Applied Microbiology and Biotechnology最新文献

Arsenic (As) is a toxic metalloid widespread in the environment, and many organisms have evolved mechanisms to mitigate its toxic effects. Bioinformatic analyses revealed that acr3 genes are predominantly distributed in mushrooms, highlighting their evolutionary and functional importance in eukaryotic arsenic metabolism. In this study, two homologous genes, HbACR3 and HsACR3, from the mushrooms Hebeloma bulbiferum and Hebeloma sinapizans were identified and functionally characterized. Both encode 399-amino-acid membrane proteins showing 99% sequence identity to each other and substantial similarity to previously characterized ACR3-type arsenite transporters from plants, yeasts, and bacteria. Heterologous expression of HbACR3 and HsACR3 in a Saccharomyces cerevisiae arr3Δ mutant restored resistance to arsenite and arsenate and significantly reduced intracellular arsenic accumulation. Fluorescence microscopy of GFP-tagged HbACR3 and HsACR3 confirmed their localization to the plasma membrane, consistent with an efflux transport function. Exposure of H. bulbiferum and H. sinapizans mycelia to arsenate led to a significant but differential transcriptional upregulation of both genes. This work provides new insight into the evolution, distribution, and physiological significance of ACR3 transporters in eukaryotic arsenic homeostasis.

Biocatalytic approaches have gained increasing attention as sustainable alternatives to metal-catalyzed asymmetric reductions of ketones to obtain enantiopure alcohols, important intermediates for pharmaceutical synthesis. For example, enzyme-catalyzed reduction of substituted benzophenone analogs to produce chiral diaryl methanols has attracted interest, as they are the key intermediates in the synthesis of antihistamines. However, benzophenone analogs are difficult to be reduced by enzymes due to steric hindrance. Moreover, the similarities between the two groups adjacent to the carbonyl group make achieving high enantioselectivity in reduction challenging. In this study, we examined the reduction of benzophenone and its analogs by Geotrichum candidum acetophenone reductase (GcAPRD). However, the wild type did not exhibit activity toward benzophenone due to the substrate’s bulkiness. Then, two mutants of GcAPRD (Trp288Ala and Phe56Ile/Trp288Ala) were applied to catalyze the reduction of benzophenone, resulting in high reduction yield (≥ 80%). In addition, both mutants exhibited catalytic activity toward methyl- and halogen-substituted benzophenones, especially toward 3- and 4-substituted substrates. Regarding enantioselectivity, Trp288Ala generally reduced both 3- and 4-substituted substrates to (R)-alcohols with up to 97% ee. In contrast, Phe56Ile/Trp288Ala reduced 3-substituted substrates to (R)-alcohols with up to 89% ee but reduced 4-substituted substrates to (S)-alcohols with up to 92% ee. At last, the reduction mechanism was investigated using molecular docking simulations.

• GcAPRD mutants exhibited catalytic performance toward benzophenone analogs.

• GcAPRD Phe56Ile/Trp288Ala exhibited substituent-dependent enantioselectivity.

• Introducing Phe56Ile into GcAPRD Trp288Ala resulted in a clear enantiopreference.

Saccharomyces cerevisiae is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression: PEP1 and VPS30, which both can channel proteins to the vacuole for degradation; MON2, which can lead to the re-uptake of secreted proteins; and ALG3, which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features VPS30, PEP1, and ALG3 deletions, and the small-scale fed-batch system, we obtained 2.5 µg/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies.

• Chemical chaperones and amino acid-rich medium increased secreted protein titers.

• Medium and host engineering are instrumental for improving productivity.

• Small-scale cultivation system enables production levels suitable for characterization.

