Microbial Cell Factories最新文献

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.

Extensive anthropogenic activity has led to the accumulation of organic and inorganic contaminants in diverse ecosystems, which presents significant challenges for the environment and its inhabitants. Utilizing microalgae as a bioremediation tool can present a potential solution to these challenges. Microalgae have gained significant attention as a promising biotechnological solution for detoxifying environmental pollutants. This is due to their advantages, such as rapid growth rate, cost-effectiveness, high oil-rich biomass production, and ease of implementation. Moreover, microalgae-based remediation is more environmentally sustainable for not generating additional waste sludge, capturing atmospheric CO₂, and being efficient for nutrient recycling and sustainable algal biomass production for biofuels and high-value-added products generation. Hence, microalgae can achieve sustainability's three main pillars (environmental, economic, and social). Microalgal biomass can mediate contaminated wastewater effectively through accumulation, adsorption, and metabolism. These mechanisms enable the microalgae to reduce the concentration of heavy metals and organic contaminants to levels that are considered non-toxic. However, several factors, such as microalgal strain, cultivation technique, and the type of pollutants, limit the understanding of the microalgal removal mechanism and efficiency. Furthermore, adopting novel technological advancements (e.g., nanotechnology) may serve as a viable approach to address the challenge of refractory pollutants and bioremediation process sustainability. Therefore, this review discusses the mechanism and the ability of different microalgal species to mitigate persistent refractory pollutants, such as industrial effluents, dyes, pesticides, and pharmaceuticals. Also, this review paper provided insight into the production of nanomaterials, nanoparticles, and nanoparticle-based biosensors from microalgae and the immobilization of microalgae on nanomaterials to enhance bioremediation process efficiency. This review may open a new avenue for future advancing research regarding a sustainable biodegradation process of refractory pollutants.

Background: Functional foods and dairy products are gaining global attention due to their nutritional value and health-promoting characteristics. Lactic acid bacteria (LAB) are one of the promising components included in these products, thanks to their probiotic properties and ability to produce bioactive compounds such as bacteriocins. On the other hand, ectomycorrhizal wild mushrooms (truffles) are known for their ethnomycological importance. Hence, we aimed to develop a functional dairy product using a bacteriocin-producing LAB isolate that has probiotic potentials together with the bioactive extract of a truffle mushroom.

Results: Screening for bacteriocin-producing LAB led to the selection of four safe isolates that also showed promising probiotic potentials. Isolate No. 7 was selected due to its wider antimicrobial spectrum and was identified as Lactiplantibacillus plantarum strain GA7. Out of resulting bands from Tricine SDS-PAGE analysis, a band (its molecular mass was approximately 7 kDa) exhibited antimicrobial activity. Amino acid sequencing of this active band detected 62 amino acid residues with 100% identity to plantaricin ASM1 bacteriocin. Simultaneously, an ethyl acetate extract was prepared from a truffle sample identified as Tirmania pinoyi. Safety of this truffle was confirmed and its extract exerted promising antioxidant and hypocholesterolemic activity. Prepared functional dairy products (Labneh) fortified with L. plantarum GA7 and nano-encapsulated T. pinoyi extract exhibited superior physicochemical, sensory and antioxidant properties compared to control. Moreover, an increase in probiotic count was observed in presence of T. pinoyi extract. Furthermore, prepared Labneh using the bacteriocin-producing L. plantarum GA7 and nano-encapsulated T. pinoyi extract remained unspoiled for over 60 days, compared to control, which spoiled after 21 days.

Conclusion: Besides improving Labneh physicochemical, sensory and antioxidant properties, the presence of the bacteriocin-producing L. plantarum GA7 has contributed in significantly extending its shelf life, while T. pinoyi extract showed prebiotic influence on probiotic count. As far as we know this is the first study describing production of a functional synbiotic dairy product fortified with bacteriocin-producing probiotic LAB and bioactive T. pinoyi truffle extract.

Background: Streptomyces roseochromogenes NRRL 3504 produces clorobiocin, an aminocoumarin antibiotic that inhibits DNA replication. No other natural products have been isolated from this bacterium so far, despite the presence of a rich repertoire of specialized metabolite biosynthesis gene clusters (smBGCs) within its genome. Heterologous expression of smBGCs in suitable chassis speeds up the discovery of the natural products hidden behind these sets of genes.

Results: In this work we focus on one intriguing smBGC of NRRL 3504 bearing some similarity to gene clusters involved in production of manumycin family polyketides. Through heterologous expression in Streptomyces chassis strains S. albus Del14 and S. lividans ΔYA9, this smBGC (hereafter referred to as lim BGC) was shown to direct the production of unusual polyketide limocrocin (LIM) known for its ability to interfere with viral reverse transcriptases. The organization of lim BGC, data on the structures of revealed metabolites as well as manipulations of lim genes allowed us to put forward an initial hypothesis about a biosynthetic pathway leading to LIM. We provide initial data on two LIM derivatives as well as updated NMR spectra for the main product.

Conclusion: This study reveals the genetic control of biosynthesis of LIM that remained hidden for the last 70 years. This, in turn, opens the door to biological routes towards overproduction of LIM as well as generation of its derivatives.

