Applied Microbiology and Biotechnology最新文献

The phylum Planctomycetota is changing our understanding of bacterial metabolism, driving critical biogeochemical processes through the transformation of complex polymeric substrates into valuable bioactive compounds. Sophisticated methods for cultivation, genome sequencing and genetic strain engineering developed in the last two decades have stimulated detailed studies on cell propagation, metabolic capabilities and potential applications of phylum members beyond the mere isolation and characterization of novel taxa. This review synthesizes recent advances in understanding the Planctomycetota physiology with a focus on the degradation of phototroph-derived polysaccharides, anaerobic ammonium oxidation (anammox) and biosynthesis of secondary metabolites. New data especially collected over the last 5 years justifies more intensive research of the yet uncharacterized pathways of substrate uptake and utilization, as well as genome mining-assisted bioprospection to exploit the phylum's chemical repertoire.

• Planctomycetes can degrade high-molecular-weight sugars produced by algae

• Anaerobic ammonium oxidation (anammox) is used in technical applications

• The first secondary metabolites were discovered in the last 5 years

Enzymatic depolymerization of seaweed polysaccharides aroused great interest in the production of functional oligosaccharides and fermentable sugars. Alginate lyase Alg0392, a potential novel member of the polysaccharide lyase PL17 family, was cloned from Alteromonas sp. A1-6. The enzymatic properties, kinetic parameters, and hydrolytic products of Alg0392 were systematically characterized. Especially, the recombinant enzyme Alg0392 showed excellent tolerance to organic reagents. When treated with 5 mmol/L of TritonX-100 or 20%(v/v) of methanol, its relative enzyme activity could be maintained at more than 70%. The recombinant enzyme has a substrate preference for poly (β-D-mannuronic acid). The products of alginate hydrolysis catalyzed by Alg0392 are mainly monosaccharides, disaccharides, and trisaccharides. The products generated by the degradation of polymannuronic acid (polyM) are mainly monosaccharides. So Alg0392 is a polymannuronate cleaving enzyme. It has excellent organic solvent-tolerance and possesses both endo- and exo-glycosidase activities towards alginate. These unique properties make the recombinant enzyme Alg0392 more advantageous for the future industrial production of biofuels and the preparation of alginate oligosaccharides.

• Alg0392 is a bifunctional alginate lyase with exolytic and endolytic cleavage activity.

• Alg0392 exhibits excellent organic solvent tolerance.

• The enzymatic hydrolysates of Alg0392 exhibit antioxidant activity.

The industrial production of menaquinone-7 (MK-7) by Bacillus subtilis has been historically constrained by significant challenges in bioprocess efficiency. To address these limitations, we explored an innovative immobilization strategy utilizing a porous thin-film hydrogel system. Specifically, we developed a novel porous thin-film PVA + B@Ca hydrogel immobilization method that fundamentally transforms cell encapsulation and fermentation dynamics. The comparison between PVA + B@Ca hydrogel immobilized cells and free cells in fermentation demonstrated a significant increase in MK-7 yield from 32.76 ± 1.92 to 48.33 ± 2.92 mg/L, as well as a reduction of the fermentation duration from 48 to 24 h. Additionally, the immobilized cells demonstrated good stability during continuous fermentation, resulting in a space–time yield of MK-7 that increased to 2.0 mg/L·h, which was five times higher than that achieved with free-cell fermentation. Mechanistic insights revealed through microscopic analysis highlight the transformative nature of the hydrogel immobilization: The PVA + B@Ca hydrogel’s porous structure creates a protective microenvironment that mitigates cellular stress and maintains optimal metabolic conditions. These findings represent a paradigm shift in understanding cellular immobilization, demonstrating how strategic encapsulation can fundamentally enhance MK-7 fermentation biotechnology.

• A novel hydrogel immobilization method was developed for MK- 7 production.

• The use of immobilized cells gave a fivefold improvement in the space–time yield.

