Microbial Cell Factories最新文献

Nowadays, biofuels, especially bioethanol, are becoming increasingly popular as an alternative to fossil fuels. Zymomonas mobilis is a desirable species for bioethanol production due to its unique characteristics, such as low biomass production and high-rate glucose metabolism. However, several factors can interfere with the fermentation process and hinder microbial activity, including lignocellulosic hydrolysate inhibitors, high temperatures, an osmotic environment, and high ethanol concentration. Overcoming these limitations is critical for effective bioethanol production. In this review, the stress response mechanisms of Z. mobilis are discussed in comparison to other ethanol-producing microbes. The mechanism of stress response is divided into physiological (changes in growth, metabolism, intracellular components, and cell membrane structures) and molecular (up and down-regulation of specific genes and elements of the regulatory system and their role in expression of specific proteins and control of metabolic fluxes) changes. Systemic metabolic engineering approaches, such as gene manipulation, overexpression, and silencing, are successful methods for building new metabolic pathways. Therefore, this review discusses systems metabolic engineering in conjunction with systems biology and synthetic biology as an important method for developing new strains with an effective response mechanism to fermentation stresses during bioethanol production. Overall, understanding the stress response mechanisms of Z. mobilis can lead to more efficient and effective bioethanol production.

Background: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.

Results: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.

Conclusions: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

Background: Volatile compounds are key elements in the interaction and communication between organisms at both interspecific and intraspecific levels. In complex bacterial communities, the emission of these fast-acting chemical messengers allows an exchange of information even at a certain distance that can cause different types of responses in the receiving organisms. The changes in secondary metabolism as a consequence of this interaction arouse great interest in the field of searching for bioactive compounds since they can be used as a tool to activate silenced metabolic pathways. Regarding the great metabolic potential that the Actinobacteria group presents in the production of compounds with attractive properties, we evaluated the reply the emitted volatile compounds can generate in other individuals of the same group.

Results: We recently reported that volatile compounds released by different streptomycete species trigger the modulation of biosynthetic gene clusters in Streptomyces spp. which finally leads to the activation/repression of the production of secondary metabolites in the recipient strains. Here we present the application of this rationale in a broader bacterial community to evaluate volatiles as signaling effectors that drive the activation of biosynthesis of bioactive compounds in other members of the Actinobacteria group. Using cocultures of different actinobacteria (where only the volatile compounds reach the recipient strain) we were able to modify the bacterial secondary metabolism that drives overproduction (e.g., granaticins, actiphenol, chromomycins) and/or de novo production (e.g., collismycins, skyllamycins, cosmomycins) of compounds belonging to different chemical species that present important biological activities.

Conclusions: This work shows how the secondary metabolism of different Actinobacteria species can vary significantly when exposed in co-culture to the volatile compounds of other phylum-shared bacteria, these effects being variable depending on strains and culture media. This approach can be applied to the field of new drug discovery to increase the battery of bioactive compounds produced by bacteria that can potentially be used in treatments for humans and animals.

Background: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct.

Results: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway.

Conclusions: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Background: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.

Results: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.

Conclusions: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Introduction: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential.

Methods: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml^-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene.

Result: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR.

Conclusion: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Background: The objectives of the current study were to extract pyocyanin from Pseudomonas aeruginosa clinical isolates, characterize its chemical nature, and assess its biological activity against different bacteria and cancer cells. Due to its diverse bioactive properties, pyocyanin, being one of the virulence factors of P. aeruginosa, holds a promising, safe, and available therapeutic potential.

Methods: 30 clinical P. aeruginosa isolates were collected from different sources of infections and identified by routine methods, the VITEK 2 compact system, and 16 S rRNA. The phenazine-modifying genes (phzM, phzS) were identified using polymerase chain reaction (PCR). Pyocyanin chemical characterization included UV-Vis spectrophotometry, Fourier Transform Infra-Red spectroscopy (FTIR), Gas Chromatography-Mass Spectrometry (GC-MS), and Liquid Chromatography-Mass Spectrometry (LC-MS). The biological activity of pyocyanin was explored by determining the MIC values against different clinical bacterial strains and assessing its anticancer activity against A549, MDA-MB-231, and Caco-2 cancer cell lines using cytotoxicity, wound healing and colony forming assays.

Results: All identified isolates harboured at least one of the phzM or phzS genes. The co-presence of both genes was demonstrated in 13 isolates. The UV-VIS absorbance peaks were maxima at 215, 265, 385, and 520 nm. FTIR could identify the characteristic pyocyanin functional groups, whereas both GC-MS and LC-MS elucidated the chemical formula C₁₁H₁₈N₂O₂, with a molecular weight 210. The quadri-technical analytical approaches confirmed the chemical nature of the extracted pyocyanin. The extract showed broad-spectrum antibacterial activity, with the greatest activity against Bacillus, Staphylococcus, and Streptococcus species (MICs 31.25-125 µg/mL), followed by E. coli isolates (MICs 250-1000 µg/mL). Regarding the anticancer activity, the pyocyanin extract showed IC₅₀ values against A549, MDA-MB-231, and Caco-2 cancer cell lines of 130, 105, and 187.9 µg/mL, respectively. Furthermore, pyocyanin has markedly suppressed colony formation and migratory abilities in these cells.

Conclusions: The extracted pyocyanin has demonstrated to be a potentially effective candidate against various bacterial infections and cancers. Hence, the current findings could contribute to producing this natural compound easily through an affordable method. Nonetheless, future studies are required to investigate pyocyanin's effects in vivo and analyse the results of combining it with other traditional antibiotics or anticancer drugs.

Background: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme.

Results: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA^[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source.

Conclusions: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.

Background: Fibroblast growth factor 21 (FGF21) is a promising candidate for treating metabolic disorder diseases and has been used in phase II clinical trials. Currently, metabolic diseases are prevalent worldwide, underscoring the significant market potential of FGF21. Therefore, the production of FGF21 must be effectively improved to meet market demand.

Results: Herein, to investigate the impact of vectors and host cells on FGF21 expression, we successfully engineered strains that exhibit a high yield of FGF21. Surprisingly, the data revealed that vectors with various copy numbers significantly impact the expression of FGF21, and the results showed a 4.35-fold increase in expression levels. Furthermore, the performance of the double promoter and tandem gene expression construction design surpassed that of the conventional construction method, with a maximum difference of 2.67 times.

Conclusion: By exploring engineered vectors and host cells, we successfully achieved high-yield production of the FGF21 strain. This breakthrough lays a solid foundation for the future industrialization of FGF21. Additionally, FGF21 can be easily, quickly and efficiently expressed, providing a better tool and platform for the research and application of more recombinant proteins.