Microbial Cell Factories最新文献

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.

Background: The presence of inorganic pollutants and heavy metals in industrial effluents has become a serious threat and environmental issues. Fungi have a remarkable ability to exclude heavy metals from wastewater through biosorption in eco-friendly way. Tannase plays an important role in bioconversion of tannin, a major constituent of tannery effluent, to gallic acid which has great pharmaceutical applications. Therefore, the aim of the current study was to exploit the potential of tannase from Aspergillus glaucus and fungal biomass waste for the bioremediation of heavy metals and tannin.

Results: Tannase from A. glaucus was partially purified 4.8-fold by ammonium sulfate precipitation (80%). The enzyme was optimally active at pH 5.0 and 40 °C and stable at this temperature for 1 h. Tannase showed high stability at different physiological conditions, displayed about 50% of its activity at 60 °C and pH range 5.0-6.0. Immobilization of tannase was carried out using methods such. as entrapment in Na-alginate and covalent binding to chitosan. The effects of Na-alginate concentrations on the beads formation and enzyme immobilization revealed that maximum immobilization efficiency (75%) was obtained with 3% Na-alginate. A potential reusability of the immobilized enzyme was showed through keeping 70% of its relative activity up to the fourth cycle. The best bioconversion efficiency of tannic acid to gallic acid by immobilized tannase was at 40 °C with tannic acid concentration up to 50 g/l. Moreover, bioremediation of heavy metal (Cr³⁺, Pb²⁺, Cu²⁺, Fe³⁺, and Mn²⁺) from aqueous solution using A. glaucus biomass waste was achieved with uptake percentage of (37.20, 60.30, 55.27, 79.03 and 21.13 respectively). The biomass was successfully used repeatedly for removing Cr³⁺ after using desorbing agent (0.1 N HCl) for three cycles.

Conclusion: These results shed the light on the potential use of tannase from locally isolated A. glaucus in the bioremediation of industrial tanneries contained heavy metals and tannin.

The remediation of emerging contaminants presents a pressing environmental challenge, necessitating innovative approaches for effective mitigation. This review article delves into the untapped potential of soil microbial communities in the bioremediation of emerging contaminants. Bioremediation, while a promising method, often proves time-consuming and requires a deep comprehension of microbial intricacies for enhancement. Given the challenges presented by the inability to culture many of these microorganisms, conventional methods are inadequate for achieving this goal. While omics-based methods provide an innovative approach to understanding the fundamental aspects, processes, and connections among microorganisms that are essential for improving bioremediation strategies. By exploring the latest advancements in omics technologies, this review aims to shed light on how these approaches can unlock the hidden capabilities of soil microbial communities, paving the way for more efficient and sustainable remediation solutions.

Background: Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries.

Results: To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris.

Conclusion: These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-L-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications.

Background: (R,R)-2,3-butanediol (BDO) is employed in a variety of applications and is gaining prominence due to its unique physicochemical features. The use of glycerol as a carbon source for 2,3-BDO production in Klebsiella pneumoniae has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation.

Results: In this study, the inactivation of the budC gene in K. pneumoniae increased the production rate of (R,R)-2,3-BDO from 21.92 ± 2.10 to 92.05 ± 1.20%. The major isomer form of K. pneumoniae (meso-2,3-BDO) was shifted to (R,R)-2,3-BDO. The purity of (R,R)-2,3-BDO was examined by agitation speed, and 98.54% of (R,R)-2,3-BDO was obtained at 500 rpm. However, as the cultivation period got longer, the purity of (R,R)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400 rpm after 24 h) and over-expression of the dhaD gene involved in (R,R)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (R,R)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47 g/L of (R,R)-2,3-BDO (1.69 g/L of meso-2,3-BDO), productivity of 1.24 g/L/h, and yield of 0.35 g/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher.

Conclusions: This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (R,R)-2,3-BDO with a high purity in Klebsiella sp. strains. In addition, to the best of our knowledge, this is the first study to produce (R,R)-2,3-BDO using glycerol as the sole carbon source.

The global demand for plant oil has reached unprecedented levels and is relevant in all industrial sectors. Driven by the growing awareness for environmental issues of traditional plant oils and the need for eco-friendly alternatives, microbial oil emerges as a promising product with significant potential. Harnessing the capabilities of oleaginous microorganisms is an innovative approach for achieving sustainable oil production. To increase economic feasibility, it is crucial to explore feedstocks such as agricultural waste streams as renewable resource for microbial bioprocesses. The fungal model Ustilago maydis is one promising organism in the field of microbial triglyceride production. It has the ability to metabolize a wide variety of carbon sources for cell growth and accumulates high amounts of triglycerides intracellularly. In this study we asked whether this large variety of usable carbon sources can also be utilized for triglyceride production, using corn stover saccharides as a showcase.Our experiments revealed metabolization of the major saccharide building blocks present in corn stover, demonstrating the remarkable potential of U. maydis. The microorganism exhibited the capacity to synthesize triglycerides using the saccharides glucose, fructose, sucrose, xylose, arabinose, and galactose as carbon source. Notably, while galactose has been formerly considered as toxic to U. maydis, we found that the fungus can metabolize this saccharide, albeit with an extended lag phase of around 100 hours. We identified two distinct methods to significantly reduce or even prevent this lag phase, challenging previous assumptions and expanding the understanding of U. maydis metabolism.Our findings suggest that the two tested methods can prevent long lag phases on feedstocks with high galactose content and that U. maydis can produce microbial triglycerides very efficiently on many different carbon sources. Looking forward, exploring the metabolic capabilities of U. maydis on additional polymeric components of corn stover and beyond holds promise for innovative applications, marking a significant step toward environmentally sustainable bioprocessing technologies.

Background: Over the last two decades, hybridization has been a powerful tool used to construct superior yeast for brewing and winemaking. Novel hybrids were primarily constructed using at least one Saccharomyces cerevisiae parent. However, little is known about hybrids used for other purposes, such as targeted flavor production, for example, 2-phenylethanol (2-PE). 2-PE, an aromatic compound widely utilised in the food, cosmetic, and pharmaceutical industries, presents challenges in biotechnological production due to its toxic nature. Consequently, to enhance productivity and tolerance to 2-PE, various strategies such as mutagenesis and genetic engineering are extensively explored to improved yeast strains. While biotechnological efforts have predominantly focused on S. cerevisiae for 2-PE production, other Saccharomyces species and their hybrids remain insufficiently described.

Results: To address this gap, in this study, we analysed a new interspecies yeast hybrid, II/6, derived from S. uvarum and S. kudriavzevii parents, in terms of 2-PE bioconversion and resistance to its high concentration, comparing it with the parental strains. Two known media for 2-PE biotransformation and three different temperatures were used during this study to determine optimal conditions. In 72 h batch cultures, the II/6 hybrid achieved a maximum of 2.36 ± 0.03 g/L 2-PE, which was 2-20 times higher than the productivity of the parental strains. Our interest lay not only in determining whether the hybrid improved in productivity but also in assessing whether its susceptibility to high 2-PE titers was also mitigated. The results showed that the hybrid exhibited significantly greater resistance to the toxic product than the original strains.

Conclusions: The conducted experiments have confirmed that hybridization is a promising method for modifying yeast strains. As a result, both 2-PE production yield and tolerance to its inhibitory effects can be increased. Furthermore, this strategy allows for the acquisition of non-GMO strains, alleviating concerns related to additional legislative requirements or consumer acceptance issues for producers. The findings obtained have the potential to contribute to the development of practical solutions in the future.