The heat shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in Escherichia coli. However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered E. coli strain (JU15), designed for d-lactic acid production, to enable cellular growth and fermentation of glucose at 45 °C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45 °C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved a controlled stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures.

• The temperature upshift response of a thermally adapted strain in continuous culture was studied for the first time.

• Genomic analyses revealed the presence of a double point mutation in the spoT gene.

• The thermally adapted strain maintained underexpression of the spoT gene at high temperatures.

• Supplementation of 0.15 g/L of hydrolyzed protein favored thermal adaptation at 45 °C.

Administering beneficial bacteria as probiotics to restore the intestinal microbiota and its metabolic functions, such as butyrogenesis, is a promising treatment strategy in ulcerative colitis (UC). This study aimed to investigate the effect of a combination of probiotics, consisting of the lactic acid bacterium Weizmannia coagulans SANK70258 and the lactate-utilizing butyrate-producing bacteria Anaerostipes caccae or Clostridium butyricum, on the colonic environment using an in vitro colonic microbiota culture model with fecal inoculums from seven patients with UC. Co-inoculated W. coagulans and A. caccae neither inhibited each other’s growth nor significantly affected the relative abundance of other bacterial species; however, the growth of W. coagulans was significantly inhibited when co-inoculated with C. butyricum. The relative abundance of pro-inflammatory bacteria (Escherichia sp. and unclassified Enterobacteriaceae) and Bifidobacterium spp. significantly decreased in W. coagulans-C. butyricum co-inoculated cultures. Inoculation with any of the probiotics alone did not increase butyrate production, whereas co-inoculation of W. coagulans with A. caccae or C. butyricum significantly increased the butyrate levels. Overall, the results suggested that W. coagulans and lactate-utilizing butyrate-producing bacteria in combination have synergistic effects through cross-feeding and can effectively restore butyrogenesis in the colonic environment of patients with UC.

• Effects of probiotics were evaluated using in vitro microbiota model of UC colon.

• W. coagulans and lactate-utilizing butyrate producers have synergistic effects.

• Co-inoculation of W. coagulans with A. caccae or C. butyricum enhanced butyrogenesis.

The use of fetal bovine serum (FBS) in cell culture applications causes high costs and unacceptable animal suffering when FBS is extracted from fetal calves. Despite efforts, the exact composition of FBS still remains partially unresolved. Native proteins in FBS, such as growth factors, and their binding to cell receptors seem to be crucial for cell proliferation and differentiation. Recently, algal extracts with high protein content were considered to reduce the FBS demand. Algae extracts yielded promising results as growth serum in mammalian cell culture. Nevertheless, the dependence on residual FBS and the undefined composition of algae extracts are challenges. In this study, we aimed to yield highly concentrated extracts of native proteins from mixotrophically grown Galdieria sulphuraria to replace FBS in mammalian cell culture. Crude extracts and native proteins were concentrated by ammonium sulfate precipitation, and all extracts underwent heat inactivation (HI) for selective protein inactivation. The remaining proteins’ native conformation was verified by enzyme activity assays. All extracts were used to replace FBS during the cultivation of Chinese hamster ovary (CHO) cells, and proliferation was tested. We found that G. sulphuraria crude and protein extracts depended on HI to promote CHO cell growth to a similar extent as FBS. CHO cells grown with 5% or 10% heat-treated algal extracts had a relative proliferation of 260 to 230% compared to FBS controls with 210% and 300%, respectively. We anticipate our findings will help replace FBS in mammalian cell culture, increasing sustainability and consumer acceptance.

