Applied Microbiology and Biotechnology最新文献

Human skin wounds primarily heal through reparative wound healing without pilosebaceous units or other appendages, rather than regenerative wound healing. Hair follicle (HF) regeneration is a significant challenge for skin wound healing. The effects and underlying mechanisms of Isaria cicadae Miquel rice fermentation extract (IMFRE) remain unclear, although it has anti-inflammatory, antioxidant, and reparative effects on oxidative damage in keratinocytes. We assessed the regenerative wound healing ability of IMFRE and its related molecular mechanisms through experimental validation and network pharmacology analysis. Our findings suggest that IMFRE could be an important potential solution for regenerative wound healing of skin hair follicle by utilizing the Hippo pathway regulatory mechanism.

• IMFRE was found to significantly enhance the wound healing rate of mouse skin.

• CK15 and CD34 were significantly increased by high-dose IMFRE intervention.

• IMFRE could inhibit EGFR, GPCR, and Integrin expression.

Filamentous fungi or mycelia are a valuable bioresource to produce several biomolecules and enzymes, especially because of their biodegradation potential and for their key role of enablers of a circular bioeconomy. Filamentous fungi can be grown in submerged cultivation to maximise the volumetric productivity of the bioprocess, instead of using the more established and time-consuming solid-state cultivation. Multicellular mycelia are sensitive to shear stresses induced by mechanical agitation, and this aspect greatly affects their morphology in submerged cultivation (pelletisation) and the connected volumetric productivity. An efficient compromise is the growth of filamentous fungi in airlift bioreactors (ALR) where the volumetric oxygen transfer (K_La) is optimal, but the shear stress is reduced. In this review, we critically analysed the advantages and disadvantages of ALR-based cultivation of filamentous fungi, comparing these bioreactors also with stirred tank reactors and bubble column reactors; we focused on scientific literature that highlights findings for the cultivation of filamentous fungi for both the production of enzymes and the production of myco-biomass in ALR; we included studies for the control of the pelletisation of the fungal biomass in batch and semi-continuous cultivation, highlighting the interlinked hydrodynamics; finally, we included studies regarding the modifications of ALR in order to enhance filamentous fungi production.

• ALR are efficient for batch and prolonged continuous cultivation of filamentous fungi.

• ALR show both optimal gas hold-up and K_La with an airflow that has high superficial velocity and critical bubble diameter (1–6 mm).

• Suspended mycelia aggregates (pellet) maintain a fluidised motion in ALR if their size/density can be controlled.

Immune checkpoint inhibitors (ICIs) have significantly advanced the field of cancer immunotherapy. However, clinical data has shown that many patients have a low response rate or even resistance to immune checkpoint inhibitor alone. The underlying reasons for its poor efficacy include the deficiency of immune infiltration and effective CD28/CD80 costimulatory signal in tumor. Discoidin domain receptor 1 (DDR1) has been reported to be negatively related to immune cell infiltration in tumors. Herein, we constructed a soluble fusion protein using CD80, the natural ligand of CD28, in combination with DDR1 inhibitor. Our results demonstrated that CD80-Fc effectively activated T cells and inhibited tumor growth in vivo, even in tumors with poor efficacy of ICIs. Importantly, CD80-Fc fusion protein had a milder affinity against the targets which suggested a potential higher safety than CD28 agonists. Further, in order to promote tumor immune infiltration, we attempted to combine CD80-Fc fusion protein with DDR1 inhibitor for treatment. Our results indicated that using CD80-Fc fusion protein along with DDR1 inhibitor significantly promoted T cell infiltration in tumor microenvironment and more strongly inhibited tumor growth. Therefore, the combination use of CD80 fusion protein and DDR1 inhibitor could become an effective tumor immunotherapy strategy, potentially benefiting a larger number of patients.

• We successfully constructed, expressed, and purified the recombinant CD80-Fc fusion protein

• We demonstrated that CD80-Fc fusion protein has good safety and anti-tumor activity

• We demonstrated that using CD80-Fc fusion protein along with DDR1 inhibitor can significantly promote immune infiltration of T cells in tumor microenvironment and more strongly inhibit tumor growth

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies, efficient and scalable expansion procedures must be developed. For other adherent human cell types, the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study, a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings, indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion (({N}_{s1u})), thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics, computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner, improved process understanding allowed an expansion factor of ≈ 26 to be achieved, yielding more than 3 × 10⁹ cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability, identity, and differentiation potential throughout cultivation.

• ({N}_{s1u}) can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors

• Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion

• Perfusion is advantageous and supports the cultivation of hiPSCs

Pollutant-derived risks to human and environmental health are exacerbated by slow natural attenuation rates, often driven by pollutant toxicity to microorganisms that can degrade them or limitations to the ability of microorganisms to metabolise them. This review explores mechanisms employed by bacteria to protect themselves from pollutant toxicity in the context of the evolution of pollutant-degrading abilities. The role of promiscuous enzymes in pollutant transformation is subsequently reviewed, highlighting the emergence of novel metabolic pathways and their transcriptional regulation in response to pollutant exposure, followed by the gene transcription regulation to optimise the cellular component synthesis for adaptation on the novel substrate. Additionally, we discuss epistatic interactions among mutations vital for this process both at macromolecular and at cellular levels. Finally, evolutionary constraints towards enhanced fitness in the context of pollutant degradation are considered, the constraints imposed by the epistasis from mutations on both enzyme level and cellular level, concluding with challenges and emerging opportunities to develop sustainable contaminated site remediation technologies.

