Applied Microbiology and Biotechnology最新文献

Aloe vera (L.) Burm.f. is a traditional Chinese medicine known for treating various ailments, including fungal infections. Aloin is one of the major components from A. vera, but its antifungal mechanism and therapeutic potential against oral candidiasis are not clear. This study aimed to examine the mechanism of aloin against Candida albicans and its inhibitory activity against oral candidiasis. In this study, we for the first time found that aloin could induce the formation of abnormal hyphae with smaller hyphal diameters and fewer branching points in C. albicans including 11 clinical isolates without growth inhibition. The transcriptome and further cell wall contents analysis indicated that aloin remodeled the cell wall to increase the contents of β-1,3-glucan and furtherly showed an antagonistic effect with micafungin. Aloin also significantly inhibited the cell damage of oral epithelial cells and oral candidiasis in mice infected by C. albicans due to its inhibitory actions on the hyphal development and expressions of virulence factors, including candidalysin (coded by ECE1). Our results suggest that aloin is a promising antifungal agent for controlling candidiasis and targeting hyphal development and pathogenesis represents a practical strategy for developing new antifungal drugs.

• Aloin remodels the C. albicans cell wall to form avirulent hyphae.

• Aloin inhibits C. albicans infections in oral epithelial cells and mouse mucosa without toxicity.

• Aloin is a promising antifungal agent with therapeutic potential against C. albicans infections.

Metagenomes present a source for novel enzymes, but under 1% of environmental microbes are cultivatable. Because of its useful properties, Escherichia coli has been used as a host organism in functional genomic screens. However, due to differing expression machineries in the expression host compared to the source organism of the DNA sequences, screening outcomes can be biased. Here, we focused on one of the limiting processes—translation initiation. To that end, we created an operon-like screening system in E. coli to select mutants of the ribosomal protein S1 with more relaxed sequence requirements for 5’-untranslated regions of mRNAs. We created two mutation libraries of the ribosomal protein S1, one covering domains 3 and 4 (D3-D4) and the second covering domains 3 to 5 (D3-D5). Most mutants from library D3-D4 proofed to be specific for a particular UTR sequence and improved only expression from a single construct. Only mutant 3 from library D3-D4 led to increased expression of four different reporters improving fluorescence levels by up to 21%. Mutants isolated from D3-D5 library led up to 90% higher expression compared to the control, though the mutants with highest improvements exhibited a specialist phenotype. The most promising mutant, mutant 4, exhibited a generalist phenotype and showed increased expression in all six reporter strains compared to the control. This could indicate the potential for a more promiscuous translation initiation of metagenomic sequences in E. coli although at the price of smaller increases compared to specialist mutants.

• An operon-like selection system allowed to isolate generalist and specialist S1 mutants.

• S1 mutants improved translation of mRNAs with 5'-UTRs from metagenomic sequences.

• Use of S1 mutants could increase coverage from metagenomic libraries in functional screens.

Second-generation (2G) bioethanol production, derived from lignocellulosic biomass, has emerged as a sustainable alternative to fossil fuels by addressing growing energy demands and environmental concerns. Fungal sugar transporters (STs) play a critical role in this process, enabling the uptake of monosaccharides such as glucose and xylose, which are released during the enzymatic hydrolysis of biomass. This mini-review explores recent advances in the structural and functional characterization of STs in filamentous fungi and yeasts, highlighting their roles in processes such as cellulase induction, carbon catabolite repression, and sugar signaling pathways. The review also emphasizes the potential of genetic engineering to enhance the specificity and efficiency of these transporters, overcoming challenges such as substrate competition and limited pentose metabolism in industrial strains. By integrating the latest research findings, this work underscores the pivotal role of fungal STs in optimizing lignocellulosic bioethanol production and advancing the bioeconomy. Future prospects for engineering transport systems and their implications for industrial biotechnology are also discussed.

Identifying hormone-like quorum sensing (QS) molecules in streptomycetes is challenging due to low production levels but is essential for understanding secondary metabolite biosynthesis and morphological differentiation. This work reports the discovery of a novel γ-butenolide-type signaling molecule (SFB1) via overexpressing its biosynthetic gene (orf18) in Streptomyces fradiae. SFB1 was found to be essential for production of tylosin through dissociating the binding of its receptor TylP (a transcriptional repressor) to target genes, thus activating the expression of tylosin biosynthetic gene cluster (tyl). Meanwhile, SFB1 biosynthesis is negatively regulated by TylQ (another transcriptional repressor); the disruption of its coding gene tylQ led to increased production of SFB1, which in turn increased the yield of tylosin. Using tylQ disrupted mutant as chassis cell, co-overexpressing transcriptional activators TylR and TylS further increased tylosin yield to 3926 ± 110 mg/L, representing a 2.93-fold improvement over the wild-type strain. Since the quorum sensing signaling system can affect the biosynthesis of many secondary metabolites, thereby this strategy may also be readily applied for improving the titers of other microbial metabolites.

