Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range that had similar spectra shape across all probes was first constructed, which improved the prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales into the model, reducing the prediction bias. External parameter orthogonalization also mitigated the interference from feeding, which further improved the model performance. The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. In addition, incorporation of at-line spectra following orthogonal design into the calibration set is more likely to generate robust calibration models than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectra collection is easier, faster, and more material-sparing than in-line spectra collection. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%.
Raman spectroscopy has gained popularity to monitor multiple process indicators simultaneously in biopharmaceutical processes. However, robust and specific model calibration remains a challenge due to insufficient analyte variability to train the models and high cross-correlation of various media components and artefacts throughout the process. Therefore, a systematic Raman calibration workflow for perfusion processes enabling highly specific and fast model calibration was developed. A harvest library consisting of frozen harvest samples from multiple CHO cell culture bioreactors collected at different process times was established, capturing process variability as widely as possible. Model calibration was subsequently performed in an offline setup using a flow cell by spiking process harvest with various sugars known to modulate glycosylation patterns of monoclonal antibodies. In a screening phase, Raman spectroscopy was proven capable not only to distinguish glucose, raffinose, galactose, mannose, and fructose in perfusion harvest, but also to quantify them independently in process relevant concentrations. In a second phase, a robust and highly specific calibration model for simultaneous glucose (RMSEP = 0.32 g/L) and raffinose (RMSEP = 0.17 g/L) real-time monitoring was generated and verified in a third phase during a perfusion process. The proposed offline calibration workflow allowed proper Raman peak decoupling, reduced calibration time from months down to days and can potentially be applied to other analytes of interest including lactate, ammonia, amino acids, or product titer.
Protein-based condensates have been proposed to accelerate biochemical reactions by enriching reactants and enzymes simultaneously. Here, we engineered those condensates into a Photo-Activated Switch in E. coli (PhASE) to regulate enzymatic reactions via tuning the spatial correlation of enzymes and substrates. In this system, scaffold proteins undergo liquid-liquid phase separation (LLPS) to form light-responsive compartments. Tethered with a light-responsive protein, enzymes of interest (EOIs) can be recruited by those compartments from cytosol within only a few seconds after a pulse of light induction and fully released in 15 minutes. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3 folds and 1.6 folds, respectively. We also developed a quantitative model to guide the further optimization of this de-mixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli.
Immobilized enzymes have drawn widespread attention due to the enhanced stability, easy separation from reaction mixture, and the prominent recyclability. Nevertheless, it is still an ongoing challenge to develop potent immobilization techniques which are capable of stable enzyme encapsulation, minimal loss of activity, and modulability for various enzymes and applications. Here, microfibers with tunable size and composition were fabricated using a home-made microfluidic device. These microfibers were able to efficiently encapsulate bovine serum albumin (BSA), glucose oxidase (GOX) and horseradish peroxidase (HRP). But the physically adsorbed enzymes readily diffused from microfibers into the catalytic reaction system. The leakage of enzymes could be substantially inhibited by conjugating to polyacrylic acid (PAA) and incorporating into the alginate-based microfibers, enabling stable immobilization, improved recyclability, and enhanced thermostability. In addition, GOX and HRP-loaded microfibers were fabricated under the optimized conditions for the visual detection of glucose using the cascade reaction of these enzymes, showing sensitive color change to glucose with concentration range of 0-2 mM. Due to the tunability and versatility, this microfluidic-based microfiber platform may provide a valuable approach to the enzyme immobilization for the cascade catalysis and diagnoses with multiple clinical markers.
The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome (=plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations, and this will present issues with respect to selectable markers: there are a limited number of markers available, these can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative marker system for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the marker’s 3’UTR and the 3’UTR of the introduced transgene(s). We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.
Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases.
Phaeodactylum tricornutum is a marine diatom, and well-studied model of unicellular microalga. This diatom contains a wide range of high-value renewables (HVRs) with high commercial relevance owing to their importance in human nutrition and health. In this study, we screened P. tricornutum for biomass, eicosapentaenoic acid (EPA) and fucoxanthin production under photoautotrophic and mixotrophic condition with various substrate combinations. Results highlights that culture supplemented with glycerol and urea lead to enhanced biomass, biochemical and HVR production. Further continuous feeding of urea in glycerol supplemented medium results in an increase in biomass yield (0.77 g L-1) by ~ 2-fold. Additionally, continuous feeding of urea channelizes the carbon flux towards biosynthesis of fatty acids increasing FAME content by ~2-fold as compared to the control conditions. Overall EPA and fucoxanthin production was 27 mg L-1 and 11 mg L-1 (~2 & 4 fold) in urea fed cultures respectively. Present study demonstrates efficient valorization of cost-effective substrates such as glycerol and urea for the production of high-value renewables in P. tricornutum.
Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with ten different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.
The signaling molecular mechanisms in zebrafish response to electricity are unknown, so here we asked if changes to dopaminergic signaling pathways can affect their electrically-evoked locomotion. To answer this question, the effects of multiple selective and non-selective dopamine compounds on the electric response of zebrafish larvae is investigated. A microfluidic device with enhanced control of experimentation with multiple larvae is used, which features a novel design to immobilize four zebrafish larvae in parallel and expose them to electric current that induces tail locomotion. In 6 days post-fertilization zebrafish larvae, the electric induced locomotor response is quantified in terms of the tail movement duration and beating frequency to discern the effect of non-lethal concentrations of dopaminergic agonists (apomorphine, SKF-81297, and quinpirole), and antagonists (butaclamol, SCH-23390, and haloperidol). All dopamine antagonists decrease locomotor activity, while dopamine agonists do not induce similar behaviours in larvae. The D2- like selective dopamine agonist quinpirole enhances movement. However, exposure to non-selective and D1-selective dopamine agonists apomorphine and SKF-81297 cause no significant change in the electric response. Exposing larvae that were pre-treated with butaclamol and haloperidol to apomorphine and quinpirole, respectively, restores electric locomotion. The results demonstrate a correlation between electric response and the dopamine signalling pathway. We propose that the electrofluidic assay has profound application potential as a chemical screening method when investigating biological pathways, behaviors, and brain disorders.
The effects of climate change, soil depletion, a growing world population putting pressure on food safety and security are major challenges for agriculture in the 21st century. The breeding success of the green revolution has decelerated and current programs can only offset the yield affecting factors. New approaches are urgently needed and we propose, “Genome Editing accelerated Re-Domestication” (GEaReD) as a major new direction in plant breeding. By combining the upcoming technologies for phenotyping, omics and artificial intelligence with the promising new CRISPR-toolkits, this approach is closer than ever. Wild relatives of current crops are often adapted to harsh environments and have a high genetic diversity. Re-domestication of wild barley or teosinte could generate new cultivars adapted to environmental changes. De novo domestication of perennial relatives like Hordeum bulbosum could counter with soil depletion and increase soil carbon. Recent research already proved the principle of re-domestication in tomato and rice and therefore laid the foundation for GEaReD.
Food security is one of the main topics of today's agriculture especially facing challenging environmental conditions. As most humankind has a daily intake of cereal grains, current breeding programs focus on these crop plants. Within the breeders' toolbox, customised endonucleases became included after this universal application had been demonstrated. Due to technological restrictions, the main focus was on aboveground plant organs, while the essential belowground has been given only limited attention. In the present review, we summarise the knowledge on the root system architecture in cereals, the importance of phytohormones in this physiological process, and the molecular mechanisms involved. The review summarises how the use of the CRISPR methodology can improve the root system architecture to enhance crop production genetically. Finally, future research directions involving all this knowledge and technical advances are suggested.
Genome editing and gene expression engineering using CRISPR-Cas systems in plants usually rely on labor-intensive tissue culture approaches to generate stably transformed plants that express the components of the reaction. Viral vectors have demonstrated to be a quick and effective alternative to express multiple guide RNAs, DNA templates for homologous recombination, and even Cas nucleases. Here we have developed an improved vector system based on tobacco rattle virus (TRV) to simplify logistics in genome editing and gene silencing approaches. The new system consists in a single Agrobacterium tumefaciens clone co-transformed with two compatible mini binary vectors from which TRV RNA1 and an engineered version of TRV RNA2 are expressed. Sequences of recombinant proteins, gene fragments for virus-induced gene silencing (VIGS) or guide RNAs can be easily inserted by one-step digestion-ligation and homology-based cloning methods in the RNA2 plasmid to produce vectors with a size substantially smaller than usual. Using this new one-Agrobacterium TRV mini vector system, we show robust VIGS of an endogenous host gene after infiltration of bacterial suspensions at low optical densities, and efficient production of recombinant proteins in Nicotiana benthamiana. Most importantly, we also show highly efficient heritable genome editing in more than half of the seedling originating from inoculated N. benthamiana plants that express Cas9.
Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation, which is based on in-situ separation, is a promising technology for achieving high SLs productivity. In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and rapeseed oil was a key factor in sedimentation. And the hydrophobicity and density of the mixture determined SLs sedimentation rate. On this basis, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9% to 485.4% with different ratio of rapeseed oil to SLs. UEST-assisted real-time in-situ separation and semi-continuous fermentation were performed. SLs productivity and yield were 2.15 g/L/h and 0.58 g/g, respectively, simultaneously the loss ratio of cells, glucose, and rapeseed oil were significantly reduced. This study provides the new horizon for optimization of the SLs fermentation process.
Seamless modification of bacteria chromosome is widely performed both in theoretical and in practical research, for this purpose, excellent counter-selection marker genes with high selection stringency are needed. Lysis gene E from bacteriophage PhiX174 was developed and optimized as a counter-selection marker in this paper. Lysis gene E was firstly constructed under the control of pL promoter. At 42 °C, Lysis gene E could effectively kill Escherichia coli. Seamless modification using E as a counter-selection marker also successfully conducted. It also works in another Gram-negative strain Serratia marcescens under the control of Arac/PBAD regulatory system. Through combining lysis gene E and kil, the selection stringency frequency of pL-kil-sd-E cassette in E. coli arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close to the best counter-selection system, inducible toxins system. Under the control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S. marcescens arrived at the level of 10−7 at four test loci. By introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold improvement of selection stringency was observed at these loci, and a surprising low selection stringency frequency 4.9×10−9 was obtained at marR-1 locus, the lowest selection stringency frequency for counter-selection reported so far. Similarly, at araB locus of E. coli selection stringency frequency of PBAD-kil-sd-E was improved to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have developed and optimized a newly universal counter-selection marker based on lysis gene E. The best selection stringency of this new marker exceeds the inducible toxins system several fold.
Tuberculosis (TB) and its emerged drug resistance exert huge threats on the global health, therefore development of novel anti-TB antibiotics is very essential. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine-derived Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and become promising anti-TB lead compounds due to their significant anti-TB activities, but their low titer hampered the further clinical development. In this work, the statistical Plackett-Burman design (PBD) model was applied to screen out bacterial peptone as the only significant but negative factor affecting the ilamycin-E1/E2 production. Subsequent single factor optimization revealed that replacement of bacterial peptone with malt extract eliminated the accumulation of porphyrin-type competitive byproduct, and the titer of ilamycin-E1/E2 in shaking flasks was improved from original 13.6±0.8 to 142.7±5.7 mg/L for about 10.5 folds. Furthermore, a pH coordinated feeding strategy was first adopted in scaled-up production of ilamycin-E1/E2. The obtained titer of ilamycin-E1/E2 in 30L was 169.8±2.5 mg/L, while in 300L fermentor was only 131.5±7.5 mg/L due to the unsynchronization of feeding response and pH change. Therefore, the continuous pulse feeding strategy was further applied in 300L fermentor and finally achieved 415.7±29.2 mg/L ilamycin-E1/E2, which represented about 30.5 folds improvement at last. Our work provided the solid basis to achieve sufficient ilamycin-E1/E2 lead compounds and support their potential anti-TB drug development.
Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high-throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien-5α-ol and Taxadien-5α-yl-acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1L bioreactors leading to a 2.1-fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of a nutrient limitation enhanced product titers a further two-fold and titers of the critical Taxol precursors, Taxadien-5α-ol and Taxadien-5α-yl-acetate were improved to 34 and 11 mg/L, representing a three-fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five-fold using 5 L bioreactors. The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.
Background: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production has enabled candidate mAbs to be identified at a rapidly increasing rate resulting in a significant bottleneck in the characterization of ‘structural’ epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. Main methods and major results: The method is demonstrated on set of 6 candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes was unambiguously determined using two HDX mixing timepoints in 24 hours total run time, corresponding to substantial decrease in the instrument time required to map a single epitope using conventional HDX workflows. Conclusion: An accelerated HDX-MS epitope screening workflow was developed. The two-timepoint ‘screening’ workflow mapped all six mAbs and generated high confidence epitopes for five of the six mAbs assayed. The substantial improvement in the rate of data collection can advance HDX-MS for higher throughput investigations supporting the ability to evaluate a broader number of mAb candidates at an earlier stage of vaccine development.
Carotenoids and tocopherols are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. Here, we demonstrate an alternative biotechnological strategy for carotenoid and tocopherol fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated in zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (pro-vitamin A), lutein and phytoene, in both rind and flesh. Considerably higher accumulation of α- and γ-tocopherol was also detected, particularly in fruit rind. Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.