Microbial Cell Factories最新文献

Background: Non-invasive spectroscopic methods are increasingly valued in life sciences, where preserving the native state of biomolecules is essential for accurate interpretation. Traditional analyses of microbial compounds typically involve solvent-based extraction and chromatographic separation processes, which are time consuming, damaging to samples, and can alter biomolecular structures of complexes. To overcome these limitations, we developed a novel spectroscopic workflow for direct metabolite monitoring in microbial cells.

Results: In this study, we established a combined spectroscopic methodology that allows direct pigment and polyhydroxyalkanoates (PHAs) analysis in complex biological samples without requiring chemical extraction procedures. The UV-Vis spectroscopy technique using an integrating sphere enables direct monitoring of pigments even in turbid whole cell suspensions, providing detailed fingerprints of bacteriochlorophyll a and carotenoids in their natural environment. Together, these techniques provide consistent information about cellular composition. Using the photosynthetic bacterium Rhodospirillum rubrum as a model organism, we demonstrate that our combined spectroscopic approach can resolve pigment states, reveal intracellular PHA content and crystallinity, and measure carotenoids and bacteriochlorophylls directly in native whole cell suspensions. Furthermore, advanced data processing provided an improved interpretation of pigment and PHA states in different cellular forms.

Conclusions: This innovative combination of spectroscopic techniques reduces sample manipulation, preserves cellular integrity and provides rapid, precise, and environmentally friendly analysis of microbial metabolites in their natural physiological conditions. The demonstrated workflow is broadly applicable to biological samples where maintaining biomolecular integrity is crucial, and it has strong potential for applications in process analytical technology and industrial biotechnology.

Background: Microalgal biodiesel is a key fossil fuel alternative, but enhancing lipid accumulation via single metabolic gene overexpression is often ineffective. Transcription factor engineering overcomes this by coordinating multiple metabolic pathways. To address the unexplored role of LEC1-type transcription factors in diatoms, we engineered the euryhaline and psychrotolerant biodiesel candidate diatom Phaeodactylum tricornutum through heterologous expression of the key plant lipid regulators AtLEC1 and AtL1L.

Results: Codon-optimized genes driven by the endogenous fcpA promoter were integrated into the nuclear genome, with regulators localization confirmed in the nucleus. Crucially, AtL1L transformants exhibited significant redirection of carbon flux from carbohydrates toward lipids, evidenced by lipid content increasing to 29.8%-33.9% of dry weight compared to 20.9% in wild-type controls while carbohydrates decreased to 13.3%-16.5% from 23.1%. AtL1L transformants accumulated 42-64% more neutral lipids and 48-68% higher total fatty acids without compromising biomass yield or photosynthetic efficiency (Fv/Fm). Molecular analyses revealed coordinated upregulation of key lipogenic, glycolytic and pyruvate metabolism genes such as acetyl-CoA carboxylase, pyruvate kinase and malic enzyme, which were corroborated by significant increases in corresponding enzyme activities and NADPH levels. Metabolite profiling confirmed accumulation of lipid precursors including acetyl-CoA (1.7-fold elevation) concurrent with reduction of sugars like glucose to less than 39% of wild-type levels.

Conclusions: This study demonstrates the first functional transfer of plant transcription factors to diatoms, providing a transformative strategy for high-productivity microalgal biodiesel.

Background: Oleaginous filamentous fungi, such as Mucor circinelloides, are capable of accumulating high levels of single cell oil (SCO), making them attractive candidates for the production of biodiesel and other oleochemicals. Lignocellulosic feedstocks offer an abundant and cost-effective carbon source for SCO production due to their high polysaccharide content. However, most oleaginous microorganisms cannot directly utilize cellulose and hemicellulose polysaccharides, necessitating their conversion into monosaccharides. Lignocellulosic substrates can be saccharified either separately from fermentation (separate hydrolysis and fermentation; SHF) or simultaneously (simultaneous saccharification and fermentation; SSF). This study evaluated SSF using M. circinelloides, as well as SHF cultivations on two types of lignocellulosic hydrolysates, and two control fermentations, with process monitoring via four vibrational spectroscopy techniques: Fourier Transform Infrared (FTIR) spectrometer with fibre optic probe, FTIR microspectrometer, FTIR spectrometer with high throughput setting (HTS), and FT-Raman spectrometer with HTS.

