Native Promoter Research Articles

Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development

The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

Yeast Synthetic Minimal Biosensors for Evaluating Protein Production.

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer, the induction of the UPR has a significant impact on the recombinant protein production in eukaryotic cell factories, including the industrial workhorseSaccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimization of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage or lack updated systematic evaluation. To this end, we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor is only 98 bp in length, has minimal homology to other native yeast sequences and displayed superior sensor characteristics. The synthetic minimal UPR sensor was able to accurately distinguish between cells expressing different heterologous proteins and between the different secretion levels of the same protein. This work demonstrated the potential of synthetic UPR biosensors as high-throughput tools to predict the protein production capacity of strains, interrogate protein properties hampering their secretion, and guide rational engineering strategies for optimal heterologous protein production.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaCA–04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA–04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA–04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA–04 protein with high YP/S and PHB productivity. Soluble phaCA–04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA–04 from pColdTF-phaCABA–04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCABA–04 enhanced soluble protein formation to approximately 3.09−4.1 times higher than that from pColdI-phaCABA–04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCABA–04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters.

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements-notably the TATA box-as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

Droplet-based microfluidic platform for high-throughput screening of Streptomyces

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2–111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.

Heterologous expression of ZmGS5 enhances organ size and seed weight by regulating cell expansion in Arabidopsis thaliana.

Maize ZmGS5 was reported to be positively associated with kernel-related traits, however, its regulatory mechanism on plant development and seed size remains unknown. In this study, ZmGS5 was demonstrated to be widely expressed in various maize tissues with the highest expression level in developing embryos, indicating its critical roles in early kernel development process. The ZmGS5 protein was subcellularly localized to both the nucleus and cytoplasm. Transgenic Arabidopsis plants overexpressing ZmGS5 under the control of either the constitutive maize Ubiquitin1 promotor or native ZmGS5 promoter resulted in increased plant size, biomass, seed size and weight, although no significant difference was observed between transgenic lines harboring the two constructs. In contrast, the antisense-ZmGS5 transgene resulted in opposite phenotypes. Our cytological data suggested that ZmGS5 enlarged petal size through enhancing cell expansion. Quantitative RT-PCR analysis indicated that ZmGS5 might enhance cell expansion and grain filling by upregulating expression levels of particular EXPA or SWEET genes. Collectively, these findings help us further understand the biological function and regulatory mechanism of ZmGS5 in improving organ size and seed weight, which imply its great potential for high-yield breeding in the future.

Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences.

Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.

Developing specific leaf promoters tools for genetic use in transgenic plants towards food security.

Significant yields enrichments are necessitated for meeting the rapid global growth population together with the expected demanding for food, particularly major crops. Photosynthesis improvement is an unexploited opportunity in research on improving crop yields. However, the lack of sufficient molecular promoters tools leads to the need to explore and analyze native leaf-specified promoters for manipulating photosynthesis activities in plants. Two B. distachyon promoters, sedoheptulose-1, 7-bisphosphatase (SBPase) and fructose-1, 6-bisphosphate aldolase (FBPA), were isolated and cloned into an expression vector upstream of the eYFP reporter gene. The results demonstrate that both promoters actively function in N. benthamiana leaves in both agro-transiently assays, successfully regulating expression specifically to leaf-tissues. Exploring these active promoters could potentially provide new well genetic tools for any transgene expression in plants or leaves to genetically manipulate photosynthesis for yield improvement.

Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum

Corynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sensitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8–9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum.

Minimal Amelogenin Domain for Enamel Formation

Amelogenin is the most abundant matrix protein guiding hydroxyapatite formation in enamel, the durable bioceramic tissue that covers vertebrate teeth. Here, we sought to refine structure-function for an amelogenin domain based on in vitro data showing a 42 amino acid amelogenin-derived peptide (ADP7) mimicked formation of hydroxyapatite similar to that observed for the full-length mouse 180 amino acid protein. In mice, we used CRISPR-Cas9 to express only ADP7 by the native amelogenin promoter. Analysis revealed ADP7 messenger RNA expression in developing mouse teeth with the formation of a thin layer of enamel. In vivo, ADP7 peptide partially replaced the function of the full-length amelogenin protein and its several protein isoforms. Protein structure-function relationships identified through in vitro assays can be deployed in whole model animals using CRISPR-Cas9 to validate function of a minimal protein domain to be translated for clinical use as an enamel biomimetic.

