Detection and validation of cis-regulatory motifs in osmotic stress-inducible synthetic gene switches via computational and experimental approaches.

Synthetic cis -regulatory modules can improve our understanding of gene regulatory networks. We applied an ensemble approach for de novo cis motif discovery among the promoters of 181 drought inducible differentially expressed soybean (Glycine max L.) genes. A total of 43 cis motifs were identified in promoter regions of all gene sets using the binding site estimation suite of tools (BEST). Comparative analysis of these motifs revealed similarities with known cis -elements found in PLACE database and led to the discovery of cis -regulatory motifs that were not yet implicated in drought response. Compiled with the proposed synthetic promoter design rationale, three synthetic assemblies were constructed by concatenating multiple copies of drought-inducible cis motifs in a specific order with inter-motif spacing using random bases and placed upstream of 35s minimal core promoter. Each synthetic module substituted 35S promoter in pBI121 and pCAMBIA3301 to drive glucuronidase expression in soybean hairy roots and Arabidopsis thaliana L. Chimeric soybean seedlings and 3-week-old transgenic Arabidopsis plants were treated with simulated with different levels of osmotic stress. Histochemical staining of transgenic soybean hairy roots and Arabidopsis displayed drought-inducible GUS activity of synthetic promoters. Fluorometric assay and expression analysis revealed that SP2 is the better manual combination of cis -elements for stress-inducible expression. qRT-PCR results further demonstrated that designed synthetic promoters are not tissue-specific and thus active in different parts upon treatment with osmotic stress in Arabidopsis plants. This study provides tools for transcriptional upgradation of valuable crops against drought stress and adds to the current knowledge of synthetic biology.

Encoding Genetic Circuits with DNA Barcodes Paves the Way for Machine Learning-Assisted Metabolite Biosensor Response Curve Profiling in Yeast.

Genetically encoded biosensors are valuable tools used in the precise engineering of metabolism. Although a large number of biosensors have been developed, the fine-tuning of their dose-response curves, which promotes the applications of biosensors in various scenarios, still remains challenging. To address this issue, we leverage a DNA trackable assembly method and fluorescence-activated cell sorting coupled with next-generation sequencing (FACS-seq) technology to set up a novel workflow for construction and comprehensive characterization of thousands of biosensors in a massively parallel manner. An FapR-fapO-based malonyl-CoA biosensor was used as proof of concept to construct a trackable combinatorial library, containing 5184 combinations with 6 levels of transcription factor dosage, 4 different operator positions, and 216 possible upstream enhancer sequence (UAS) designs. By applying the FACS-seq technique, the response curves of 2632 biosensors out of 5184 combinations were successfully characterized to provide large-scale genotype-phenotype association data of the designed biosensors. Finally, machine-learning algorithms were applied to predict the genotype-phenotype relationships of the uncharacterized combinations to generate a panoramic scanning map of the combinatorial space. With the assistance of our novel workflow, a malonyl-CoA biosensor with the largest dynamic response range was successfully obtained. Moreover, feature importance analysis revealed that the recognition sequence insertion scheme and the choice of UAS have a significant impact on the dynamic range. Taken together, our pipeline provides a platform for the design, tuning, and profiling of biosensor response curves and shows great potential to facilitate the rational design of genetic circuits.

Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution.

Adaptive laboratory evolution (ALE) has served as a historic microbial engineering method that mimics natural selection to obtain desired microbes. The past decade has witnessed improvements in all aspects of ALE workflow, in terms of growth coupling, genotypic diversification, phenotypic selection, and genotype-phenotype mapping. The developing growth-coupling strategies facilitate ALE to a wider range of appealing traits. In vivo mutagenesis methods and multiplexed automated culture platforms open new gates to streamline its execution. Meanwhile, the application of multi-omics analyses and multiplexed genetic engineering promote efficient knowledge mining. This article provides a comprehensive and updated review of these advances, highlights newest significant applications, and discusses future improvements, aiming to provide a practical guide for implementation of novel, effective, and efficient ALE experiments.

A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.

Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.

Recent advances of integrated microfluidic suspension cell culture system.

Microfluidic devices with superior microscale fluid manipulation ability and large integration flexibility offer great advantages of high throughput, parallelisation and multifunctional automation. Such features have been extensively utilised to facilitate cell culture processes such as cell capturing and culturing under controllable and monitored conditions for cell-based assays. Incorporating functional components and microfabricated configurations offered different levels of fluid control and cell manipulation strategies to meet diverse culture demands. This review will discuss the advances of single-phase flow and droplet-based integrated microfluidic suspension cell culture systems and their applications for accelerated bioprocess development, high-throughput cell selection, drug screening and scientific research to insight cell biology. Challenges and future prospects for this dynamically developing field are also highlighted.

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters.

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

Overexpression of GmCAMTA12 Enhanced Drought Tolerance in Arabidopsis and Soybean.

Fifteen transcription factors in the CAMTA (calmodulin binding transcription activator) family of soybean were reported to differentially regulate in multiple stresses; however, their functional analyses had not yet been attempted. To characterize their role in stresses, we first comprehensively analyzed the GmCAMTA family in silico and thereafter determined their expression pattern under drought. The bioinformatics analysis revealed multiple stress-related cis-regulatory elements including ABRE, SARE, G-box and W-box, 10 unique miRNA (microRNA) targets in GmCAMTA transcripts and 48 proteins in GmCAMTAs' interaction network. We then cloned the 2769 bp CDS (coding sequence) of GmCAMTA12 in an expression vector and overexpressed in soybean and Arabidopsis through Agrobacterium-mediated transformation. The T3 (Transgenic generation 3) stably transformed homozygous lines of Arabidopsis exhibited enhanced tolerance to drought in soil as well as on MS (Murashige and Skoog) media containing mannitol. In their drought assay, the average survival rate of transgenic Arabidopsis lines OE5 and OE12 (Overexpression Line 5 and Line 12) was 83.66% and 87.87%, respectively, which was ~30% higher than that of wild type. In addition, the germination and root length assays as well as physiological indexes such as proline and malondialdehyde contents, catalase activity and leakage of electrolytes affirmed the better performance of OE lines. Similarly, GmCAMTA12 overexpression in soybean promoted drought-efficient hairy roots in OE chimeric plants as compare to that of VC (Vector control). In parallel, the improved growth performance of OE in Hoagland-PEG (polyethylene glycol) and on MS-mannitol was revealed by their phenotypic, physiological and molecular measures. Furthermore, with the overexpression of GmCAMTA12, the downstream genes including AtAnnexin5, AtCaMHSP, At2G433110 and AtWRKY14 were upregulated in Arabidopsis. Likewise, in soybean hairy roots, GmELO, GmNAB and GmPLA1-IId were significantly upregulated as a result of GmCAMTA12 overexpression and majority of these upregulated genes in both plants possess CAMTA binding CGCG/CGTG motif in their promoters. Taken together, we report that GmCAMTA12 plays substantial role in tolerance of soybean against drought stress and could prove to be a novel candidate for engineering soybean and other plants against drought stress. Some research gaps were also identified for future studies to extend our comprehension of Ca-CaM-CAMTA-mediated stress regulatory mechanisms.

Overexpression of a Novel Cytochrome P450 Promotes Flavonoid Biosynthesis and Osmotic Stress Tolerance in Transgenic Arabidopsis.

Flavonoids are mainly associated with growth, development, and responses to diverse abiotic stresses in plants. A growing amount of data have demonstrated the biosynthesis of flavonoids through multienzyme complexes of which the membrane-bounded cytochrome P450 supergene family shares a crucial part. However, the explicit regulation mechanism of Cytochrome P450s related to flavonoid biosynthesis largely remains elusive. In the present study, we reported the identification of a stress-tolerant flavonoid biosynthetic CtCYP82G24 gene from Carthamus tinctorius. The transient transformation of CtCYP82G24 determined the subcellular localization to the cytosol. Heterologously expressed CtCYP82G24 was effective to catalyze the substrate-specific conversion, promoting the de novo biosynthesis of flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation of metabolites demonstrated that the expression of CtCYP82G24 was effectively induced by Polyethylene glycol stress in transgenic Arabidopsis. In addition, the overexpression of CtCYP82G24 could also trigger expression levels of several other flavonoid biosynthetic genes in transgenic plants. Taken together, our findings suggest that CtCYP82G24 overexpression plays a decisive regulatory role in PEG-induced osmotic stress tolerance and alleviates flavonoid accumulation in transgenic Arabidopsis.