Results: Quantitative estimation of glucose in the cultivation media and lipid content in the biomass was achieved using PLSR analysis of FT-Raman measurements from the cell suspension. FT-Raman spectroscopy demonstrated exceptional capability for online and at-line process monitoring among the tested techniques. It enabled direct and rapid analysis of raw cell suspensions (containing growth media, cellulose-rich pulp substrate, and fungal biomass) without the need for sample pre-treatment, purification, or modification. FT-Raman provided comprehensive biochemical profiles, effectively detecting key chemical changes in both the cellulose-rich pulp substrates and the fungal biomass, including lipid accumulation by the oleaginous fungi. FTIR with fiber optics is effective for monitoring glucose in SHF processes, but its accuracy is limited in SSF processes due to the very low glucose concentrations. The study demonstrates that FTIR microspectroscopy is a valuable tool for lab-scale fermentation process development, as well as for investigating the bioconversion of lignocellulosic biomass into fungal biomass and metabolites.

Conclusions: FT-Raman spectroscopy is highlighted as a powerful process analytical technology (PAT) tool for real-time or near-real-time monitoring of SSF processes for intracellular SCO production. Its ability to provide rich chemical information rapidly and without extensive sample preparation holds significant promise for optimizing industrial SCO production from lignocellulosic feedstocks.

Background: Mannose has wide-ranging applications but microbial fermentation remains underdeveloped compared to biotransformation for its production. The yeast Komagataella phaffii stands out as a premier synthetic biology platform, renowned for its safety profile and exceptional suitability for high-density fermentation. This established chassis organism is ideally positioned for large-scale mannose production through targeted rewiring of its mannose biosynthetic pathway via metabolic engineering.

Results: K. phaffii was metabolically engineered for efficient mannose production using a dual carbon source system: glycerol for biomass generation and glucose for mannose synthesis. To redirect carbon flux toward fructose-6-phosphate (F6P) accumulation at the glycolytic node, glycolytic flux was attenuated by knocking out the phosphofructokinase II (pfk2) gene and downregulating phosphofructokinase I (pfk1). Simultaneously, pentose phosphate pathway flux was reduced by downregulating glucose-6-phosphate dehydrogenase (zwf1). To enhance mannose biosynthesis, conversion of F6P into mannose was promoted by suppressing phosphomannose isomerase (PAS_chr3_1115) and overexpressing the Escherichia coli-derived phosphatase gene yniC. Additionally, three genes involved in arabinitol and ribitol production (PAS_chr2-2_0019, PAS_chr4_0754, and PAS_chr4_0988) were deleted to suppress byproduct accumulation. The engineered strain achieved ~ 121.1 g/L mannose in high-cell-density, fed-batch fermentation, representing the highest reported titer via microbial fermentation to date.

Conclusions: This study achieved efficient mannose production in K. phaffii by remodeling central metabolism. It not only offers a new route for mannose biosynthesis but also establishes a model framework for engineering K. phaffii to produce other high-value bioactive compounds.

Background: Fermented bamboo shoots, locally known as Khorisa, are a traditional food widely consumed in Assam, India, valued for their distinctive tangy flavour and potential probiotic benefits. This study aimed to isolate, identify, and characterize microbial strains from Khorisa, assessing their probiotic potential, safety, and flavour-producing capabilities for application in functional food products.

Methods: Fifteen Khorisa samples were collected from five districts of Assam. Fifty-two microbial isolates were obtained and screened for proteolytic, lipolytic, and carbohydrate-fermenting activities. Six strains (Khorisa/NA/1-Khorisa/NA/6) showing positive enzymatic activity were evaluated for safety (haemolysis, DNase activity, antibiotic susceptibility) and probiotic attributes, including acid, bile, salt, and phenol tolerance, auto-aggregation, and epithelial cell adhesion. Sensory evaluation of curd and rice beverage fermented with these isolates was performed by a trained panel. Derivatized compounds were profiled using gas chromatography-mass spectrometry (GC-MS). Molecular identification was conducted via 16 S rRNA sequencing.

