster with more fluoride release than a preadapted culture due to larger cell populations. Genomic sequencing of both pre- and postadapted strain pairs revealed decreases in the defluorinase gene content. With both defluorinases, adaption produced a 56%-57% decrease in the plasmid copy number. Additionally, during adaption of the strain expressing the faster defluorinase, two plasmids were present: the original and a derivative in which the defluorinase gene was deleted. An examination of the enzyme rates in the pathway suggested that the defluorinase rate was concurrently optimised for pathway flux and minimising fluoride toxicity. The rapid alteration of plasmid copy number and mutation was consistent with other studies on microbial responses to stresses such as antibiotics. The data presented here support the idea that fluoride stress is significant during the biodegradation of organofluorine compounds and suggest engineered strains will be under strong selective pressure to decrease fluoride stress.

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.