Oleaginous yeasts are considered promising sources for lipid production due to their ability to accumulate high levels of lipids under appropriate growth conditions. The current study aimed to isolate and identify oleaginous yeasts having superior ability to accumulate high quantities of lipids; and enhancing lipid production using response surface methodology and repeated-batch fermentation. Results revealed that, twenty marine oleaginous yeasts were isolated, and the most potent lipid producer isolate was Candida parapsilosis Y19 according to qualitative screening test using Nile-red dye. Orange peels was used as substrate where C. parapsilosis Y19 produced 1.14 g/l lipids at 23.0% in batch fermentation. To enhance the lipid production, statistical optimization using Taguchi design through Response surface methodology was carried out. Total lipids were increased to 2.46 g/l and lipid content increased to 30.7% under optimal conditions of: orange peel 75 g/l, peptone 7 g/l, yeast extract 5 g/l, inoculum size 2% (v/v), pH 5 and incubation period 6 d. Furthermore, repeated-batch fermentation of C. parapsilosis Y19 enhanced lipid production where total lipids increased at 4.19 folds (4.78 g/l) compared to batch culture (before optimization). Also, the lipid content was increased at 1.7 folds (39.1%) compared to batch culture (before optimization). Fatty acid profile of the produced lipid using repeated-batch fermentation includes unsaturated fatty acids (USFAs) at 74.8% and saturated fatty acids (SFAs) at 25.1%. Additionally, in repeated-batch fermentation, the major fatty acid was oleic acid at 45.0%; followed by linoleic acid at 26.0%. In conclusion, C. parapsilosis Y19 is considered a promising strain for lipid production. Also, both statistical optimizations using RSM and repeated-batch fermentation are efficient methods for lipid production from C. parapsilosis Y19.

Background: Paenibacillus polymyxa, is a Gram-positive, plant growth promoting bacterium, known for producing 98% optically pure 2,3-butanediol, an industrially valuable chemical for solvents, plasticizers and resins. Immobilization of Paenibacillus polymyxa has been proposed to improve the cell stability and efficiency of the fermentation process, reduce contamination and provide easy separation of butanediol in the culture broth as compared to conventional bioprocesses. This research aimed to explore the potential of Paenibacillus polymyxa with immobilization technique to produce 2,3-butanediol.

Results: We investigated different immobilization methods with natural biopolymers like alginate, chitosan and carrageenan-chitosan-based immobilization. These methods were further investigated for their immobilization efficiency and yield in 2,3-butanediol production. Carrageenan-chitosan beads enabled a higher cell concentration and demonstrated superior cell retention to calcium-alginate-chitosan beads. Carrageenan-chitosan immobilization preserved 2,3-butanediol production in bacteria and increased the product formation rate.

Conclusion: Carrageenan-chitosan immobilization enables non-pathogenic Paenibacillus polymyxa to be a capable 2,3-butanediol producer with increased product formation rate, which has not been previously reported. This novel strategy offers promising alternative to traditional fermentation processes using pathogenic strains and can be further applied in co-cultivations for metabolite production, wastewater management and bioremediation.

Genome mining is a promising avenue for expanding the repertoire of microbial natural products, which are important for drug development. This approach involves predicting genetically encoded small molecules by examining bacterial genomes via accumulated knowledge of microbial biosynthesis. However, it is also important that the microbes produce the predicted molecule in practice. Here, we introduce an endophytic Streptomyces sp. N50, which was isolated from the medicinal plant Selaginella tamariscina. Upon sequencing its entire genome, 33 biosynthetic gene clusters (BGCs) were identified in a chromosome and a megaplasmid. Subsequent genome mining revealed that the new 15-deoxynaphthomycin could be produced due to the presence of an enoyl reductase domain, which is absent in the known BGC of naphthomycin, a type of ansamycin antibiotics. In addition, the engineered strain with the introduction of the global regulatory gene afsR2 into N50 successfully produced 15-deoxynaphthomycins. Furthermore, molecular network analysis via MS/MS selectively confirmed the presence of additional sulfur-containing 15-deoxynaphthomycin congeners. Eventually, six new 15-deoxynaphthomycins were isolated and elucidated from the engineered strain N50. This family of compounds is known to exhibit various biological activities. Also, the presence of quinone moieties in these compounds, which are known to activate NRF2, they were tested for their ability to activate NRF2. Among the new compounds, three (1, 5, and 6) activated the antioxidant NRF2-ARE signaling pathway. Treatment with these compounds significantly elevated NRF2 levels in HepG2 cells and further induced the expression of NRF2 target genes associated with the antioxidant response. This study suggests that the combination of genome mining, gene engineering and molecular networking is helpful for generating new small molecules as pharmaceutical candidates from microorganisms.

Background: The biosynthesis of the natural product family of the polycyclic tetramate macrolactams (PoTeMs) employs an uncommon iterative polyketide synthase/non-ribosomal peptide synthetase (iPKS/NRPS). This machinery produces a universal PoTeM biosynthetic precursor that contains a tetramic acid moiety connected to two unsaturated polyene side chains. The enormous structural and hence functional diversity of PoTeMs is enabled by pathway-specific tailoring enzymes, particularly cyclization-catalyzing oxidases that process the polyene chains to form distinct ring systems, and further modifying enzymes.

Results: Ikarugamycin is the first discovered PoTeM and is formed by the three enzymes IkaABC. Utilizing the iPKS/NRPS IkaA, we established a genetic plug-and-play system by screening eight different strong promoters downstream of ikaA to facilitate high-level heterologous expression of PoTeMs in different Streptomyces host systems. Furthermore, we applied the system on three different PoTeM modifying genes (ptmD, ikaD, and cftA), showing the general utility of this approach to study PoTeM post-PKS/NRPS processing of diverse tailoring enzymes.

Conclusion: By employing our plug-and-play system for PoTeMs, we reconstructed the ikarugamycin biosynthesis and generated five derivatives of ikarugamycin. This platform will generally facilitate the investigation of new PoTeM biosynthetic cyclization and tailoring reactions in the future.