• SFB1 is a novel γ-butenolide-type quorum sensing signaling molecule of S. fradiae.

• SFB1 regulates the production of tylosin.

• Engineering SFB1 regulatory cascade improves tylosin production.

Improving ale or lager yeasts by conventional breeding is a non-trivial task. Domestication of lager yeasts, which are hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus, has led to evolved strains with severely reduced or abolished sexual reproduction capabilities, due to, e.g. postzygotic barriers. On the other hand, S. cerevisiae ale yeasts, particularly Kveik ale yeast strains, were shown to produce abundant viable spores (~ 60%; Dippel et al. Microorganisms 10(10):1922, 2022). This led us to investigate the usefulness of Kveik yeasts for conventional yeast breeding. Surprisingly, we could isolate heterothallic colonies from germinated spores of different Kveik strains. These strains presented stable mating types in confrontation assays with pheromone-sensitive tester strains. Heterothallism was due to inactivating mutations in their HO genes. These led to amino acid exchanges in the Ho protein, revealing a known G223D mutation and also a novel G217R mutation, both of which abolished mating type switching. We generated stable MATa or MATα lines of four different Kveik yeasts, named Odin, Thor, Freya and Vör. Analyses of bud scar positions in these strains revealed both axial and bipolar budding patterns. However, the ability of Freya and Vör to form viable meiotic offspring with haploid tester strains demonstrated that these strains are haploid. Fermentation analyses indicated that all four yeast strains were able to ferment maltose and maltotriose. Odin was found to share not only mutations in the HO gene, but also inactivating mutations in the PAD1 and FDC1 genes with lager yeasts, which makes this strain POF-, i.e. not able to generate phenolic off-flavours, a key feature of lager yeasts. These haploid ale yeast-derived strains may open novel avenues also for generating novel lager yeast strains by breeding or mutation and selection utilizing the power of yeast genetics, thus lifting a block that domestication of lager yeasts has brought about.

• Haploid Kveik ale yeasts with stable MATa and MATα mating types were isolated.

• Heterothallic strains bear mutant HO alleles leading to a novel inactivating G217R amino acid change.

• One strain was found to be POF- due to inactivating mutations in the PAD1 and FDC1 gene rendering it negative for phenolic off-flavor production.

• These strains are highly accessible for beer yeast improvements by conventional breeding, employing yeast genetics and mutation and selection regimes.

Monoclonal antibodies are extensively used as biotherapeutics for treatment of a variety of diseases. Glycosylation of therapeutic antibodies is considered a critical quality attribute as it influences the effector function, circulatory half-life, immunogenicity, and eventually efficacy and patient safety. During upstream process development, media components play a significant role in determining the glycosylation profile. In this study, we have evaluated 20 media additives (metal ions, vitamins, sugars, nucleosides). Six of the additives were shortlisted for their impact and then used to modulate the glycosylation profile of an in-house produced mAb (G0 2.38 ± 0.08%, G0F 75.58 ± 0.45%, G1F 10.07 ± 0.04%, G2F 0.54 ± 0.01%, G0F-N 5.84 ± 0.32%, sialylation 1.60 ± 0.33%, mannosylation 1.56 ± 0.39%) to achieve the glycan profile of a commercially available reference product (G0 2.49 ± 0.07%, G0F 37.83 ± 0.37%, G1F 34.77 ± 0.03%, G2F 4.87 ± 0.01%, G0F-N 2.34 ± 0.12%, sialylation 9.84 ± 0.30%, mannosylation 2.86 ± 0.29%). The proposed approach yielded us a glycan profile (G0 2.10 ± 0.07%, G0F 38.00 ± 0.49%, G1F 31.92 ± 0.09%, G2F 5.26 ± 0.54%, G0F-N 1.92 ± 0.02%, sialylation 10.28 ± 1.68%, mannosylation 3.12 ± 0.29%) that was near identical to that of the reference product. Equally importantly, other quality attributes including charge variants, aggregates, titer, and viability were not found to be significantly impacted by the addition of the additives under consideration.

