Biotechnology Journal

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging...

Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator-tRNA complexes in living cells. Our strategy allows the generation of Fluo-Met-tRNAiMet complexes. These complexes can have highly chromogenic N-terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non-purified fluorescently labeled methionine (NPFM)...

Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab...

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology...

The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions.  To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. Saccharomyces cerevisiae culture was exposed to 8 pulses of 100 µs width with electric field strength magnitude 2-7 kV cm-1 ...

Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production. To identify barriers, we analyzed the purification performance and economics of 290 phase separation purifications from 168 publications. First, we found that studies using Design of Experiments for optimization achieved significantly greater mean yield and host cell protein log10 removal values than those optimizing one factor at a time (11.5% and 53% increases, respectively)...

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth...

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs...

The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, this study provides an innovative antibacterial treatment strategy by synergistically combining R12-AgNPs with H2 O2 therapy. R12-AgNPs were simply produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature...

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing...

Polyurethane (PU) is a complex polymer synthesized from polyols and isocyanates. It contains urethane bonds that resist hydrolysis, which decreases the efficiency of biodegradation. In this study, we first expressed the amidase GatA250, and then, assessed the enzymatic characterization of GatA250 and its efficiency in degrading the polyester-PU. GatA250 degraded self-synthesized thermoplastic PU film and postconsumption foam with degradation efficiency of 8.17% and 4.29%, respectively. During the degradation, the film released 14...

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification...

Natural bone tissue features a complex mechanical environment, with cells responding to diverse mechanical stimuli, including fluid shear stress (FSS) and hydrostatic pressure (HP). However, current in vitro experiments commonly employ a singular mechanical stimulus to simulate the mechanical environment in vivo. The understanding of the combined effects and mechanisms of multiple mechanical stimuli remains limited. Hence, this study constructed a mechanical stimulation device capable of simultaneously applying FSS and HP to cells...

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E...

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers...

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested...

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains...

The bacterium Sporosarcina pasteurii is the most commonly used microorganism for Microbial Induced Calcite Precipitation (MICP) due to its high urease activity. To date, no proper fed-batch cultivation protocol for S. pasteurii has been published, even though this cultivation method has a high potential for reducing costs of producing microbial ureolytic biomass. This study focusses on fed-batch cultivation of S. pasteurii DSM33. The study distinguishes between limited fed-batch cultivation and extended batch cultivation...

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20...

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+ -NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan...