Journal of Industrial Microbiology & Biotechnology最新文献

The thiopeptide GE2270A is a clinically relevant, ribosomally synthesised, and post-translationally modified peptide naturally produced by Planobispora rosea. Due to the genetically intractable nature of P. rosea, heterologous expression is considered a possible route to yield improvement. In this study, we focused on improving GE2270A production through heterologous expression of the biosynthetic gene cluster (BGC) in the model organism Streptomyces coelicolor M1146. A statistically significant yield improvement was obtained in the S. coelicolor system through the data-driven rational engineering of the BGC, including the introduction of additional copies of key biosynthetic and regulatory genes. However, despite our best efforts, the highest production level observed in the strains generated in this study is 12× lower than published titres achieved in the natural producer and 50× lower than published titres obtained using Nonomuraea ATCC 39727 as expression host. These results suggest that, while using the most genetically amenable strain as host can be the right choice when exploring different BGC designs, the choice of the most suitable host has a major effect on the achievable yield and should be carefully considered. The analysis of the multiomics data obtained in this study suggests an important role of PbtX in GE2270A biosynthesis and provides insights into the differences in production metabolic profiles between the different strains. One Sentence Summary: Data-driven rational engineering of Streptomyces coelicolor for heterologous production of the thiopeptide antibiotic GE2270A resulted in increased production but encountered unexpected challenges compared to production in the natural producer or the alternative host Nonomuraea ATCC 39727.

Achieving high-purity biohydrogen (Bio-H₂) production necessitates the suppression of hydrogenotrophic methanogens, as their activity can impede hydrogen yield. Various inoculum pretreatments have been employed to suppress methane-producing microorganisms; however, these methods can negatively impact the enzymatic activity of hydrogen-producing microorganisms, thereby reducing hydrogen production. To address this challenge, this research investigates a novel approach to enhance Bio-H₂ production by activating microbial enzymes using magnetite Fe₃O4-doped carbonized nanoparticles (NPs) derived from vegetable leaves (VLCFe₃O4-NPs) within a coupled dark fermentation-microbial Electrohydrogenesis system. Characterization results revealed that VLCFe₃O4-NPs exhibited cubic and spherical morphologies, with a small diameter of 1 ± 100 nm and a mean crystallite size of 38.1 nm, indicating high purity. Fermentation tests investigated the impact of different nanoparticle dosages on Bio-H₂ generation, hydrogenase gene expression (Fe-Fe and Ni-Fe), and microbial biodiversity. Bio-H₂ production significantly improved with 500 mg/L VLCFe₃O4-NPs, yielding 1.2-fold more than the control group, while even a low dose of 25 mg/L resulted in a 0.22-fold increase. Relative gene expression analysis using qPCR and the 2-ΔΔCT method demonstrated a 30-fold increase in Cbei 1773 (Fe-Fe hydrogenase) and a 23-fold increase in hucL (Ni-Fe hydrogenase) gene expression, along with an increase in 16S rDNA. Additionally, the abundance of biohydrogen-producing bacteria, Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_11, increased by 14.3% and 11.1%, respectively, compared to 4.9% and 3.9% in the control group. This research indicates that VLCFe₃O4-NPs offer an eco-friendly solution for boosting biohydrogen production within microbial electrohydrogenesis cells with dark fermentation systems, thereby supporting sustainable bioenergy generation. One-sentence summary: Green carbonized nanoparticles Fe3O4-doped have been shown to turn on the genes of bacteria (Fe-Fe and Ne-Fe) and increase the biodiversity of microbes, both of which are important for biohydrogen production.

Agarwood is a highly valuable non-timber forest product mainly derived from the Aquilaria genus, widely traded in the perfumery, religious items, and traditional medicine industries. Naturally, agarwood forms within the xylem as part of the tree's defense mechanism against environmental stressors and microbial infection. The escalating demand for agarwood has led to the overexploitation of Aquilaria species, with some now classified as critically endangered. Despite advancements in artificial induction methods for sustainable agarwood supply, the intricate links between physiological and molecular mechanisms governing its formation remain poorly understood. This review addresses these knowledge gaps by examining the interplay between morphological changes in xylem structure during tylose formation and molecular alterations, particularly the biosynthesis of 2-(2-phenylethyl)chromones (PECs), key compounds in agarwood. Additionally, it integrates findings from multi-omics approaches including genomics, transcriptomics, proteomics, and metagenomics to reveal how secondary metabolite biosynthesis, including PECs and terpenes, is regulated across various Aquilaria species, regions, and induction techniques. The role of microbial communities, particularly endophytes such as Fusarium, in regulating agarwood formation is also discussed, emphasizing their involvement in both natural and artificial induction strategies. Furthermore, this review explores the role of reactive oxygen species in mediating morphological and biochemical defense responses, alongside the functions of transcription factors (TFs), protein kinases, and signaling molecules in balancing defense and growth. However, the crosstalk between key genes such as chalcone synthases, MAPK, cytochromes, NADPH oxidases, TFs, and miRNAs require further study to fully understand the complex defense mechanisms in Aquilaria trees. Overall, this review aims to bridge the current knowledge gaps by linking morphological and biochemical changes in agarwood formation, particularly PEC biosynthesis, while proposing metabolite engineering using microbial hosts as a promising tool for sustainable and technology-driven agarwood production. One-Sentence Summary: This review explores the physiological and molecular processes behind agarwood formation in Aquilaria malaccensis, highlighting the roles of tyloses, microbial interactions, secondary metabolite biosynthesis particularly 2-(2-phenylethyl)chromones and the integration of biotechnology for sustainable production and metabolic engineering.

