Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

Computer Analysis and Recognition of Drosophila melanogasterGene Promoters

A new method for recognizing eukaryotic gene promoters was based on their partition and on analysis of correlations of dinucleotide frequencies for each individual fragment. The method was used to recognize the TATA-containing and TATA-less promoters of Drosophila melanogastergenes. The dinucleotide context was correlated with conformational and physicochemical DNA properties in promoter fragments. Mean values of several parameters proved to dramatically change on transition from the TATA box to its GC-rich flanks. In TATA-less promoters, specific properties were revealed in the DPE region. The method was employed in a promoter recognition program, which is available through Internet.     

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well‐established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron‐mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small‐scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron‐mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron‐mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.     

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis‐regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)‐inducible motif discovery among promoters of 18 co‐expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean–SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co‐localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN‐inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN‐inducible, leading to the discovery of 23 core motifs of 5‐ to 7‐bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W‐AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high‐throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.     

Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.     

Characteristics and diversity of microbial communities in lead–zinc tailings under heavy metal stress in north-west China

High-throughput 16S rRNA and 18S rRNA sequencing were performed to study the changes of soil microbial diversity and community structure under different heavy metal pollution levels in Chengxian lead–zinc mining area, Gansu Province. In this study, we characterized the main physicochemical properties, multiple heavy metal pollution, and microbial community structure of the soil in the tailings. The results show that the soil near the tailings pond was alkaline, barren and the heavy metals were seriously polluted. The microbial diversity and richness of S1 and S2 sites were significantly lower than that of CK2 site (P < 0·05), indicating that the heavy metal pollution could change the physicochemical properties and microbial community structure in soil. Among 97 identified core operating taxa of fungal communities, Ascomycota, Teguta and Basidiomycota were dominant at the phylum level, while among 1523 identified core operating taxa of bacterial communities, Actinomycota was dominant at the phylum level. In addition, the redundancy analysis and Spearman correlation analysis showed that the physicochemical properties and the heavy metal concentration had significant effects on the composition and distribution of soil microbial community. The basic characteristics of soil physicochemical properties, multiple heavy metal pollution and microbial community structure in the tailings were revealed, hoping to provide a basis for ecological rehabilitation of tailings by revealing the variance rule of microbial community diversity in the future.     

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.     

Comprehensive construction strategy of bidirectional green tissue‐specific synthetic promoters

Bidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue‐specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue‐specific synthetic promoters. It is the first report on bidirectional green tissue‐specific promoters that could be efficiently applied in genetic engineering.