Biotechnology Letters最新文献

Biofuels generated through fermentation of plant-derived lignocellulosic biomass are currently not competitive with fossil fuels. Here, a framework is described for the engineering of valuable coproducts to improve the economic feasibility of biofuel production. To accomplish this goal, the desirable traits of the bioenergy crop Sorghum bicolor are harnessed via modern engineering approaches. A robust analysis is provided to highlight the scientific basis, costs, regulatory challenges, and lucrative nature of coproduct development. Sustainable biofuel production could also have far-reaching societal impacts, especially in the context of our rapidly changing global climate. This perspective suggests that the path to profitable biofuel production is attainable and encourages consideration of similar approaches by the biotechnology industry.

Kluyveromyces marxianus is an attractive chassis for microbial cell factories due to its rapid growth, thermotolerance, and wide substrate spectrum. However, gene disruption in this organism is challenging primarily due the prevalence of dominant nonhomologous recombination. AsCas12f1, a hypercompact CRISPR-associated protein consisting of 422 amino acids-approximately one-third the size of Cas9 or Cas12a-enables more efficient packaging into delivery vehicles than its larger counterparts. In this study, a gene disruption method using AsCas12f1 was established in K. marxianus through a transient targeting strategy. The integration of tRNA-gRNA into the gRNA construct increased gene disruption efficiency. Additionally, disrupting KmKU70 or KmLIG4 further increased this efficiency, achieving nearly 100%. By combining the disruption of KmKU70 with the AsCas12f1 system, the length of the homologous arm was shortened to 200 bp while maintaining a disruption efficiency of 87.5%. The implementation of the gRNA-tRNA-array system resulted in the successful generation of three single-gene knockout strains from a single transformation, resulting an overall efficiency of 86.4%. This approach leverages the transient transformation of fragments, eliminates the need for extensive time investment in constructing gRNA expression vectors and negates the requirement for the removal of the CRISPR-AsCas12f1 system after gene disruption. This study presents a novel strategy for gene disruption in K. marxianus and demonstrates the applicability of Cas12f in yeast systems.

Currently, the shift to a greener economy requires the prospection of new industrial processes to reduce greenhouse-gas emissions. Enzymatic catalysis is considered a green alternative to traditional industrial processes. Among enzymes of industrial relevance, starch-degrading enzymes, such as α-amylases, have received attention because of their enormous potential to hydrolyze starch-based materials, generating smaller sugars that can be used for the biosynthesis of chemicals of industrial relevance, such as ethanol. In the present study, a new isolate of Paenibacillus barengoltzii was obtained from cow rumen and its potential to produce amylases was evaluated. Additionally, a recombinant amylase, AmyPb, was produced and biochemically characterized. AmyPb displays high activity at elevated temperatures (55 °C) and can withstand elevated temperatures. The experimentally calculated melting temperature showed that AmyPb is more stable in alkaline environments, with a Tm of 59 °C at pH 9. AmyPb hydrolyzed potato and cassava starches with hydrolysis efficiencies of 28 and 55%, respectively. The results of this study are relevant for the development of industrial processes that employ thermostable amylases.

Recently, new immunomodulatory compounds have benn sought as effective adjuvants in vaccine development. In this context, the bioactive substances from macroalgae stand out, as they can satisfactorily activate an immune response against infectious diseases, such as caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis. This study aimed to evaluate the adjuvant activity of Iridea cordata and Sacorpletis skotibergii lipid extracts associated with the protein rCP01850 against infection by C. pseudotuberculosis in a murine model. Five groups of ten BALB/c mice each were inoculated with 0.9% saline (G1), rCP01850 (G2), rCP01850 + saponin (G3), rCP01850 + Iridea cordata (G4), and rCP01850 + Sacorpletis skotibergii (G5). Two doses of vaccine were administered with a 21-day interval between doses. After that, the animals were challenged with 2 × 10⁴ UFC of the MIC-6 strain. Experimental groups G4 and G5 presented protection rates of 60 and 70%, respectively. The production levels of total IgG and its IgG1 and IgG2a isotypes were significantly increased in G4 and G5 after the forty-second day of immunization. In addition, the expression of the cytokines IL-4, IL-12, IL-10, and IFN-γ significantly increased in G4 and G5 when compared to the negative control (G1). In turn, IL-17 and TNF-α had significant expression levels in G4 when compared to the other experimental groups (p < 0.05). The results show that subantarctic macroalgae extracts associated with rCP01850 induced substantial levels of humoral and cellular immune response and protected immunized animals against the challenge.

Purpose: Currently, a robust process using healthy Chinese hamster ovary (CHO) cells, the dominating host for biopharmaceutical protein therapeutics, is crucial for high productivity and successful scaling-up. However, issues which are originated from cell performance and detrimental to protein products such as dramatic viability drop and accumulation of toxic metabolites still exist. Aurintricarboxylic acid (ATA), reported as an antioxidant chelator to prevent apoptosis and boost cell growth, was applied as an additive in this study to address the above problems.

Methods: Two CHO cells were cultivated by fed-batch culture with ATA of different concentrations and adding strategies supplemented. The reactive oxygen species (ROS) test and confocal microscopy have been used to illustrate the working mechanism of ATA.

Results: ATA was proved to be effective in maintaining viability and improving lactate performance in the late fed-batch culture stage for both tested clones. Briefly, the harvest viability was increased by at least 10% after ATA was introduced, and the suppression of lactate accumulation was observed with ATA addition. Moreover, besides adding ATA in the fed-batch stage, a novel and favorable process to involve ATA in seed train was developed, which improved the performance of seed and further benefitted the cell performance and productivity during fed-batch culture. And the ATA was detected to penetrate into CHO cells and suppress the ROS generation intracellularly.

