Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids.

Biosensors are powerful tools for detecting, real-time imaging, and quantifying molecules, but rapidly constructing diverse genetically encoded biosensors remains challenging. Here, we report a method to rapidly convert enzymes into genetically encoded circularly permuted fluorescent protein-based indicators to detect organic acids (GECFINDER). ANL superfamily enzymes undergo hinge-mediated ligand-coupling domain movement during catalysis. We introduce a circularly permuted fluorescent protein into enzymes hinges, converting ligand-induced conformational changes into significant fluorescence signal changes. We obtain 11 GECFINDERs for detecting phenylalanine, glutamic acid and other acids. GECFINDER-Phe3 and GECFINDER-Glu can efficiently and accurately quantify target molecules in biological samples in vitro. This method simplifies amino acid quantification without requiring complex equipment, potentially serving as point-of-care testing tools for clinical applications in low-resource environments. We also develop a GECFINDER-enabled droplet-based microfluidic high-throughput screening method for obtaining high-yield industrial strains. Our method provides a foundation for using enzymes as untapped blueprint resources for biosensor design, creation, and application.

A homolog of AtCBFs, SmDREB A1-4, positively regulates salt stress tolerance in Arabidopsis thaliana and Salix matsudana.

CBFs (C-repeat binding factors) have multiple functions in abiotic stress adaption; functional research of these genes will provide precious gene resources for plant genetic improvement. In this study, a homolog of AtCBFs, SmDREB A1-4 was cloned and its role in salt tolerance was explored. SmDREB A1-4 is a member of DREB A1 subgroup with 10 members. SmDREB A1-4 localized in nuclei and cytoplasm and expressed ubiquitously in different tissue and organs. The expression level of SmDREB A1-4 could be induced by NaCl treatment and the TC-rich repeat and DREB motif on the SmDREB A1-4 gene promoter may mediate the NaCl-induced expression pattern. Overexpression of the SmDREB A1-4 gene in Arabidopsis enhanced the salt tolerance of transgenic Arabidopsis lines, while down-regulated the expression level in Salix plantlets by Virus induce gene silencing decreased the salt tolerance capacity in VIGS Salix plantlets. Experiments data from both sides confirmed that SmDREB A1-4 is a positive regulatory factor in salt stress tolerance. qRT-PCR and luciferase reporter assays revealed that SOS1 and DREB2A are downstream genes of SmDREB A1-4. Through upregulating the expression of SOS1 and DREB2A, SmDREB A1-4 enhanced plant tolerance to salinity by regulating ion homeostasis, reduction of Na+/K+ ratio, and improvement of proline biosynthesis. This research offers a potentially valuable gene resource for the stress-resistant varieties breeding of Salix matsudana in the future.

Whole-Cell Biosensor and Producer Co-cultivation-Based Microfludic Platform for Screening Saccharopolyspora erythraea with Hyper Erythromycin Production.

Actinomycetes are versatile secondary metabolite producers with great application potential in industries. However, industrial strain engineering has long been limited by the inefficient and labor-consuming plate/flask-based screening process, resulting in an urgent need for product-driven high-throughput screening methods for actinomycetes. Here, we combine a whole-cell biosensor and microfluidic platform to establish the whole-cell biosensor and producer co-cultivation-based microfluidic platform for screening actinomycetes (WELCOME). In WELCOME, we develop an MphR-based Escherichia coli whole-cell biosensor sensitive to erythromycin and co-cultivate it with Saccharopolyspora erythraea in droplets for high-throughput screening. Using WELCOME, we successfully screen out six erythromycin hyper-producing S. erythraea strains starting from an already high-producing industrial strain within 3 months, and the best one represents a 50% improved yield. WELCOME completely circumvents a major problem of industrial actinomycetes, which is usually genetic-intractable, and this method will revolutionize the field of industrial actinomycete engineering.

[Advances in automated high-throughput editing and screening of engineered strains].

One of the revolutionary features of synthetic biology is that the standardization and modularization of biological experimental objects, methods, technologies and processes can be combined with various software and hardware to forge into an automated high-throughput synthetic biology biofoundry. Disrupting the conventional labor-intensive research paradigm, biofoundry represents a novel research paradigm with highly enhanced technical iteration capabilities, and remarkably promotes the development and industrial applications of synthetic biology. On the occasion of the 10th anniversary of the founding of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, this review summarized a series of important achievements in the field of automated high-throughput editing and screening of industrial strains. These achievements range from automated editing technologies such as gene cloning, genome editing, and editing sequence design, to high-throughput screening technologies such as fluorescence activated cell sorting, fluorescence activated droplet sorting, and genome-scale gene perturb sequencing. Moreover, we prospected future development of this field, hoping to provide overall support for intelligent, automated and full chain integrated creation of excellent industrial strains with intellectual property rights.

Hydroxysafflor Yellow A Promoted Bone Mineralization and Inhibited Bone Resorption Which Reversed Glucocorticoids-Induced Osteoporosis.