Tissue-Specific Regulation of Gma-miR396 Family on Coordinating Development and Low Water Availability Responses.

Previously, it was reported that miR396s interact with growth-regulating factors (GRFs) to modulate plant growth, development, and stress resistance. In soybean, 11 gma-miR396 precursors (Pre-miR396a-k) were found, and 24 GmGRFs were predicted as targets of seven mature gma-miR396s (gma-miR396a/b/c/e/h/i/k). To explore the roles of the miR396-GRF module in low water availability response of soybean, we analyzed the expression of Pre-miR396a-k, and found that Pre-miR396a/i/bdgk/e/h were up-regulated in leaves and down-regulated in roots; on the contrary, GmGRF5/6/7/8/15/17/21 were down-regulated in leaves and GmGRF1/2/17/18/19/20/21/22/23/24 were up-regulated in roots of low water potential stressed soybean. Any one of gma-miR396a/b/c/e/h/i/k was able to interact with 20 GmGRFs (GmGRF1/2/6-11/13-24), confirming that this module represents a multi-to-multi network interaction. We generated Arabidopsis plants over-expressing each of the 11 gma-miR396 precursors (Pre-miR396a-k), and seven of them (miR396a/b/c/e/h/i/k-OE transgenic Arabidopsis) showed altered development. The low water availability of miR396a/b/c/e/h/i/k-OE was enhanced in leaves but reduced in seeds and roots. Contrary to previous reports, miR396a/b/c/i-OE seedlings showed lower survival rate than WT when recovering after rewatering under soil drying. In general, we believe our findings are valuable to understand the role of gma-miR396 family in coordinating development and low water availability responses, and can provide potential strategies and directions for soybean breeding programs to improve seed yield and plant drought tolerance.

Normalization for Relative Quantification of mRNA and microRNA in Soybean Exposed to Various Abiotic Stresses.

Plant microRNAs are small non-coding, endogenic RNA molecule (containing 20-24 nucleotides) produced from miRNA precursors (pri-miRNA and pre-miRNA). Evidence suggests that up and down regulation of the miRNA targets the mRNA genes involved in resistance against biotic and abiotic stresses. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful technique to analyze variations in mRNA levels. Normalizing the data using reference genes is essential for the analysis of reliable RT-qPCR data. In this study, two groups of candidate reference mRNAs and miRNAs in soybean leaves and roots treated with various abiotic stresses (PEG-simulated drought, salinity, alkalinity, salinity+alkalinity, and abscisic acid) were analyzed by RT-qPCR. We analyzed the most appropriate reference mRNA/miRNAs using the geNorm, NormFinder, and BestKeeper algorithms. According to the results, Act and EF1b were the most suitable reference mRNAs in leaf and root samples, for mRNA and miRNA precursor data normalization. The most suitable reference miRNAs found in leaf and root samples were 166a and 167a for mature miRNA data normalization. Hence the best combinations of reference mRNAs for mRNA and miRNA precursor data normalization were EF1a + Act or EF1b + Act in leaf samples, and EF1a + EF1b or 60s + EF1b in root samples. For mature miRNA data normalization, the most suitable combinations of reference miRNAs were 166a + 167d in leaf samples, and 171a + 156a or 167a + 171a in root samples. We identified potential reference mRNA/miRNAs for accurate RT-qPCR data normalization for mature miRNA, miRNA precursors, and their targeted mRNAs. Our results promote miRNA-based studies on soybean plants exposed to abiotic stress conditions.