The global interest in fatty acids is steadily rising due to their wealth of industrial potential ranging from cosmetics to biofuels. Unfortunately, certain fatty acids, such as monounsaturated lauric acid with a carbon atom chain length of twelve (C12 fatty acids), cannot be produced cost and energy-efficiently using conventional methods. Biosynthesis using microorganisms can overcome this drawback. However, rewiring a microbe’s metabolome for increased production remains challenging. To overcome this, sophisticated genome-wide metabolic network models have become available. These models predict the effect of genetic perturbations on the metabolism, thereby serving as a guide for metabolic pathways optimization. In this work, we used constraint-based modeling in combination with the algorithm Optknock to identify gene deletions in Escherichia coli that improve C12 fatty acid production. Nine gene targets were identified that, when deleted, were predicted to increase C12 fatty acid titers. Targets play a role in anaplerotic reactions, amino acid synthesis, carbon metabolism, and cofactor-balancing. Subsequently, we constructed the corresponding (combinatorial) deletion mutants to validate the in silico predictions in vivo. Our highest producer (ΔmaeB Δndk ΔpykA) reaches a titer of 6.7 mg/L, corresponding to a 7.5-fold increase in C12 fatty acid production. This study demonstrates that model-guided metabolic engineering is a useful tool to improve C12 fatty acid production.

•Escherichia coli as a promising biofactory for unsaturated C12 fatty acids.

•Optknock to identify non-obvious gene deletions for increased C12 fatty acids.

•7.5-fold higher C12 fatty acid production achieved by deleting maeB, ndk, and pykA.

A wide array of pharmaceutical and industrial products available in today’s market stems from bioreactors. Meeting the escalating demand for these products necessitates significant enhancements in biotechnological processes. This study focuses on developing cost-effective scaffolds designed explicitly for use within bioreactors, employing commonly used polymers such as gelatin and collagen. Bacterial proliferation assays involving Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were conducted to assess the effectiveness of these scaffolds. The scaffolds were produced by electrospinning polymeric solutions with varying concentrations of gelatin and collagen and were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. Results revealed that scaffolds with 15% gelatin increased the 24-h proliferation of S. aureus, P. aeruginosa, and E. coli by 52%, 35%, and 20%, respectively. In the case of E. coli, scaffolds with lower gelatin concentrations (1–10%) were more effective, leading to 35–55% proliferation growth. These findings highlight the potential application of gelatin/collagen scaffolds in fabricating industrial products derived from these bacteria.

• GEL/COL fibers boost S. aureus growth by 128%

• Offers scalable biotech applications

The agro-food chain produces an impressive amount of waste, which includes not only lignocellulosic biomass, but also plastic, used for both protective films and packaging. Thanks to advances in enzymatic hydrolysis, it is now possible to imagine an upcycling that valorizes each waste through microbial fermentation. With this goal in mind, we first explored the ability of the oleaginous red yeast Rhodotorula toruloides to catabolize ethylene glycol (EG), obtained by the hydrolysis of polyethylene terephthalate (PET), in the presence of glucose in batch bioreactor experiments. Secondly, we focused on the physiology of EG catabolism in the presence of xylose as a sole carbon source, and in a mixture of glucose and xylose. Our results show that EG is metabolized to glycolic acid (GA) in all tested conditions. Remarkably, we report for the first time that the consumption of EG improves xylose bioprocess, possibly alleviating a cofactor imbalance by regenerating NAD(P)H. Consumption of EG in the presence of glucose started after the onset of the nitrogen limitation phase, while no significant differences were observed with the control; a 100% mol mol⁻¹ yield of GA was obtained, which has never been reported for yeasts. Finally, a putative EG oxidative pathway was proposed by in silico analyses supported with the existing omics data. Our results propose R. toruloides as a promising candidate for the production of GA from EG that could be exploited simultaneously for the sustainable production of microbial oils from residual hemicellulosic biomasses.

• Ethylene glycol (EG) is not assimilated as a carbon source by Rhodotorula toruloides

• With glucose, EG is oxidized to glycolic acid (GA) with a yield of 100% (mol mol⁻¹)

• With xylose, EG to GA is associated with improved growth and xylose uptake rate