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.

Yangpumicins (YPMs), eg. YPM A, F, and G, are newly discovered enediynes from Micromonospora yangpuensis DSM 45577, which could be exploited as promising payloads of antibody-drug conjugates. However, the low yield of YPMs in the wild-type strain (~1 mg/L) significantly hampers their further drug development. In this study, a combined ribosome engineering and fermentation optimization strategy has been used for yield improvement of YPMs. One gentamycin-resistant M. yangpuensis DSM 45577 strain (MY-G-1) showed higher YPMs production (7.4 ± 1.0 mg/L), while it exhibits delayed sporulation and slender mycelium under scanning electron microscopy. Whole genome re-sequencing of MY-G-1 reveals several deletion and single nucleotide polymorphism mutations, which were confirmed by PCR and DNA sequencing. Further Box–Behnken experiment and regression analysis determined that the optimal medium concentrations of soluble starch, mannitol, and pharmamedia for YPMs production in shaking flasks (10.0 ± 0.8 mg/L). Finally, the total titer of YPM A/F/G in MY-G-1 reached to 15.0 ± 2.5 mg/L in 3-L fermenters, which was about 11-fold higher than the original titer of 1.3 ± 0.3 mg/L in wild-type strain. Our study may be instrumental to develop YPMs into a clinical anticancer drug, and inspire the use of these multifaceted strategies for yield improvement in Micromonospora species.

Many transgenic animals have been produced using CRISPR–Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique in cattle. The goal of this study was to produce trait-improved cattle using the genome editing technology CRISPR–Cas9. Myostatin (MSTN) was selected as a target locus and synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in vitro fertilized embryos. As a result, 17 healthy calves were born and 3 of these showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had biallelic mutation (-12bp) and a doubling muscle growth phenotype. In conclusion, we showed that the genome editing technology CRISPR–Cas9 can produce genetically modified calves with improved traits.

In the development of personalized medicine, the ultrasensitive detection of point mutations that correlate with diseases is important to improve the efficacy of treatment and guide clinical medication. In this study, locked nucleic acid (LNA) was introduced as an amplification suppressor of a massive number of wild-type alleles in an amplification refractory mutation system (ARMS) to achieve the detection of low-abundance mutations with high specificity and sensitivity of at least 0.1%. By integrating the length of clamp, base type, number and position of LNA modifications, we have established a “shortest length with the fewest LNA bases” principle from which each LNA base would play a key role in the affinity and the ability of single base discrimination could be improve. Finally, based on this LNA design guideline, a series of the most important single point mutation sites of epidermal growth factor receptor (EGFR) was verified to achieve the optimal amplification state which as low as 0.1% mutation gene amplification was not affected under the wild gene amplification was completely inhibited, demonstrating that the proposed design principle has good applicability and versatility and is of great significance for the detection of circulating tumor DNA.

Abstract: 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important industrial derivative of L-ascorbic acid (AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O- glycoside hydrolases, may be used in glycosylation reactions to synthesize AA-2G. Here, one α-glucosidase from Oryza sativa (rAGL) was recombinantly produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The specific activity of purified rAGL is 49.83 U/mg at 37 °C and pH 4.0. The optimal temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the basis for the industrial application of recombinant α-glucosidase. Keywords: α-Glucosidase; Oryza sativa; 2-O-α-D-glucopyranosyl-L-ascorbic acid; Transglycosylation; Pichia pastoris

Recent noteworthy advances in the development of high-performing microbial and mammalian strains have enabled the sustainable production of bio-economically valuable substances such as bio-compounds, biofuels, and biopharmaceuticals. However, to obtain an industrially viable mass-production scheme, much time and effort are required. The robust and rational design of fermentation processes requires analysis and optimization of different extracellular conditions and medium components, which have a massive effect on growth and productivity. In this regard, knowledge- and data-driven modeling methods have received much attention. Constraint-based modeling (CBM) is a knowledge-driven mathematical approach that has been widely used in fermentation analysis and optimization due to its capabilities of predicting the cellular phenotype from genotype through high-throughput means. On the other hand, machine learning (ML) is a data-driven statistical method that identifies the data patterns within sophisticated biological systems and processes, where there is inadequate knowledge to represent underlying mechanisms. Furthermore, ML models are becoming a viable complement to constraint-based models in a reciprocal manner when one is used as a pre-step of another. As a result, more predictable models are produced. This review highlights the applications of CBM and ML independently and the combination of these two approaches for analyzing and optimizing fermentation parameters.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety Level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of ≥ 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2-3 days versus 4-7 months.

Because of their excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

We herein describe an ingenious centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was automatically regulated by utilizing ferrowax microvalves systematically integrated into the channels within the specially designed metering structure. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and ten-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within five minutes, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. The centrifugal microfluidic system would serve as a powerful elemental tool to realize the fully automated diagnostic microsystem involving the serial dilution process.

Difference between two strong cation-exchange resins, namely sulfonium type and sulfate type regarding both their salt tolerance and hydrophobicity were investigated. There is only tiny variation between sulfate and sulfonic group and at the first glance it seems unlikely that it could be the reason for changed selectivity and salt tolerance that was detected in our preliminary experiments. For that reason salt tolerance and hydrophobicity of both ligands was investigated by using two representative polymethacrylate-based ion exchangers as for the sulfonium type TOYOPEARL GigaCap S-650M and for the sulfate type TOYOPEARL Sulfate-650F. In addition some in-silico calculations were performed for model substances representing the sulfonium and sulfate group, and significant differences were calculated regarding their hydrophobicity. These experiments confirmed the working hypothesis that salt tolerance and higher affinity and selectivity for some human plasma derived vitamin K dependent clotting factors and inhibitors are interrelated and dependent from the presence of the sulfate group. The affinity for these proteins was experimentally verified by separation of clotting factor IX from the prothrombin complex concentrate. Presented results show that a simple and fast separation between clotting factor IX and other vitamin K dependent clotting factors II, VII and X is possible, only if the resin with the sulfate, and not with sulfonic acid ligand was applied. Consequently, an immediate application of undiluted feedstock or the eluate from previous isolation step to sulfate resin is possible, and a significant optimization of downstream process can be achieved.