New biotechnology最新文献

Poly(3-hydroxyalkanoate) (PHA), a bacteria-synthesized biodegradable polyester, is a useful alternative to fossil resources, and current systems for its production rely predominantly on edible resources, raising concerns about microbial competition for nutrients. Therefore, we investigated mechanisms underlying PHA production from non-edible resources by Piscinibacter gummiphilus strain NS21^T. Strain NS21^T can utilize natural rubber as a carbon source on solid media and potentially produces PHA. Gas chromatography and nuclear magnetic resonance analyses of NS21^T cell extracts revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate) from natural rubber and glucose, respectively. Transcriptional analysis suggested that phaC is involved in PHA production. An increased PHBV accumulation rate under nitrogen-limiting conditions indicates the potential of this strain to be used as a PHBV production enhancement strategy. Furthermore, the disruption of PHA depolymerase genes resulted in enhanced PHA production, indicating the involvement of these genes in PHA degradation. These findings highlight the potential of NS21^T for PHBV production from natural rubber, a non-edible resource.

Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development.

Research on the biodegradation of polyethylene (PE), polystyrene (PS) and related polymers has become popular and the number of publications on this topic is rapidly increasing. However, there is no convincing evidence that the frequently claimed biodegradability of these so-called “plastics” really exists. Rather, a diffuse definition of the term “biodegradability” has led to the publication of reports showing either marginal weight losses of hydrocarbon polymers by the action of isolated bacterial strains or mechanical disintegration and polymer surface modification in case of hydrocarbon polymer-consuming insect larvae. Most of the data can be alternatively explained by the utilization of polymer impurities/additives, by the utilization of low molecular weight oligomers, and/or by physical fragmentation and subsequent loss of small fragments. Evidence for a (partial) biotic and/or abiotic oxidation of the amorphous polymer fraction and of surface-exposed hydrocarbon side chains is not sufficient to claim that PE is biodegradable. To the best of my knowledge, no report has been so far published in which substantial biodegradation and mineralization of PE or related (long chain length) hydrocarbon polymers to carbon dioxide has been convincingly demonstrated by the determination of the fate of carbon atoms in isotope-labeled polymers. It is disappointing that publications with a critical view on biodegradation of hydrocarbon polymers are not cited in most of these reports. The possibility should be considered that the rapidly expanding research field of hydrocarbon polymer biodegradation is chasing rainbows.

Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng.

Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7 µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation.

The discovery of unspecific peroxygenases (UPOs) completely changed the paradigm of enzyme-based oxyfunctionalization reactions, as these enzymes can transform a wide variety of substrates with a relatively simple reaction mechanism. The fact that UPO can exert both peroxygenative and peroxidative activity in either aromatic or aliphatic carbons, represents a great potential in the production of high value-added products from natural antioxidants. In this work, the flavonoid rutin has been considered as possible substrate for UPO from Agrocybe aegerita, and its peroxygenation or its peroxidation and successive oligomerization have been studied. Different experiments were performed in order to reduce the range of process variables involved and gaining insight on the behavior of this enzyme, leading to a multivariable optimization of UPO-based rutin modification. While trying to preserve enzyme activity this optimization aimed for maximizing the production of more soluble antioxidants. Reusability of the enzyme was evaluated recovering UPO using an enzymatic membrane reactor, revealing challenges in enzyme stability due to inactivation during the filtration stages. The influence of the radical scavenger ascorbic acid on product formation was investigated, revealing its role in directing the reaction towards hydroxylated rutin derivatives, hence indicating a shift towards more soluble and bioactive products.

Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles’ function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5’ UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch – a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Polyhydroxyalkanoates (PHAs) are biopolymers produced by microorganisms under nutrient limiting conditions and in the presence of excess carbon source. PHAs have gained popularity as a sustainable alternative to traditional plastics. However, large scale production of PHAs is economically challenging due to the relatively high costs of organic carbon. Alternative options include using organisms capable of phototrophic or mixotrophic growth. This study aimed at the production of poly(3-hydroxybutyrate) P(3HB), a type of PHA, at pilot scale using the freshwater cyanobacterium Synechocystis sp. PCC6803. First, to identify optimal conditions for P(3HB) production from Synechocystis sp. PCC6803, different supplemental carbon source concentrations and salinity levels were tested at laboratory scale. The addition of 4 g/L acetate with no added NaCl led to P(3HB) accumulation of 10.7 % dry cell weight on the 28th day of cultivation. Although acetate additions were replicated in an outdoor 400 L serpentine photobioreactor, P(3HB) content was lower, implying uncontrolled conditions impact on biopolymer production efficiency. An optimized P(3HB) extraction methodology was developed to remove pigments, and the biopolymer was characterized and subjected to 3D printing (fused deposition modelling) to confirm its processability. This study thus successfully led to the large-scale production of P(3HB) using sustainable and environmentally friendly cyanobacterial fermentation.