Glucocorticoids intake is the most common cause of secondary osteoporosis. Clinical studies have shown that 50% patients develop glucocorticoids-induced osteoporosis (GCIOP) after taking glucocorticoids for more than 6 months. Hydroxysafflor yellow A (HYA) is one main active ingredient in Carthamus tinctorius L. Previous studies have shown that HYA promoted bone marrow mesenchymal stem cells to differentiate into osteoblasts which promoted bone formation. Therefore, we speculated that HYA has a therapeutic effect on GCIOP. However, there is no in vivo evidence about the anti-GCIOP effect of HYA. In this paper, the effect of HYA (0.1, 1.0, and 10.0 µM) on bone formation in normal zebrafish was investigated firstly. Secondly, the reversal effect of HYA on GCIOP was also evaluated by zebrafish model. It is demonstrated that HYA not only promoted bone formation in normal zebrafish (compared to Control group), but also reversed glucocorticoid induced bone loss (compared to Prednisolone group) according to the intervention of HYA in upregulating the area of mineralized bones (p < 0.01), increasing cumulative optical density (p < 0.01), promoting bone formation related gene expression (AKP, Type I, Runx2, OPG, and OCN, p < 0.01), inhibiting bone resorption related gene expression (TRACP, p < 0.01), and elevating whole-body trace mineral elements (Ca, P, K, Mg, Zn, and Fe) levels (p < 0.01). In conclusion, HYA had the potential to prevent and heal GCIOP by promoting bone mineralization, osteoblasts viability, and bone collagen expression and inhibiting bone resorption.

An Alfin-like gene from Atriplex hortensis enhances salt and drought tolerance and abscisic acid response in transgenic Arabidopsis.

Alfin-like (AL) is a small plant-specific gene family with prominent roles in root growth and abiotic stress response. Here, we aimed to identify novel stress tolerance AL genes from the stress-tolerant species Atriplex hortensis. Totally, we isolated four AhAL genes, all encoding nuclear-localized proteins with cis-element-binding and transrepression activities. Constitutive expression of AhAL1 in Arabidopsis facilitated plants to survive under saline condition, while expressing anyone of the other three AhAL genes led to salt-hypersensitive response, indicating functional divergence of AhAL family. AhAL1 also conferred enhanced drought tolerance, as judged from enhanced survival, improved growth, decreased malonaldehyde (MDA) content and reduced water loss in AhAL1-expressing plants compared to WT. In addition, abscisic acid (ABA)-mediated stomatal closure and inhibition of seed germination and primary root elongation were enhanced in AhAL1-transgenic plants. Further analysis demonstrated that AhAL1 could bind to promoter regions of GRF7, DREB1C and several group-A PP2C genes and repress their expression. Correspondingly, the expression levels of positive stress regulator genes DREB1A, DREB2A and three ABFs were all increased in AhAL1-expressing plants. Based on these results, AhAL1 was identified as a novel candidate gene for improving abiotic stress tolerance of crop plants.

Photoenhanced gene transfection by a curcumin loaded CS-g-PZLL micelle.

The codelivery of drug and gene is a promising method for cancer treatment. In our previous works, we prepared a cationic micelles based on chitosan and poly-(N-3-carbobenzyloxylysine) (CS-g-PZLL), but transfection ratio of CS-g-PZLL to Hela cell was low. Herein, to improve the transfection efficiency of CS-g-PZLL, curcumin was loaded in the CS-g-PZLL micelle. After irradiation, the obtained curcumin loaded micelle showed a better transfection, and the p53 protein expression in Hela cells was higher. The apoptosis assay showed that the complex could induce a more significant apoptosis to Hela cells than that of curcumin or p53 used alone, and the curcumin loaded micelle inducing apoptosis was best after irradiation. Therefore, CS-g-PZLL is a safe and effective carrier for the codelivery of drug/gene, and curcumin could be used as a photosensitizer to induce a photoenhanced gene transfection, which should be encouraged in improving transfection and tumor therapy.

Soybean NIMA-Related Kinase1 Promotes Plant Growth and Improves Salt and Cold Tolerance.

NEK (NIMA-related kinase) is known as a family of serine/threonine kinases which mainly participate in microtubule-related mitotic events in fungi, mammals and other eukaryotes. Our previous studies found that Arabidopsis NEK6 plays an important role in plant response to abiotic stress. We further investigated roles of the NEK family in soybean and found that at least eight members can respond to abiotic stresses. Among them, only GmNEK1, a novel NEK member which is distantly related to Arabidopsis NEK6, enhanced plant growth and promoted salt and cold tolerance in transgenic Arabidopsis plants. The growth of soybean plants harboring GmNEK1-overexpressing hairy roots under saline condition was also improved. A series of stress-related genes including RH3, CORI3 and ALDH10A8 were found to be up-regulated in GmNEK1-overexpressing Arabidopsis plants and soybean hairy roots. Moreover, soybean plants with GmRH3-overexpressing hairy roots exhibited increased salt tolerance, while soybean plants with GmRH3-RNAi (RNA interference) roots were more sensitive to salt stress than the wild-type plants. Our study uncovers a novel role for GmNEK1 in promoting plant adaptive growth under adverse conditions at least partially through up-regulation of GmRH3. Manipulation of these genes in soybean or other crops may improve growth and production under stress conditions.

Preliminary assessment of two non-destructive instrumental techniques for quality evaluation of Lobelia chinensis Lour.

Two non-destructive instrumental methods, infrared spectroscopy (IR) and X-ray diffraction (XRD), were studied for quality evaluation of Lobelia chinensis Lour. (L. chinensis). We obtained the IR spectra and XRD patterns of L. chinensis collected from different sources. The similarity of samples was analyzed by calculating the cosine coefficient. The cosine values were in the range of 0.83-0.90, indicating that the main components of L. chinensis samples are similar. Sample L1 and L6 showed a slightly lower similarity than that of L2, L3, L4, L5 detected by the two methods, which revealed that IR and XRD methods exhibited analogous detection ability for quality evaluation of L. chinensis. The two methods could be highly recommended as simple and rapid detection means for quality evaluation of L. chinensis.