Microbial Cell Factories最新文献

Environmental concerns are rising the need to find cost-effective alternatives to fossil oils. In this sense, short-chain fatty acids (SCFAs) are proposed as carbon source for microbial oils production that can be converted into oleochemicals. This investigation took advantage of the outstanding traits of recombinant Yarrowia lipolytica strains to assess the conversion of SCFAs derived from real digestates into odd-chain fatty acids (OCFA). High yeast OCFAs content was aimed by using two engineered strains (Y. lipolytica JMY7780 and JMY7782). Batch and two-step batch fermentations were performed, reaching high lipid content (40.8% w/w) and lipid yield (0.07 g/g) with JMY7782, which overexpresses propionyl-CoA synthase. Fed-batch fermentation with an acetic acid pulse after 24 h was also carried out to promote SCFAs consumption and OCFAs production. In this case, SCFAs consumption rate increased and JMY7782 was able to accumulate up to 60.4% OCFAs of the total lipids produced from food waste-derived carbon sources.

Background: Ribosome pausing slows down translation and can affect protein synthesis. Improving translation efficiency can therefore be of commercial value. In this study, we investigated whether ribosome pausing occurs during production of the α-amylase AmyM by the industrial production organism Bacillus subtilis under repeated batch fermentation conditions.

Results: We began by assessing our ribosome profiling procedure using the antibiotic mupirocin that blocks translation at isoleucine codons. After achieving single codon resolution for ribosome pausing, we determined the genome wide ribosome pausing sites for B. subtilis at 16 h and 64 h growth under batch fermentation. For the highly expressed α-amylase gene amyM several strong ribosome pausing sites were detected, which remained during the long fermentation despite changes in nutrient availability. These pause sites were neither related to proline or rare codons, nor to secondary protein structures. When surveying the genome, an interesting finding was the presence of strong ribosome pausing sites in several toxins genes. These potential ribosome stall sites may prevent inadvertent activity in the cytosol by means of delayed translation.

Conclusions: Expression of the α-amylase gene amyM in B. subtilis is accompanied by several ribosome pausing events. Since these sites can neither be predicted based on codon specificity nor on secondary protein structures, we speculate that secondary mRNA structures are responsible for these translation pausing sites. The detailed information of ribosome pausing sites in amyM provide novel information that can be used in future codon optimization studies aimed at improving the production of this amylase by B. subtilis.

Biomanufacturing offers a potentially sustainable alternative to deriving chemicals from fossil fuels. However, traditional biomanufacturing, which uses sugars as feedstocks, competes with food production and yields unfavourable land use changes, so more sustainable options are necessary. Cupriavidus necator is a chemolithoautotrophic bacterium capable of consuming carbon dioxide and hydrogen as sole carbon and energy sources, or formate as the source of both. This autotrophic metabolism potentially makes chemical production using C. necator sustainable and attractive for biomanufacturing. Additionally, C. necator natively fixes carbon in the form of poly-3-hydroxybutyrate, which can be processed to make biodegradable plastic. Recent progress in development of modelling and synthetic biology tools have made C. necator much more usable as a biomanufacturing chassis. However, these tools and applications are often limited by a lack of consideration for the unique physiology and metabolic features of C. necator. As such, further work is required to better understand the intricate mechanisms that allow it to prioritise generalization over specialization. In this review, progress toward physiology-informed engineering of C. necator across several dimensions is critically discussed, and recommendations for moving toward a physiological approach are presented. Arguments for metabolic specialization, more focus on autotrophic fermentation, C. necator-specific synthetic biology tools, and modelling that goes beyond constraints are presented based on analysis of existing literature.

Background: Sesquiterpene ( +)-valencene is a characteristic aroma component from sweet orange fruit, which has a variety of biological activities and is widely used in industrial manufacturing of food, beverage and cosmetics industries. However, at present, the content in plant sources is low, and its yield and quality would be influenced by weather and land, which limit the supply of ( +)-valencene. The rapid development of synthetic biology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products.