Conclusion: Overall, this study advances current knowledge with respect to ATA's capability of enhancing cell performances for CHO cell manufacturing and introduces a novel process of hyper seed train.

With the continuous advancement of technologies such as microbial cultivation, DNA sequencing, bioinformatics, and genetic engineering, in vivo mutagenesis methods based on perturbation factors are now widely utilized. We identified a RecJ enzyme (BaRecJ) with endonuclease and exonuclease activities from Bacillus alcalophilus, and established a broad-spectrum mutagenic method based on the endonuclease and exonuclease activities of BaRecJ. The BaRecJ mutagenesis method was applied to S cerevisiae to enhance its ethanol or acetic acid tolerance, resulting in mutant strains with improved fermentation performance. Genomic resequencing analysis summarized genes possibly associated with the tolerance of mutants. BaRecJ mutagenesis method not only holds immense potential in microbial mutagenesis breeding and adaptive evolution but also, when coupled with genomic resequencing, allows for the rapid identification of candidate genetic loci associated with specific traits.

Objective: The study employed response surface methodology (RSM) to optimize physicochemical variables for extracellular collagenase production by gram negative bacterial strain Chryseobacterium contaminans KU665299 under submerged fermentation. It is also revealing the ability of collagenase to degrade collagen, main structural protein in human blood.

Result: The study successfully enhanced collagenase activity by 1.2 folds through Response Surface Methodology (RSM) and 5.33 folds through purification of enzyme using ammonium sulfate precipitation and DEAE-Sepharose chromatography (specific activity with 538.0 U/mg). SDS-PAGE analysis identified its molecular weight as 32 kDa. Optimal conditions for the enzyme's activity were pH 7.5 and 40 °C. Kinetic studies of collagenase KU665299 revealed specificity for collagen, with K_m and V_max values of 0.059 mg/l and 588.24 µmol/min/mg, respectively. Zinc and calcium ions enhanced activity, while EDTA and DTT strongly inhibited it. The purified collagenase demonstrated remarkable efficiency in digesting blood clots, fully dissolving 1 ml clots within 40 min at 37 °C, showcasing significant thrombolytic potential.

Conclusion: The study successfully optimized and characterized a novel collagenase from C. contaminans KU665299, revealing its high specificity, stability, and efficiency in degrading collagen and its promising ability to rapidly digest blood clots for potential thrombolytic properties.

The CRISPR/Cas9 system facilitates precise genome editing in various organisms. In this study, a single-vector CRISPR/Cas9 system was developed for Saccharomyces cerevisiae, employing a type II Cas9 enzyme from Streptococcus pyogenes and a single-guide RNA cassette targeting CAN1.Y locus on chromosome V. This system is broadly applicable across yeast strains, as it utilizes G418 selection, eliminating the need for auxotrophic markers. The efficiency of the CRISPR/Cas9 system was demonstrated, with editing efficiencies ranging from 70 to 100%. This system was utilized to integrate a cassette encoding secretory pectate lyase (PL) from Bacillus subtilis 168 into the yeast genome. The engineered S. cerevisiae strain secreted active PL, which exhibited pectin-degrading activity characterized by significant reductions in residual pectin and increased production of reducing sugars. Since pectin constitutes a major component of coffee mucilage, the secreted PL was applied to coffee beans for mucilage removal. The treated beans presented noticeably reduced residual mucilage, a purer green color, and decreased viscosity. These findings suggest the potential of the engineered S. cerevisiae strain for applications in coffee processing, particularly in efficient mucilage removal.

Baculovirus bacmids have been widely used in over-expression and gene deletion. Traditionally, baculovirus bacmids are developed by inserting an 8.6 kbp bacterial DNA cassette into baculovirus genomes either through homologous recombination in cultured cells or via in vitro cloning. In this study, by introducing Bsu36i-attached egfp to the 8.6 kbp bacterial DNA cassette, we develop a novel method for generating baculovirus bacmids. An 11.6 kbp bacterial DNA cassette containing the introduced egfp was used to generate an intermediate bacmid. With the EGFP reporter, purification was performed in cultured cells, increasing the proportions of recombinants. The intermediate bacmid containing the 11.6 kbp bacterial DNA cassette was obtained by transforming DH10B competent cells with viral DNA after 3 rounds of purification. The intermediate bacmid DNA was linearized by digestion with Bsu36i and then was co-transfected with the PCR-amplified 8.6 kbp bacterial cassette into BmN cells, where homologous recombination occurred between them. The final BmNPV bacmid was obtained by transforming DH10B competent cells with viral DNA. Capable of increasing the proportions of recombinants via purification and linearization, this method has great potential to be used for bacmid generation for baculoviruses, especially those that are not capable of producing high titers of viruses.

Purpose: High-throughput screening methods for cytochrome P450 enzymes (P450s), such as colorimetric, mass spectrometric, and fluorescence-based assays, often face limitations in throughput, real-time monitoring, and versatility.

Methods: To address these challenges, we developed a novel biosensor leveraging glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation for real-time monitoring of intracellular NADP⁺ levels, enabling P450 activity detection. The sensor was applied to monitor P450 activity by tracking intracellular NADP⁺ dynamics, as P450s catalyze diverse substrate reactions and convert NADPH to NADP⁺ via their electron transport system. To enhance detection precision, intracellular NADP⁺ synthesis was reduced by knocking down NADPH-dependent aldehyde reductase (YqhD), minimizing background fluorescence interference.

Results: The sensor exhibited a linear NADP⁺ detection range of 1 μM to 10 mM, suitable for P450 assays. The sensor's performance was validated by comparing P450 activities in engineered strains with traditional gas chromatography.

Conclusion: The developed biosensor demonstrates its potential as a robust, real-time screening tool for P450 enzyme studies.