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Summary Root architecture remodelling is critical for forage moisture in water‐limited soil. DEEPER ROOTING 1 (DRO1) in Oryza, Arabidopsis, and Prunus has been reported to improve drought avoidance by promoting roots to grow downward and acquire water from deeper soil. In the present study, we found that ZmDRO1 responded more strongly to abscisic acid (ABA)/drought induction in Zea mays ssp. mexicana, an ancestral species of cultivated maize, than in B73. It was proposed that this is one of the reasons why Zea mays ssp. mexicana has a more noticeable change in the downward direction angle of the root and fewer biomass penalties under water‐deficient conditions. Thus, a robust, synthetic ABA/drought‐inducible promoter was used to control the expression of ZmDRO1B73 in Arabidopsis and cultivated maize for drought‐resistant breeding. Interestingly, ABA‐inducible ZmDRO1 promoted a larger downward root angle and improved grain yield by more than 40% under water‐limited conditions. Collectively, these results revealed that different responses to ABA/drought induction of ZmDRO1 confer different drought avoidance abilities, and we demonstrated the application of ZmDRO1 via an ABA‐inducible strategy to alter the root architecture of modern maize to improve drought adaptation in the field.

Summary Characterization of the function of stress‐related genes helps to understand the mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI‐5 gene, encoding a stress‐responsive homeodomain–leucine zipper class I (HD‐Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI‐5 expression by low temperatures, and the elevated TaHDZipI‐5 levels of expression in flowers and early developing grains in the absence of stress, suggests that TaHDZipI‐5 is involved in the regulation of frost tolerance at flowering. The TaHDZipI‐5 protein behaved as an activator in a yeast transactivation assay, and the TaHDZipI‐5 activation domain was localized to its C‐terminus. The TaHDZipI‐5 protein homo‐ and hetero‐dimerizes with related TaHDZipI‐3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI‐5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress‐inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI‐5 overexpression.

An inducible promoter region, PTTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of PTTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of PTTMP were determined using site-directed mutagenesis. Furthermore, PTTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

Advances in systematic computational biology and rapid elucidation of synergistic interplay between cis and trans factors governing transcriptional control have facilitated functional annotation of gene networks. The generation of data through deconstructive, reconstructive and database assisted promoter studies, and its integration to principles of synthetic engineering has started an era of designer promoters. Exploration of natural promoter architecture and the concept of cis engineering have not only enabled fine tuning of single or multiple transgene expression in response to perturbations in the chemical, physiological and environmental stimuli but also provided researchers with a unique answer to various problems in crop improvement in the form of bidirectional promoters.

A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, P xylT . The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene ( nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis.

We identified a gene, designated TRM2, responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. The encoded protein Trm2p contains two C₂H₂-type zinc finger motifs near the N terminus and shows high similarity to Saccharomyces cerevisiae Adr1p and Pichia pastoris Mxr1p. A C. boidinii gene-disrupted strain (trm2Δ) could not grow on methanol or oleate, but could grow on glucose or ethanol. Trm2p was necessary for the activation of five methanol-inducible promoters tested. Trm2p was localized to the nucleus during growth on nonfermentable carbon sources, but to the cytosol during growth on glucose. A chromatin immunoprecipitation assay revealed that Trm2p specifically bound to the promoters of the alcohol oxidase gene (AOD1) and the dihydroxyacetone synthase gene in cells grown on methanol or oleate, but did not bind to these promoters in cells grown on glucose. The derepressed level of expression of AOD1, which was observed in the trm1Δ strain (the TRM1 gene encodes a transcription factor responsible for methanol-specific gene activation), was decreased in the trm1Δtrm2Δ strain to a level similar to that observed in the trm2Δ strain. These results suggest that Trm2p-dependent derepression is essential for the Trm1p-dependent methanol-specific gene activation in C. boidinii.

In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications.

Abstract A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a −10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (P lacUV5 ), which is leakier and more active than wild-type lac promoter (P lacWT ) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced P lacUV5 with other inducible promoters respectively, including arabinose promoter (P araBAD ), rhamnose promoter (P rhaBAD ), tetracycline promoter (P tet ), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.