Microbial Cell Factories最新文献

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.

Background: E. coli still remains the most commonly used organism to produce recombinant proteins in research labs. This condition is mirrored by the attention that researchers dedicate to understanding the biology behind protein expression, which is then exploited to improve the effectiveness of the technology. This effort is witnessed by an impressive number of publications, and this review aims to organize the most relevant novelties proposed in recent years.

Results: The examined contributions address several of the known bottlenecks related to recombinant expression in E. coli, such as improved glycosylation pathways, more reliable production of proteins whose folding depends on the formation of disulfide bonds, the possibility of controlling and even benefiting from the formation of aggregates or the need to overcome the dependence of bacteria on antibiotics during bacterial culture. Nevertheless, the majority of the published papers aimed at identifying the conditions for optimal control of the translation process to achieve maximal yields of functional exogenous proteins.

Conclusions: Despite community commitment, the critical question of what really is the metabolic burden and how it affects both host metabolism and recombinant protein production remains elusive because some experimental results are contradictory. This contribution aims to offer researchers a tool to orient themselves in this complexity. The new capacities offered by artificial intelligence tools could help clarifying this issue, but the training phase will probably require more systematic experimental approaches to collect sufficiently uniform data.

Background: Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense is a soil-borne fungal disease. Especially, tropical Race 4 (Foc TR4) can infect almost Cavendish subgroup and has a fatal threat to banana industry. Use of antagonistic microbes to manage soil-borne pathogen is viewed as a promising strategy.

Results: Strain XZ11-1 isolated from tropical rainforest has the production ability of high siderophore. By the analysis of physiological and biochemical profiles, construction of phylogenetic tree, and comparative results from the NR database, strain XZ11-1 was identified as Trichoderma virens. A relative content of 79.45% siderophores was produced in the optimized fermentation solution, including hydroxamate and carboxylate-type siderophores. Siderophores were key for inhibiting the growth of Foc TR4 by competing for environmental iron. Similarly, T. virens XZ11-1 also had antagonistic activities against 10 phytopathogenic fungi. Pot experiments demonstrated that T. virens XZ11-1 could colonize in the root system of banana plants. The symbiotic interaction not only improve plant resistance to Foc TR4, but also enhance iron absorption of roots to promote plant growth by secreting siderophores.

Conclusions: T. virens XZ11-1 with the high-yield siderophores was isolated and identified. The strain could effectively inhibit the infection of Foc TR4 in banana roots and promote plant growth. It is a promising biocontrol agent for controlling fungal disease.

Background: Aspergillus niger is an important industrial filamentous fungus used to produce organic acids and enzymes. A wide dynamic range of promoters, particularly strong promoters, are required for fine-tuning the regulation of gene expression to balance metabolic flux and achieve the high yields of desired products. However, the limited understanding of promoter architectures and activities restricts the efficient transcription regulation of targets in strain engineering in A. niger.

Results: In this study, we identified two functional upstream activation sequences (UAS) located upstream of the core promoters of highly expressed genes in A. niger. We constructed and characterized a synthetic promoter library by fusing the efficient UAS elements upstream of the strong constitute PgpdA promoter in A. niger. It demonstrated that the strength of synthetic promoters was fine-tuned with a wide range by tandem assembly of the UAS elements. Notably, the most potent promoter exhibited 5.4-fold higher activity than the strongest PgpdA promoter reported previously, significantly extending the range of strong promoters. Using citric acid production as a case study, we employed the synthetic promoter library to enhance citric acid efflux by regulating the cexA expression in A. niger. It showed a 1.6-2.3-fold increase in citric acid production compared to the parent strain, achieving a maximum titer of 145.3 g/L.

Conclusions: This study proved that the synthetic promoter library was a powerful toolkit for precise tuning of transcription in A. niger. It also underscores the potential of promoter engineering for gene regulation in strain improvement of fungal cell factories.