Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Schaffert L, Schneiker-Bekel S, Gierhake J, et al. Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. Applied Microbiology and Biotechnology. 2020;104:5395–5408.Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium

On the AAVenue to success: Advances in technologies for AAV production

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Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications.

BACKGROUND: Biosafety is a key aspect in the international Genetically Engineered Machine (iGEM) competition, which offers student teams an amazing opportunity to pursue their own research projects in the field of Synthetic Biology. iGEM projects often involve the creation of genetically engineered bacterial strains. To minimize the risks associated with bacterial release, a variety of biosafety systems were constructed, either to prevent survival of bacteria outside the lab or to hinder horizontal or vertical gene transfer. MAIN BODY: Physical containment methods such as bioreactors or microencapsulation are considered the first safety level. Additionally, various systems involving auxotrophies for both natural and synthetic compounds have been utilized by iGEM teams in recent years. Combinatorial systems comprising multiple auxotrophies have been shown to reduced escape frequencies below the detection limit. Furthermore, a number of natural toxin-antitoxin systems can be deployed to kill cells under certain conditions. Additionally, parts of naturally occurring toxin-antitoxin systems can be used for the construction of 'kill switches' controlled by synthetic regulatory modules, allowing control of cell survival. Kill switches prevent cell survival but do not completely degrade nucleic acids. To avoid horizontal gene transfer, multiple mechanisms to cleave nucleic acids can be employed, resulting in 'self-destruction' of cells. Changes in light or temperature conditions are powerful regulators of gene expression and could serve as triggers for kill switches or self-destruction systems. Xenobiology-based containment uses applications of Xeno-DNA, recoded codons and non-canonical amino acids to nullify the genetic information of constructed cells for wild type organisms. A 'minimal genome' approach brings the opportunity to reduce the genome of a cell to only genes necessary for survival under lab conditions. Such cells are unlikely to survive in the natural environment and are thus considered safe hosts. If suitable for the desired application, a shift to cell-free systems based on Xeno-DNA may represent the ultimate biosafety system. CONCLUSION: Here we describe different containment approaches in synthetic biology, ranging from auxotrophies to minimal genomes, which can be combined to significantly improve reliability. Since the iGEM competition greatly increases the number of people involved in synthetic biology, we will focus especially on biosafety systems developed and applied in the context of the iGEM competition

High Quality de Novo Transcriptome Assembly of Croton tiglium

Haak M, Vinke S, Keller W, et al. High Quality de Novo Transcriptome Assembly of Croton tiglium. Frontiers in Molecular Biosciences. 2018;5: 62

Factores que influyen en la dispersión espacio-temporal de broca del café Hypothenemus hampei (Coleoptera: Curculionidae)

La broca del café, Hypothenemus hampei (Ferrari), es la plaga más importante del café, Coffea spp., en el mundo. