Applied Microbiology and Biotechnology最新文献

Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). The knockdown of ATM, ATR, and PDEδ in HEK293 cells increased HIV-1 VLP titers in the supernatant by 3.4-, 2.1-, and 2.2-fold, respectively. Also, possible metabolic synergies between plasmids were investigated by statistical design of experiments (DoE), enabling us to identify the optimal production strategy, that was further demonstrated at lab-scale stirred tank bioreactor operated in perfusion, significantly increasing both VLPs specific and volumetric productivities to 8.3 × 10³ VLPs/cellxday and 7.5 × 10¹² VLPs/Lxday, respectively.

• ATM, ATR, and PDEδ knockdowns increased VLP production in HEK293 cells.

• Knockdown of ATM increased budding efficiency and extracellular vesicle concentration.

• ATM knockdown could be intensified to bioreactor scale operated in perfusion.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG⁺ recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs.

• Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus.

• Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences.

• Efficient genome editing protocol for safer molecular breeding in mushroom fungus.

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class—it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

• Transcriptomics revealed further insights into the photobiology of X. dendrorhous, specifically metabolic nodes that are transcriptionally regulated by light.

• A modular genetic part collection was developed, including 26 constitutive and regulated promoters derived from the transcriptomics of X. dendrorhous.

• Omics-to-parts can be applied to nonconventional microbes for rapid “onboarding”.

Campylobacter jejuni, a major cause of foodborne zoonotic infections worldwide, shows a paradoxical ability to survive despite its susceptibility to environmental and food-processing stressors. This resilience is likely due to the bacterium entering a viable but non-culturable state, often within biofilms, or even initiating biofilm formation as a survival strategy. This study presents an innovative application of NanoLuc bioluminescence to accurately monitor the development of C. jejuni biofilms on various substrates, such as polystyrene plates, mucin-coated surfaces, and chicken juice matrices. Introduction of NanoLuc luciferase in a pathogenic C. jejuni strain enables rapid non-invasive holistic observation, capturing a spectrum of cell states that may comprise live, damaged, and viable but non-culturable populations. Our comparative analysis with established biofilm quantification methods highlights the specificity, sensitivity, and simplicity of the NanoLuc assay. The assay is efficient and offers precise cell quantification and thus represents an important complementary or alternative method to conventional biofilm monitoring methods. The findings of this study highlight the need for a versatile approach and suggest combining the NanoLuc assay with other methods to gain comprehensive insight into biofilm dynamics.

• Innovative NanoLuc bioluminescence assay for sophisticated biofilm quantification.

• Holistic monitoring of C. jejuni biofilm by capturing live, damaged and VBNC cells.

• Potential for improving understanding of biofilm development and structure.

Azospirillum argentinense Az19 is an osmotolerant plant growth-promoting bacterium that protects maize plants from drought. In this work, we explored the role of trehalose in the superior performance of Az19 under stress. The trehalase-coding gene treF was constitutively expressed in Az19 through a miniTn7 system. The resulting recombinant strain, Az19F, did not accumulate trehalose, was affected in its capacity to cope with salt-, osmotic-, and UV-stress, and showed higher reactive oxygen species levels. Physiological alterations were also observed under normal conditions, such as increased growth in biofilms, higher motility, and decreased auxin secretion. Even so, the capacity of Az19F to colonize maize roots was not affected, either under normal or drought conditions. When inoculated in maize, both Az19 and Az19F strains promoted plant growth similarly under normal irrigation. However, unlike Az19, the trehalose-deficient strain Az19F could not improve the height, aerial fresh weight, or relative water content of maize plants under drought. Notably, Az19F triggered an exacerbated oxidative response in the plants, resulting in higher levels of antioxidant and phenolic compounds. We conclude that the role of trehalose metabolism in A. argentinense Az19 transcends stress tolerance, being also important for normal bacterial physiology and its plant growth-promoting activity under drought.

• Trehalose is required by Az19 for full tolerance to salt-, osmotic-, and UV-stress.

• A restriction in trehalose accumulation alters Az19 normal cell physiology.

• Trehalose contributes to Az19-induced maize growth promotion under drought.