Biocides are crucial in industrial applications to minimize microbial growth and prevent product spoilage. Water-based construction coatings are susceptible to microbial contamination during manufacturing and storage and this adversely impacts product properties, reduces shelf-life, and leads to substantial commercial losses. The future trend to lower the biocide concentrations in water-based coatings raises concerns about the emergence of biocide-resistant microbes. This study aims to identify and characterize the biocide-resistant microbe isolated from construction water-based coating materials to better understand its mechanisms of resistance. A total of 63 samples were collected from spoiled products, raw materials, and water from a manufacturing facility, and Pseudomonas oleovorans P4A were identified in all biocides-treated samples. A comparison between a P. oleovorans reference strain, 1045, and the P4A isolate revealed distinct colony morphology, growth rate and sensitivity to biocides and antibiotics. The P4A isolate was threefold more resistant to 5-chloro-2-methyl-isothiazolin-3-one and 1.5-fold more resistant to benzothiazolinone (BIT) compared to the reference strain. Conversely, it was 1.4-fold more sensitive to methylisothiazolinone (MIT) compared to the reference strain. No cross-resistance to antibiotics was observed. Metabolomic analysis using liquid chromatography combined with high-resolution mass spectrometry of lipids and polar metabolites showed that P4A had a relatively higher amount of lipids, while 1045 had a relatively higher amount of polar metabolites identified. A significant difference in lipid composition, specifically in diacylglycerol, phosphatidic acid, phosphatidylcholine, and phosphatidylserine was observed between P. oleovorans strains 1045 and P4A. These distinctions highlight increased lipid metabolism in P. oleovorans P4A and this may contribute to its adaptation to biocides. Microbial resistance can directly affect the effectiveness of these products, leading to an increased need for frequent maintenance and replacement, safety concerns, and environmental implications. One-Sentence Summary: Biocide-resistant Pseudomonas oleovorans isolate exhibited reduced growth rate and increased lipid levels relative to the reference strain.

β-Carotene, a key provitamin A carotenoid, is widely used as an antioxidant and natural pigment. Due to animals' inability to synthesize carotenoids, dietary sources are essential. This study utilized low-cost sorghum syrup for β-carotene production via Rhodotorula glutinis fermentation. Bioprocess optimization using response surface methodology was conducted in shake flasks, then scaled to 300 mL and 7 L fermentations. The optimized medium (9.18% sorghum syrup, 0.96% yeast extract, 0.07% KH₂PO4, 0.13% (NH4)₂SO4, 0.42% MgSO4) yielded a predicted 1 003 µg/g β-carotene after 10 days. Scale-up achieved 1 153 µg/g (300 mL) and 1 753.33 µg/g (7 L). Nutritional analysis showed the presence of chelated minerals, vitamins, proteins, and glucosamine, enhancing biomass value. These results highlight sorghum syrup as an effective, sustainable substrate for β-carotene production with applications in food, feed, and nutraceutical sectors. One Sentence Summary: Using sorghum syrup as a low-cost substrate, we optimized β-carotene production by Rhodotorula glutinis via response surface methodology and validated at 7 L scale (up to 1,753 μg/g), while profiling the nutrient-dense biomass (protein, minerals, glucosamine) for food/feed applications.

Industrial bioprocess optimization has significantly increased the productivity of biomass and biologics in upstream production. Such process improvement in fermentation often translates to challenges in recovering intracellularly expressed recombinant proteins due to increased matrix complexity, resulting in a higher performance burden in midstream. Tangential flow filtration (TFF) is a popular industry standard for buffer exchange and protein separation from cellular debris. However, due to variations in the physicochemical properties of recombinant proteins, solutions for E. coli-based protein clarification remain challenging and often necessitate extensive exploration and process optimization. With growing options in filtration-based technologies, the identification of a near-universal clarification platform is desirable to accelerate bioprocess development overall. In this study, three TFF modalities, hollow fibre (HF), flat-sheet cassette (CAS), and vibro membrane filtration (VMF), were assessed in parallel to evaluate their clarification performance for three E. coli recombinant proteins with different biochemical properties. Reverse phase liquid chromatography data showed target protein recovery was uniformly higher for VMF than HF at equivalent loading. Cell density and lysate protein load were comparable for HF and VMF, and lower for CAS. These results support the choice of VMF and HF as easily optimized and operated TFF modalities for clarification of recombinant protein from complex crude bacterial matrix, where either can be efficiently performed with ease and minimum supervision. Both TFF applications were successfully demonstrated in primary cell harvest, cell wash and cell lysate clarification, for E. coli-based recombinant proteins.