Inonotus hispidus is a traditional medicinal mushroom in China with significant potential for development of health products and future foods, owing to its diverse functional components and pharmacological activities. Recent advancements in cultivation techniques, coupled with growing market demand, have expanded the production scale of I. hispidus. Breeding superior strains is essential for industry progress, but the absence of clamp connections in I. hispidus complicates mating system studies, making accurate identification of homokaryotic strains a critical step. In this study, we first confirmed the multinucleate nature of both heterokaryotic and homokaryotic mycelia, revising the traditional concepts of monokaryotic and dikaryotic mycelia in this species. Additionally, the mating type loci of I. hispidus were identified through genome sequencing and homologous gene BLAST analysis. Homokaryotic and heterokaryotic strains were distinguished based on sequence differences at the mating type loci between different mating types, which also allowed for differentiation of the mating types themselves. Furthermore, by combining traditional mating tests, we clearly elucidated the bipolar mating system of I. hispidus, refuting previous reports of a tetrapolar system. The growth rate of mycelium, its performance on a wheat grain substrate, as well as the antagonism between the homokaryotic strain and the heterokaryotic parent strain have been demonstrated to be useful for distinguishing the homokaryons. This study established a reliable method for identifying homokaryotic strains and systematically characterized the mating system of I. hispidus for the first time. These findings provide scientific foundation for uncovering the life cycle and presents methods for creating new germplasms.

• First confirmation of a bipolar mating system in Inonotus hispidus

• Single-spore isolates are multinucleate homokaryons

• The significant growth rate differences provide method for homokaryon identification

Marine macroalgae, particularly their complex polysaccharides, are an untapped renewable source of high-quality monosaccharides and related building blocks. To utilize this feedstock for industrial applications, the enzymatic depolymerization by marine microorganisms has been shown to be effective. A prime example is the common green alga Ulva, with its storage polysaccharide ulvan, which contains high quantities of L-rhamnose and D-glucuronic acid. As suitable high-throughput methods for analyzing the enzymatic degradation of complex polysaccharides are still lacking, a transcription factor–based biosensor is described here that utilizes the P_rhaBAD promoter native to E. coli, which is specific for L-rhamnose. This biosensor exhibited a linear response, enabling the quantification of L-rhamnose within a concentration range of 10–1000 µM. The introduction of a T7 stem-loop improved the performance, and various fluorescent reporter genes were studied. The optimized system was then used to evaluate various stages of the ulvan degradation cascade in terms of L-rhamnose release, confirming its applicability to complex sugar mixtures. A detectable fluorescence signal was only generated when all the necessary enzymes for breaking down the polymer into undecorated monosaccharides were present, highlighting the biosensor’s specificity. The application of this method to the degradation of Ulva sp. biomass samples of various origins was also successfully demonstrated. This establishes the biosensor as a promising method for further high-throughput investigations.

• Development of an improved transcription factor-based biosensor for L-rhamnose.

• Biosensor application for the analysis of enzymatic polysaccharide degradation.

• Reliable quantification of L-rhamnose in complex carbohydrate mixtures.

Microcin B17 (MccB17) is a ribosomally synthesized and post-translationally modified peptide (RiPP) with antibacterial activity, natively produced by Escherichia coli. Despite its unique gyrase-inhibiting activity, therapeutic development has been limited, primarily due to its low solubility and narrow spectrum of activity. Here, we employed a genome mining approach to identify and functionally characterize novel MccB17-like biosynthetic gene clusters (BGCs) from uncharacterized environmental Pseudomonas isolates. Four BGCs sharing substantial sequence similarity were cloned and heterologously expressed in E. coli. Regardless of their similarity, the clusters triggered congener-dependent growth retardation and cell elongation, despite lacking detectable antibacterial activity in overlay assays. Transcription and production of modified MccB17-like peptides or their unmodified precursors were confirmed by reverse transcription-quantitative PCR (RT-qPCR) and liquid chromatography-mass spectrometry (LC–MS), respectively. Transcriptome profiling indicated differential induction of stress responses and metabolic shifts in host cells, with limited overlap among the different BGCs. Our findings suggest that structurally similar MccB17 congeners can elicit distinct physiological responses. This study expands the known range of MccB17-like RiPPs and highlights the importance of functionally exploring RiPP diversity beyond known antimicrobial activity, even among congeners.

• In silico identification of uncharacterized MccB17 congeners from Pseudomonas spp.