•Pollutants can exert toxicity on cellular membrane, enzyme and gene transcription.

•Bacteria can patch promiscuous enzymes into novel pathway to degrade pollutants.

•The evolution trajectory is constrained by epistasis from mutations on enzyme and cellular level.

The evolution of the bacterial community in an up-flow anaerobic reactor with silicone support, continuously fed with pure glycerol (day 0–293) and crude glycerol (day 294–362), was studied. Biomass from a former glycerol-degrading reactor was used as inoculum. The maximum yield and productivity of 1,3-propanediol (PDO) (0.62 mol.mol-gly⁻¹ and 14.7 g.L⁻¹.d⁻¹, respectively) were obtained with crude glycerol. The inoculum had low diversity, with dominance of Lactobacillus (70.6%) and Klebsiella/Raoultella (23.3%). After 293 days of feeding with pure glycerol, the abundance of both taxa decreased to less than 10%, either in the attached biofilm or in the biomass growing in suspension. The genus Clostridium and members of the Ruminococcaceae family then became the majority. In the period after feeding with crude glycerol, Clostridium remained as the majority genus in the biofilm; however, it was partially replaced in the suspension by Eubacterium, a non-glycerol degrading bacterium. This fact, together with the prevalence of other glycerol-degrading genera in the biofilm, such as Caproiciproducens and Lactobacillus, indicated that the bacteria attached to the silicone support were responsible for converting glycerol into 1,3-PDO. Therefore, to increase the 1,3-PDO productivity, a good approach would be to maximize the amount of reactor support. Other genera that do not degrade glycerol, such as Anaerobacter and Acetomaculum, thrived at the expense of cellular decay material. The Canonical Correspondence Analysis demonstrated that the origin of glycerol is an important variable to consider during the bioreactor operation for producing 1,3-PDO, while the glycerol loading rate is not.

• Microbial community showed robustness in a range of operational conditions.

• A significantly high 1,3-propanediol yield can be achieved using crude glycerol.

• The attached biofilm appears to be key to the high production of 1,3-propanediol.

The lignocellulosic biomass (LCB) is an attractive, sustainable, and environmentally friendly alternative to fossil sources to produce biofuel, biomaterials, and biochemicals. However, its recalcitrant and heterogenous structure challenges its biodegradation and valorization. The gut microbiome of soil invertebrate species has emerged as a rich source of LCB-degrading bacteria and enzymes in terrestrial ecosystems. The primary objective of this investigation was to identify the bacterial communities within the Porcellio dilatatus gut (Crustacea: Isopods), to obtain enriched cultures, and to identify bacterial isolates with LCB-degrading activity. A total of 112 enriched cultures were screened, all exhibiting xylanolytic activity. Among them, 94 displayed cellulolytic activity, 30 showed chitinolytic activity, and 21 demonstrated ligninolytic activity. Four enriched cultures were selected, and 128 bacteria with cellulolytic, xylanolytic, chitinolytic, or ligninolytic activity were isolated and taxonomically classified. The obtained results reinforce the potential of bacterial communities within the digestive tract of soil invertebrates as a valuable source of lignocellulose-degrading microorganisms. Thirty-one isolates underwent in-depth enzymatic characterization, and five were selected and functionally evaluated. An artificial bacterial consortium was constructed to assess the potential benefits of using consortia to achieve enhanced LCB degradation. The positive results of this proof-of-concept artificial consortium (PdG-AC) can be used in future applications and is a valuable tool for enzymatic and microbial consortia engineering by, e.g., changing growth conditions for enhanced LCB-degrading abilities.

• The gut microbiome of Porcellio dilatatus was characterized.

• Porcellio dilatatus gut hosts many lignocellulose-degrading bacteria.

• Developed an artificial bacterial consortium for lignocellulose degradation.

One strategy for CO₂ mitigation is using photosynthetic microorganisms to sequester CO₂ under high concentrations, such as in flue gases. While elevated CO₂ levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO₂ concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m⁻² s⁻¹ and a gas phase containing 30% CO₂ was 2.7-fold lower compared to 4% CO₂. Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO₂. Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO₂, while repression of key regulators of photosynthesis (pmgA) and CO₂ capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO₂. Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO₂ and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO₂ compared to the WT. These findings suggest that enhanced fitness under very high CO₂ involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO₂. This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications.

• Synechocystis growth was inhibited under very high CO₂.

• Inhibition of growth under very high CO₂ was light dependent.

• Repression of photosynthesis genes improved growth under very high CO₂.

The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain.

• Quantification of heterologous promoters enables rational choice for genetic engineering.

• CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans.

• Conjugation protocol and type II REase deletion enhance genetic accessibility.