Results: Strain Khorisa/NA/3 was identified as Bacillus sp. strain FPIK1. It exhibited outstanding probiotic-like potential, maintaining 59.2% survival at pH 2, 92.5% at pH 4, and 40% bile salt tolerance. It demonstrated notable halotolerance (20.6% viability at 8% NaCl), phenol resistance (97% at 0.4%), high auto-aggregation (29%), and strong epithelial adhesion (69%). Thermal adaptability was evident with 91.5% viability at 37 °C and 83.4% at 40 °C. Safety evaluation confirmed a non-haemolytic, DNase-negative phenotype with broad antibiotic susceptibility, showing resistance to only one tested antibiotic. Sensory trials revealed that curd and rice beverage fermented with FPIK1 achieved the highest scores for flavour, aroma, and overall acceptability. GC-MS profiling revealed a diverse array of esters, alcohols, ketones, and organic acids that imparted sweet and lemony-sour notes. These volatiles were absent in both uninoculated controls and products prepared with non-flavour-producing strains, underscoring the strain's inherent ability to enhance organoleptic quality through natural flavour biosynthesis.

Conclusion: Bacillus sp. FPIK1, isolated from traditional Khorisa, combines robust probiotic-like attributes, a favourable safety profile, and diverse flavour-producing capabilities, underscoring its suitability as a natural starter culture for probiotic-like, flavour-enhanced fermented foods. These findings support its potential incorporation into functional dairy and non-dairy products, offering both sensory and health benefits while valorizing an indigenous fermented food resource.

Background: Oxalate oxidase (OxOx) is an important enzyme commonly used in the determination of urine oxalate concentration. It is naturally expressed by a variety of plants and microorganisms and recombinant OxOx enzyme has been successfully expressed in yeast, but not in Escherichia coli (E. coli) due to its aggregation in insoluble inclusion bodies. This study aimed to (1) develop a method for purification of active OxOx following expression in E. coli and (2) demonstrate the feasibility of using filter paper coated with recombinant OxOx as a method to measure oxalate concentration in solution.

Results: A synthetic gene optimized for E. coli codon bias expression was derived from barley and cloned into the pRSET expression vector. Phenyl-Sepharose CL-4B and Q-Sepharose Fast Flow chromatography purified the active enzyme to over 90% purity. Using these methods, 21 mg of purified active OxOx enzyme was obtained from a 1 L culture. The purified OxOx exhibited an activity of 3.4 U/mg with the minimum oxalate concentration needed for visual detection on filter paper of 25 µM (range 25-500 µM).

Conclusion: Our study highlights the feasibility of using E. coli for the expression and purification of OxOx, providing a promising approach for future applications in the field of healthcare. Our study has also laid the foundation for further developing an oxalate test strip for urinary oxalate assays based on OxOx.

Background: Microbial anti-inflammatory molecule (MAM) is a key effector of the next-generation probiotic Faecalibacterium duncaniae A2-165, a species whose depletion in the gut microbiota is strongly linked to inflammatory bowel disease (IBD) and other conditions. Despite its importance, the direct anti-inflammatory effects of purified MAM have never been evaluated in vitro or in intestinal inflammation models. Prior studies have relied on bacterial supernatants, synthetic peptides, or DNA delivery systems, each with inherent limitations.

Results: In this study, we produced and purified recombinant MAM (R-MAM) under denaturing conditions and, for the first time, demonstrated its direct anti-inflammatory activity in vitro and its protective effects in a colitis murine model. Despite numerous attempts, we were not able to obtain a non-aggregated R-MAM. Therefore, we can assume that the R-MAM used here is partly or totally denatured. Nevertheless, in vitro assays with human intestinal epithelial cells (HT-29) and peripheral blood mononuclear cells (PBMCs) confirmed the ability of MAM to induce an anti-inflammatory cytokine profile. In addition, in a DNBS-induced colitis model, oral administration of R-MAM significantly prevented weight loss and reduced colon weight and thickness, key macroscopic indicators of inflammation.

Conclusions: These findings provide a critical validation step for the therapeutic potential of MAM in intestinal inflammation, despite its purification under denaturing conditions. Future studies should focus on optimizing protein stability and conformational integrity to increase its therapeutic potential as a biotherapeutic agent.