• Functional analysis of novel variants reveals phenotypic effects on expression host

• Transcriptomics reveals congener-specific effects despite similarity to E. coli MccB17

Bacterial cellulose (BC) production is limited by the high cost of refined nitrogen sources such as yeast extract and tryptone. While carbon source substitution has been widely studied, approaches for nitrogen replacement remain underexplored. Here, we evaluated cereal and pseudo-cereal flours as low-cost nitrogen alternatives for Komagataeibacter xylinus DSMZ 2325 under static fermentation under static fermentation using two nitrogen substitution strategies: constant total nitrogen (CTN) and constant nitrogen source mass (CNSM). Thirteen flour variants were screened, with maximum BC yields of 3.40 g/L (soy), 3.17 g/L (teff), 2.74 g/L (quinoa), and 1.94 g/L (triticale) under the CTN strategy, in several cases surpassing the modified HS control. Structural analyses confirmed that flour-derived BC retained the defining characteristics of high-quality cellulose, including high crystallinity, robust fibrillar networks, and thermal stability. Soy consistently produced the highest volumetric yields, whereas triticale exhibited the greatest fold-increase compared to the control, highlighting the distinct nutritional and compositional contributions. These results demonstrate that flour-based media provide a viable strategy to reduce production costs while tailoring BC properties through substrate choice. By shifting the focus from carbon to nitrogen optimization, this study introduces a sustainable and scalable approach to bioprocessing using agro-derived raw materials.

• Flour-based nitrogen sources replace costly yeast extract and tryptone in BC media

• Triticale, teff, quinoa, and soy influence BC yield and cellulose fibril formation

• Agro-flour substrates offer low-cost, sustainable bacterial cellulose production

Carotenoids are known for their antioxidant and antiapoptotic properties, which make them effective in reducing cellular damage caused by UV radiation and improving cryopreservation outcomes in various cell types. Among them, bacterioruberin (BR) stands out as a potent antioxidant carotenoid produced by Haloarchaea, such as Haloferax mediterranei. This study evaluates the effects of a bacterioruberin-rich carotenoid extract (BRCE) on peripheral blood mononuclear cells (PBMCs) under these conditions. Fluorescence microscopy and flow cytometry revealed that pre-incubation with BRCE at concentrations of 1–2.7 µg/mL significantly increased PBMC viability after UV irradiation, reducing apoptosis and cell death. Higher BRCE concentrations (≥ 25 µg/mL) diminished these protective effects, with 75 µg/mL further increasing apoptosis and cell death rates. For cryopreserved PBMCs, BRCE concentrations of 1–2.7 µg/mL improved cell viability, reduced apoptosis, and minimized cell death, with similar adverse effects observed at higher concentrations. These findings underscore BRCE’s dual role in mitigating UV-induced cellular damage and enhancing cryopreservation efficiency, with optimal concentrations identified for both processes. This highlights BR’s potential in biomedical applications requiring immune cell preservation and protection.

• BRCE enhances PBMC viability after UV exposure and cryopreservation.

• Low BRCE doses (1–2.7 µg/mL) are protective; high doses increase cell death.

• BRCE shows biomedical potential as an antioxidant and natural UV filter.

Plant growth-promoting rhizobacteria (PGPR) of the genus Variovorax facilitate plant growth through beneficial microbe-plant interplay. Unlike most PGPRs, Variovorax boronicumulans CGMCC 4969 utilizes indole-3-acetonitrile as a precursor to generate indole-3-acetic acid (IAA), which is subsequently metabolized by itself. In this study, IAA enhanced the growth of V. boronicumulans CGMCC 4969 in minimal salt medium (MSM), whereas it inhibited bacterial growth when glucose was added to the MSM broth. IAA was rapidly degraded within 12 h in MSM broth despite glucose appeared or not. Notably, in LB broth, the cell growth was significantly inhibited by IAA concentration beyond 1 mmol/L, while the IAA degradation capability of CGMCC 4969 was significantly increased following exposure to IAA-dosed LB medium. V. boronicumulans CGMCC 4969 degraded IAA to yield a new intermediate 3-hydroxy-anthranilate. An iad gene cluster was identified in V. boronicumulans CGMCC 4969, and co-expression of the iadD and iadE genes endows Escherichia coli with the capacity to degrade IAA. This degradation efficiency is augmented when the iadC gene is expressed simultaneously. Subsequent proteomics and bioinformatics analyses highlighted that the addition of IAA induced a significant up-regulation of ABC transporter proteins, in particular IadK3 and IadK2. Interestingly, there was also a significant increase in protein expression associated with group-sensing metabolism. Collectively, this research helps our understanding of the intricate regulatory mechanisms of IAA within Variovorax own metabolism and expands our knowledge of its complex role in plant-microbe interactions.