Results: In this study, we first introduced the ( +)-valencene synthase into Komagataella phaffii by CRISPR/Cas9 system, and successfully constructed a ( +)-valencene producer with the initial yield of 2.1 mg/L. Subsequently, the ( +)-valencene yield was increased to 8.2 mg/L by fusing farnesyl pyrophosphate synthase with ( +)-valencene synthase using the selected ligation linker. High expression of key genes IDI1, tHMG1, ERG12 and ERG19 enhanced metabolic flux of MVA pathway, and the yield of ( +)-valencene was further increased by 27%. Besides, in-situ deletion of the promoter of ERG9 increased the yield of ( +)-valencene to 48.1 mg/L. Finally, we optimized the copy number of farnesyl pyrophosphate synthase and ( +)-valencene synthase fusion protein, and when the copy number reached three, the yield of ( +)-valencene achieved 173.6 mg/L in shake flask level, which was 82-fold higher than that of the starting strain CaVAL1.

Conclusions: The results obtained here suggest that K. phaffii has the potential to efficiently synthesize other terpenoids.

Outer membrane vesicles (OMVs), shed by Gram-negative bacteria, are spherical nanostructures that play a pivotal role in bacterial communication and host-pathogen interactions. Comprising an outer membrane envelope and encapsulating a variety of bioactive molecules from their progenitor bacteria, OMVs facilitate material and informational exchange. This review delves into the recent advancements in OMV research, providing a comprehensive overview of their structure, biogenesis, and mechanisms of vesicle formation. It also explores their role in pathogenicity and the techniques for their enrichment and isolation. Furthermore, the review highlights the burgeoning applications of OMVs in the field of biomedicine, emphasizing their potential as diagnostic tools, vaccine candidates, and drug delivery vectors.

Background: Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved.

Results: In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha.

Conclusions: This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.

The bacterium Streptomyces sp. KN37 was isolated from the soil of Kanas, Xinjiang. The broth dilution of strain KN37 has a strong inhibitory effect against a variety of crop pathogenic fungi. However, in practical applications, its effective biological activity is limited by medium formulations and fermentation conditions. In this study, we used the response surface method to optimize the fermentation medium and conditions of the strain KN37, for investigating the reasons for the enhanced biological activity at both the metabolic and transcriptomic levels. The results of the Plackett-Burman design showed that millet, yeast extract, and K₂HPO₄ were the key factors influencing its antifungal activity. Subsequently, optimization by the response surface methodology yielded the final fermentation conditions as: millet 20 g/L, yeast extract 1 g/L, K₂HPO₄ 0.5 g/L, rotation speed 150 r/min, temperature 25 °C, initial pH 8, fermentation time 9 d, inoculation amount 4%, liquid volume 100 mL. The antifungal effect of the optimized strain fermentation dilution was significantly enhanced, and the antifungal rate of R. solani increased from 27.33 to 59.53%, closely aligning with the predicted value of 53.03%. The results of HPLC-MS/MS and transcriptomic analysis revealed that the content of some secondary metabolic active substances in the fermentation broth of KN37 was significantly different from that of the original fermentation broth. Notably, the content of 4- (diethylamino) salicylaldehyde (DSA) was significantly increased by 16.28-fold while the yield of N- (2,4-dimethylphenyl) formamide (NDMPF) was increased by 6.35 times. Transcriptomic analysis further elucidated molecular mechanisms behind these changes with the expression of salicylic acid dehydrogenase (SALD) was significantly down-regulated, which was only 0.48 times compared to that before optimization. This research successfully optimized the fermentation process of strain KN37 providing a strong foundation for the actual production and application of strain KN37 in agriculture.

Background: Hyaluronic acid (HA) is extensively employed in various fields such as medicine, cosmetics, food, etc. The molecular weight (MW) of HA is crucial for its biological functions. Streptococcus zooepidemicus, a prominent HA industrial producer, naturally synthetizes HA with high MW. Currently, few effective approaches exist for the direct and precise regulation of HA MW through a one-step fermentation process, and S. zooepidemicus lacks metabolic regulatory elements with varying intensities. The ratio of HA's precursors, UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA), is critical for the extension and release of HA. An imbalance in the precursor proportions for HA synthesis leads to a significant decrease in HA MW, indicating that controlling the precursor ratio may serve as a potential method for regulating HA MW.