• Screened 20 media additives to evaluate their effect on glycosylation of mAbs.

• Developed glycosylation indices models to evaluate the effect of various additives.

• Additive concentrations were optimized to target the reference product profile.

Butenyl-spinosyn, derived from Saccharopolyspora pogona, is a broad-spectrum and effective bioinsecticide. However, the regulatory mechanism affecting butenyl-spinosyn synthesis has not been fully elucidated, which hindered the improvement of production. Here, a high-production strain S. pogona H2 was generated by Cobalt-60 γ-ray mutagenesis, which showed a 2.7-fold increase in production compared to the wild-type strain S. pogona ASAGF58. A comparative transcriptomic analysis between S. pogona ASAGF58 and H2 was performed to elucidate the high-production mechanism that more precursors and energy were used to synthesize of butenyl-spinosyn. Fortunately, a PurR family transcriptional regulator TF00350 was discovered. TF00350 overexpression strain RS00350 induced morphological differentiation and butenyl-spinosyn production, ultimately leading to a 5.5-fold increase in butenyl-spinosyn production (141.5 ± 1.03 mg/L). Through transcriptomics analysis, most genes related to purine metabolism pathway were downregulated, and the butenyl-spinosyn biosynthesis gene was upregulated by increasing the concentration of c-di-GMP and decreasing the concentration of c-di-AMP. These results provide valuable insights for further mining key regulators and improving butenyl-spinosyn production.

• A high production strain of S. pogona H2 was obtained by ⁶⁰Co γ-ray mutagenesis.

• Positive regulator TF00350 identified by transcriptomics, increasing butenyl-spinosyn production by 5.5-fold.

• TF00350 regulated of butenyl-spinosyn production by second messengers.

L-valine holds wide-ranging applications in medicine, food, feed, and various industrial sectors. Escherichia coli, a pivotal strain in industrial L-valine production, features a concise fermentation period and a well-defined genetic background. This study focuses on mismatch repair genes (mutH, mutL, mutS, and recG) and genes associated with mutagenesis (dinB, rpoS, rpoD, and recA), employing a high-glucose adaptive culture in conjunction with metabolic modifications to systematically screen for superior phenotypes. This approach enhances the spontaneous survival rate of stress cells and facilitates the enrichment of positive mutations. Leveraging a multi-fragment seamless recombination technique, we successfully assembled the ilvBN, ilvC, ilvE, and ilvD pathway enzyme genes, transforming E. coli from a non-producer into a proficient L-valine producer capable of generating up to 6.62 g/L. Through a synergistic application of self-evolution engineering and metabolic engineering strategies, the engineered E. coli strain exhibited significantly enhanced tolerance and demonstrated heightened accumulation of L-valine.

• The innovation centered on mutated genes and mismatch repair genes

• By integrating modification with adaptive culture, a superior phenotype was attained

• Double plasmids expressing enzymes for L-valine production in E. coli were obtained

A new strategy has been developed to successfully produce the active component danshensu ex vivo. For this purpose, phenylalanine dehydrogenase from Bacillus sphaericus was combined with the novel hydroxyphenylpyruvate reductase from Mentha x piperita, thereby providing an in situ cofactor regeneration throughout the conversion process. The purified enzymes were co-immobilized and subsequently employed in batch biotransformation, resulting in 60% conversion of 10 mM L-dopa within 24 h, with a catalytic amount of NAD⁺ as cofactor. Furthermore, the bienzymatic system was implemented as a packed-bed reactor in continuous flow, achieving a conversion rate up to 80% with 60 min retention time. The process was further intensified by implementing a 48-h flow bioreaction. The biocatalysts demonstrated remarkable stability, retaining 62% of their initial activity at the end of the process. The final productivity of the isolated compound (96% purity) was calculated to be 1.84 g L⁻¹ h⁻¹ yielding a sustainable synthesis of danshensu.

• Characterization of the hydroxyphenylpyruvate reductase from Mentha x piperita

• Bi-enzymatic system in a cascade reaction to produce danshensu

• Purification and isolation of the active compound danshensu

The animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilize in silico tools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism.

• Gut microbial secondary metabolites have therapeutic and biotechnological potential

• Culture- and (meta)genome-based workflows drive gut anaerobe metabolite discovery

• Anaerobic biofoundries enable high-throughput workflows for metabolite discovery