Malaria, a parasitic disease caused by Plasmodium spp. and transmitted by Anopheles mosquitoes, remains a major global health issue, with an estimated 249 million cases and 608,000 deaths in 2022. Rapid and accurate diagnosis and treatment are crucial for malaria control and elimination. However, limited access to sensitive molecular tests means that microscopic examination and rapid diagnostic tests (RDT) are the most used methods in endemic areas, despite their lower diagnostic accuracy. Therefore, there is a need for developing sensitive, simple, accurate, and rapid diagnostic tools suitable for field conditions. Herein, we aimed to explore the potential of the enzymatic recombinase amplification assay (ERA® Technology) as a remote laboratory test by evaluating and validating the GENEYE® ERA Plasmodium detection kit in Brazilian endemic areas. A cross-sectional cohort study was conducted between June and August of 2023 in the Brazilian Amazon. The study enrolled 323 participants residing in three malaria-affected regions: Cruzeiro do Sul and Mâncio Lima (Acre State) and Guajará (Amazonas State). The participants were tested for malaria by microscopy, rapid diagnostic tests (RDT), nested PCR (nPCR), quantitative real-time PCR (qPCR), and ERA. The sensitivity, specificity, and predictive values were assessed using nPCR as a gold standard. Plasmodium prevalence was 21.7%, 18.8%, 19.2%, 21.7%, and 21.7% by nPCR, microscopy, RDT, qPCR, and ERA respectively. Using nPCR as the standard, qPCR, and ERA showed a sensitivity of 100%. In comparison, microscopy and RDT showed a sensitivity of 87.1% and 88.6%, a negative predictive value (NPV) of 96.56 and 96.93, and kappa values of 0.91 and 0.92, respectively. For Plasmodium falciparum, the sensitivity of qPCR and ERA was 100% while the sensitivity of microscopy and RDT was 96.9% and 93.7%, and the NPV was 99.66 and 99.32, respectively. For Plasmodium vivax, only ERA showed the same sensitivity of nPCR. The sensitivity, NPV, and kappa values were 78.85%, 97.27, and 0.87 for qPCR and microscopy, and 84.21%, 97.94, and 0.9 for RDT. The data presented here show that the GENEYE® ERA Plasmodium detection kit offers a promising alternative to traditional malaria diagnostic methods. Its high sensitivity, specificity, fast processing time, and operational simplicity position it as a valuable point-of-care diagnostic tool, particularly in resource-limited and remote malaria-endemic areas.

• GENEYE® ERA kit detects Plasmodium in under 25 min, no DNA purification needed.

• The kit matches or exceeds the compared methods in sensitivity and specificity.

• The kit is suitable for accurate testing in low-infrastructure, point-of-care settings.

Chiral diaryl alcohols, such as (4-chlorophenyl)(pyridin-2-yl)methanol, are important intermediates for pharmaceutical synthesis. However, using alcohol dehydrogenases (ADHs) in the asymmetric reduction of diaryl ketones to produce the corresponding alcohols is challenging due to steric hindrance in the substrate binding pockets of the enzymes. In this study, the steric hindrance of the ADH from Geotrichum candidum NBRC 4597 (G. candidum acetophenone reductase, GcAPRD) was eliminated by simultaneous site-directed mutagenesis of Phe56 (in the large pocket) and Trp288 (in the small pocket). As a result, two double mutants, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala, exhibited much higher specific activities towards 2-(4′-chlorobenzoyl)pyridine (4.5 μmol/min/mg and 3.4 μmol/min/mg, respectively) than the wild type (< 0.2 μmol/min/mg). In whole-cell-catalyzed asymmetric reductions of diaryl ketones, Phe56Ile/Trp288Ala significantly increased the isolated yields, which were over 90% for the reactions of most of the tested substrates. Regarding enantioselectivity, Phe56Ile/Trp288Ala and Phe56Ala/Trp288Ala, and Trp288Ala generally exhibited similar selectivity to produce (R)-alcohols with up to 97% ee.

• Phe56 in Geotrichum reductase (GcAPRD) was mutated to eliminate steric hindrance.

• Mutation at Phe56 increased enzymatic activity and expanded substrate specificity.

• Phe56Ile/Trp288Ala showed high activity and (R)-selectivity towards diaryl ketones.