Results: In this study, the type and concentration of carbon sources were manipulated to disrupt the balance of precursor supply. Based on the results, it was speculated that the transcription level of hasE, which may connect the two HA synthesis precursors, is positively correlated with HA MW. Consequently, an endogenous expression component library for S. zooepidemicus was constructed, comprising 32 constitutive and 4 inducible expression elements. The expression of hasE was subsequently regulated in strain SE0 (S12 ΔhasE) using two constitutive promoters of differing strengths. The recombinant strain SE1, in which hasE was controlled by the stronger promoter PR31, produced HA with a MW of 1.96 MDa. In contrast, SE2, utilizing the weaker promoter PR22, synthesized shorter HA with a MW of 1.63 MDa, thereby verifying the hypothesis. Finally, to precisely regulate HA MW according to specific demands, an efficient sucrose-induced expression system was screened and employed to control the transcription level of hasE, obtaining recombinant strain SE3. When induced with sucrose concentrations of 3, 5-10 g/L, the HA MW of SE3 reached 0.78 to 1.77 MDa, respectively.

Conclusions: Studies on regulating the balance of the HA precursor substances indicate that an oversupply of either UDP-GlcNAc or UDP-GlcUA can reduce HA MW. The hasE gene serves as a crucial regulator for maintaining this balance. Precise regulation of hasE transcription was achieved through an efficient inducible expression system, enabling the customized production of HA with specific MW. The HA MW of strain SE3 can be accurately manipulated by adjusting sucrose concentration, establishing a novel strategy for customized HA fermentation.

Background: The composition of anaerobically digested sludge is inherently complex, enriched with structurally complex organic compounds and nitrogenous constituents, which are refractory to biodegradation. These characteristics limit the subsequent rational utilization of resources from anaerobically digested sludge. White-rot fungi (WRF) have garnered significant research interest due to their exceptional capacity to degrade complex and recalcitrant organic pollutants. However, the exploration of WRF in the context of sludge treatment remains an under-investigated area within the scientific community. The present investigation explores the application of WRF in the treatment of anaerobically digested sludge, offering a novel approach for the valorization of sludge resources.

Results: In this study, WRF enzymes, manganese peroxidase (MnP) and lignin peroxidase (LiP), exhibited sustained high activities of approximately 102 U/L and 26 U/L, respectively, within the anaerobically digested sludge under a controlled pH of 5.5 within the growth system. These conditions were found to significantly enhance the treatment efficacy of the anaerobic sludge. The removal of soluble chemical oxygen demand (COD) and Total COD by Trametes versicolor powder was better than that of Phanerochaete chrysosporium powder. The treatment of sludge samples with WRF, specifically Phanerochaete chrysosporium powder, resulted in a significant reduction of ultraviolet radiation (UV₂₅₄). Fourier-transform infrared spectroscopy (FTIR) analysis revealed that the application of Trametes versicolor powder exerted a notably pronounced impact on the functional groups present in sludge samples. Specifically, there was a significant decrease in the peak intensities corresponding to the C-O bonds, indicative of saccharide degradation, alongside an observable increase in the intensities of amide peaks, which is suggestive of protein synthesis enhancement. Microbial community analysis demonstrated that Phanerochaete chrysosporium was the predominant fungal species, exerting a significant regulatory role within the sludge ecosystem.

Conclusion: In conclusion, this research furnishes a robust scientific foundation for the utilization of WRF in the treatment of anaerobic digestion sludge. It elucidates the fungi's capacity to ameliorate the physicochemical attributes and microbial community composition within the sludge. Furthermore, the study offers a certain reference for the subsequent use of WRF in the treatment of other types of sludge.