One-sentence summary: High-density E. coli microfiltration and lysate clarification were tested for three diverse recombinant proteins, where hollow fibre and vibro membrane filtration outperformed flat sheet cassette in terms of process time, suspended solid loading, and target protein recovery.

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2-4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 103 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6-7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (Pclo1313_1194) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass. One-Sentence Summary: A highly efficient site-specific recombination system was created for Clostridium thermocellum, which enabled the rapid characterization of a large collection of genetic parts for controlled gene expression.

This study assesses a plant-based Soytone Medium as an alternative to the animal-derived standard de Man, Rogosa, and Sharpe (MRS) Broth for the cultivation of Lactobacillaceae. The application focuses on five isolates that have shown probiotic potential for bacterial vaginosis treatment. Cultivation was performed in 300 mL bench-scale bioreactors, monitored for cell density, pH, lactate production, and glucose consumption. The media's carbon and nitrogen concentrations and costing were quantified. Though the medium's carbon concentrations were identical, the Soytone Medium had a higher carbon-to-nitrogen ratio than MRS (8.1 vs. 6.6). Four strains achieved higher cell densities and maximum specific growth rates in the Soytone Medium. The greatest benefit was shown for L. crispatus 70.6PA, which had a 45% higher final cell density. A cost analysis showed that the Soytone Medium was 44% cheaper than MRS Broth. It was thus confirmed that the proposed plant-based Soytone Medium is a viable and less expensive alternative for Lactobacillaceae cultures in which exposure to animal products was also avoided. One-Sentence Summary: This study presents a plant-based Soytone Medium as a cost-effective alternative to standard MRS Broth for the high-density biomass cultivation of vaginal Lactobacillaceae, demonstrating enhanced growth performance and supporting their application in probiotic-based treatment of bacterial vaginosis in LMICs like South Africa.

Phosphopantetheinyl transferases (PPTases) play an essential role in primary and secondary metabolism. These enzymes facilitate the posttranslational activation of acyl carrier proteins (ACPs) central to the biosynthesis of fatty acids and polyketides. Modulation of ACP-PPTase interactions is a promising approach to both increase access to desired molecular outputs and disrupt mechanisms associated with disease progression. However, such an approach requires understanding the molecular principles that govern ACP-PPTase interactions across diverse synthases. Through a multiyear, course-based undergraduate research experience (CURE), 17 ACPs representing a range of putative type II polyketide synthases, from actinobacterial and nonactinobacterial phyla, were evaluated as substrates for three PPTases (AcpS, Sfp, and vulPPT). The observed PPTase compatibility, sequence-level analyses, and predictive structural modeling suggest that ACP selectivity is driven by amino acids surrounding the conserved, modified serine on the ACP. We propose that vulPPT and Sfp interactions with ACPs are driven primarily by hydrophobic contacts, whereas AcpS may favor ACPs that exhibit high net-negative charge density, as well as a broad electronegative surface distribution. Furthermore, we report a plausible, hitherto unreported hydrophobic interaction between vulPPT and a conserved ACP crease upstream of the invariant serine, which may facilitate docking. This work provides a catalog of compatible and incompatible ACP-PPTase partnerships, highlighting specific regions on the ACP and/or PPTase that show promise for future strategic engineering and inhibitor development efforts. One-Sentence Summary: Seventeen acyl carrier proteins from diverse type II polyketide synthases were evaluated for their compatibility with three phosphopantetheinyl transferases; results, along with sequence level-analyses and predictive structural modeling, reveal specific regions that can guide future strategic engineering efforts.

In the last decade, the global warming and plastic pollution issue have driven research on developing a more sustainable platform for chemicals production from alternative feedstocks. Ethylene glycol (EG), a monomer of polyethylene terephthalate (PET) plastic, has a potential to become a renewable substrate for microbial production of value-added chemicals. This study presents a biotransformation platform using Corynebacterium glutamicum to produce glycolic acid (GA) from EG. C. glutamicum was engineered to express a heterologous EG oxidation pathway. Subsequent promoter engineering yielded strain FA4, producing 10.6 g/L GA from EG in 48 h. Implementation of a two-stage biotransformation strategy using resting cells further enhanced the GA production, reaching a cumulative GA titer of 98.8 g/L after a 72-h production. Finally, applying this platform to a simulated EG mixture from PET-degradation achieved a cumulative GA titer of 67.3 g/L over 72 h, highlighting the potential for valorizing plastic waste through this biotransformation platform. These findings establish C. glutamicum as an efficient biotransformation chassis for sustainable GA production from EG and offer a promising route for PET waste valorization into value-added chemicals. One Sentence Summary: High yield production of GA from EG.