In the present investigation, 13% ± 0.84 of the extracted and purified phycocyanin from Phormidium versicolor was obtained, with a purity of 0.69 following dialysis. FT-IR analysis of purified phycocyanin revealed stretching vibration peaks in the profiles of the functional groups of N–H, O–H, C = O, N–H, C = O, and C = NH⁺. The phycocyanin had a significant DPPH radical scavenging ability (IC₅₀ = 0.6 ± 0.02 mg mL⁻¹) confirmed with FRAP assay, and it exhibited microbiological activity between 1.25 and 2.5 mg mL⁻¹ against Candida albicans, Klebsiella pneumoniae, and Enterococcus faecalis. Phycocyanin showed no cytotoxic and improved the viability of HEK-293. It was added to skin cream at a rate of 6 mg g⁻¹ because of its significant yield extraction and biological activity. At 10 mg mL⁻¹, a bactericidal activity has been noted, inhibiting the growth of bacteria responsible for inflammatory skin conditions. For 60 days, the emulsion’s stability was monitored at room temperature, 25 °C, and 45 °C. The appearance of the batch kept at 45 °C was changed to beige after 7 days, while the others were kept for 15 days. Skin creams enhanced with phycocyanin were found to be stable over the course of storage at both room temperature and 25 °C, based on centrifugation stability analysis. But starting on the fifteenth day, the items kept at 45 °C were unstable. Thus, the current study’s findings are in favor of using phycocyanin as an antioxidant in cosmetic products. However, further investigation is required before using it in clinical trials.

• Phycocyanin extraction field (13%) is particularly significant compared to other cyanobacteria.

• Phycocyanin at 0.6 μg g−1 in skin cream fights bacteria in skin inflammation.

• Phycocyanin-enriched cream was stable at room temp, 25 °C, and unstable at 45 °C after day 15.

Nonstructural protein 3C, a master protease of Picornaviridae, plays a critical role in viral replication by directly cleaving the viral precursor polyprotein to form the viral capsid protein and antagonizing the host antiviral response. Additionally, 3C protease, as a tool enzyme, is involved in regulating polyprotein expression. Here, the 3C mutant gene (3Cm), fused with a small ubiquitin-like modifier (SUMO) tag at the N-terminal and featuring a mutation at position 127, was inserted into the cold-shock plasmid pCold of Escherichia coli for expression. Meanwhile, the P1-∆2A plasmid was constructed for expression in Pichia pastoris. The expressions of 3C protein and P1 precursor protein were confirmed by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (SDS-PAGE), and western blot (WB) analysis. The results showed that the wild-type 3C protease is toxic to the host, not only inhibiting protein expression but also inducing the degradation of the host. Moreover, mutation of the 127th amino acid from leucine (L) to proline (P) on the β-ribbon of 3C enhanced the overexpression capacity of 3C in E. coli while maintaining enzymatic activity. Subsequently, 100 µg P1 protein was utilized as a substrate to investigate the cleavage efficiency of 3C protease at various concentrations, temperatures, durations, and pH levels. The results showed that the target protein was cleaved when the protease reached 8 μg. We also found that the presence of the N-terminal SUMO tag did not affect the cleavage activity of 3Cm. The optimal cleavage activity was observed between 25 and 37 °C, with the peak cleavage efficiency of 89% at 30 °C for 2 h. More than 50% of the substrate was degraded within 1 h at 30 °C. Its optimal pH range is between 7 and 8. Remarkably, the P1 protein, cleaved by 3Cm protease, can further form virus-like particles (VLPs) in vitro.

• Expression and purification of toxic protein 3C protease in E. coli

• Cleavage efficiency assessment of 3C protease at various temperatures, durations, and pH

• Assembly of virus-like particles of FMDV by cleaving the precursor polyprotein in vitro

Syngas fermentation to ethanol has reached industrial production. Further improvement of this process would be aided by quantitative understanding of the influence of imposed reaction conditions on the fermentation performance. That requires a reliable model of the microbial kinetics. Data were collected from 37 steady states in chemostats and from many batch experiments that use Clostridium authoethanogenum. Biomass-specific rates from CO conversion experiments were related to each other according to simple reaction stoichiometries and the Pirt equation, with only the ratio of ethanol to acetate production remaining as degree of freedom. No clear dependency of this ratio on dissolved concentrations, such as CO or acetic acid concentration, was found. This is largely caused by the lack of knowledge about the dependency of the CO uptake rate (and hence all other rates) on the CO concentration. This knowledge gap is caused by a lack of dissolved CO measurements. For dissolved H₂, a similar gap applies. Modelling H₂ consumption adds more degrees of freedom to the system, so that more structured experiments with H₂ is needed. The inhibition of gas consumption by acetate and ethanol is partly known but needs further study.

• Set of Clostridium autoethanogenum syngas fermentation data from chemostats.

• Unstructured kinetic models can relate most biomass-specific rates to dilution rates.

• Lack of dissolved gas measurements limits deeper understanding.