Complementation Studies of the Pah1‐encoded Phosphatidate Phosphatase in Yarrowia lipolytica

The PAH1 gene encodes for phosphatidate (PA) phosphatase which catalyzes the dephosphorylation of PA to produce diacylglycerol that can be further converted to triacylglycerol (TAG). Our recent studies in the oleaginous yeast Yarrowia lipolytica showed that the pah1Δ mutation caused a significant decrease in total lipid content and TAG levels during the lipogenic phase, where cell growth ceases and lipid biosynthesis predominates. Also, the expression of the Pah1 protein is regulated under these conditions. To gain a better understanding of this regulation, integrative vectors that drive the expression of PAH1 under the control of its native promoter and either the LIP2 or the PEX20 terminator were constructed and transformed into pah1Δ cells. The expression of Pah1 in the transformants was confirmed by immunoblot analysis using antibodies directed against the protein, and its biological function was examined by analyzing the PA phosphatase activity, lipid content, and TAG levels. The results showed that the levels of the Pah1 protein expressed under theLIP2 and the PEX20 terminators were similar to the wild type levels. Also, the vector expressed Pah1 was able to restore the TAG levels in pah1Δ cells. The constructed vectors will be further used to study the function of Pah1 by mutating its catalytic motif using site-directed mutagenesis. We hypothesize that the catalytically inactive Pah1 protein will not complement the phenotype of the pah1Δ cells, thereby confirming that the catalytic activity of Pah1 is responsible for the phenotypical changes observed in the pah1Δ cells.

Topologies of synthetic gene circuit for optimal fold change activation.

Computations widely exist in biological systems for functional regulations. Recently, incoherent feedforward loop and integral feedback controller have been implemented into Escherichia coli to achieve a robust adaptation. Here, we demonstrate that an indirect coherent feedforward loop and mutual inhibition designs can experimentally improve the fold change of promoters, by reducing the basal level while keeping the maximum activity high. We applied both designs to six different promoters in E. coli, starting with synthetic inducible promoters as a proof-of-principle. Then, we examined native promoters that are either functionally specific or systemically involved in complex pathways such as oxidative stress and SOS response. Both designs include a cascade having a repressor and a construct of either transcriptional interference or antisense transcription. In all six promoters, an improvement of up to ten times in the fold change activation was observed. Theoretically, our unitless models show that when regulation strength matches promoter basal level, an optimal fold change can be achieved. We expect that this methodology can be applied in various biological systems for biotechnology and therapeutic applications.

In Vivo Function of Flow-Responsive Cis-DNA Elements of eNOS Gene: A Role for Chromatin-Based Mechanisms.

eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Characterization of a fowl adenovirus 9 (FAdV-9) early promoter and its application in generating dual expression FAdV-9s

The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73−574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.

Evaluation of an oxygen\u2010dependent self\u2010inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD

A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter PsrfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters PnarG and PnasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfA expression. As hypothesized, activity of PnarG and PnasD increased with lower oxygen levels and surfactin was not produced by PsrfA::PnarG as well as PsrfA::PnasD mutant strains under conditions with highest oxygen availability. PnarG showed expressions similar to PsrfA at lowest oxygen availability, while maximum value of PnasD was more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall YP/X of 1.007 g/g. This value also surpassed the YP/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.

Self-Construction in Immigration - I-Positioning through Tensional Dialogues to Powerful Foreign and Native Voices.

This paper explores proculturative semiotic dynamics underlying self-construction in emigration that reveals various forms of the self's positioning through the processes of relating to the native and foreign socio-cultural environments. The self is conceptualised as the heterogeneous entirety of voices and self-related positions which are hierarchically organised. Hierarchical organisation implies the dominance of certain voices and I-positions at the expense of silencing others. Moreover, external societal voices promote hegemonic social representations which are represented as promoter I-positions/signs in the self-structures and have the power to regulate individuals' mental activity. Therefore, it is argued that selves' relations to the environment are not always symmetrically dialogical. The compelling power of hierarchically ordered external meaning systems that are conceptualised as "objective culture" is illustrated in the best manner when a person occurs in emigration where the native organisation of voices and I-positions is being semiotically ruptured due to the meeting with a foreign configuration of a hierarchy of external I-positions and gets "attacked" by alien promoter signs. External promoter voices and I-positions have the power to take the dominant position and establish asymmetric relations with other self-related elements. They can significantly influence intra-psychological negotiations by vocalising hegemonic social representations which exist in any community. The case study of a Georgian emigrant's living in Germany vividly reveals the wave of self-transformations which she undergoes after the liberation from the pressure of native promoter signs and engagement with the German ones. Specific microgenetic experiences leading to the transformations at the ontogenetic level are highlighted. Symmetric and asymmetric forms of communication are conceived as particular instances of relating. This paper vividly reveals the significance of the exploration of the forms of dynamic relations between various components of the self and socio-cultural environment and entailing intra-psychic and external negotiations for better understanding of the nature of humans' epigenetic development.

Development of CRISPR-Cas9 genome editing system in Talaromyces marneffei

Talaromyces marneffei is an important pathogenic thermally dimorphic fungus causing systemic talaromycosis mainly prevalent in Southeast Asia. The dimorphic transition between mycelium and yeast is considered crucial for the pathogenicity of T. marneffei. However, the lack of genetic toolbox has been a major impediment for understanding its pathogenicity. Here a CRISPR-Cas9 system was developed to facilitate genetic manipulations in this organism. In this study, the CRISPR-Cas9 gene editing system uses a native U6 snRNA promoter from T. marneffei to drive the expression of sgRNA. Employing this system and PEG-mediated protoplast transformation, the sakA gene was mutated. Sanger sequencing confirmed nearly 40% site-directed mutation rate. The phenotype analysis confirmed the sakA gene function in T. marneffei dimorphic transition. Our study provided a powerful genome-manipulating tool, which could accelerate studies on T. marneffei for further revealing the mechanisms of its pathogenicity.

Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production.

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino-acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting the growth of the most susceptible bacterial strains at picomolar concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here, we report the development of pRV610gcc, an easily modifiable 11.2-kbp plasmid that enables the production of GccF in L. plantarum NC8. gcc gene expression relies on native promoters in the cloned cluster, and NC8(pRV610gcc) produces mature GccF at levels similar to KW30. Key findings are that the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence but glycosylation of the loop serine is both sequence and spatially specific, that glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation, that neither of the putative thioredoxin proteins is essential for peptide maturation, and that removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity.IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

The Sandarazols are Cryptic and Structurally Unique Plasmid-Encoded Toxins from a Rare Myxobacterium*.

Herein, we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous‐recombination‐mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A–G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α‐chlorinated ketone, an epoxyketone, and a (2R)‐2‐amino‐3‐(N,N‐dimethylamino)‐propionic acid building block. In‐depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazol C shows an IC50 value of 0.5 μm against HCT 116 cells and a MIC of 14 μm against Mycobacterium smegmatis, which points at the sandarazols’ potential function as defensive secondary metabolites or toxins.

Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements.

SummaryAbiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic‐related and salt stress‐inducible synthetic promoters for use in hybrid poplar. De novo motif‐detecting algorithms yielded 30 water‐deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water‐deficit stress motifs (SD18, SD13 and SD9) found in 16 co‐expressed gene promoters, and we discovered a well‐conserved motif for salt response (SS16). We characterized several native poplar stress‐inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five‐to‐eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18‐1, SD9‐2, SS16‐1, SS16‐2 and SS16‐3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18‐1 and SD9‐2 were induced by water‐deficit stress, whereas SS16‐1, SS16‐2 and SS16‐3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.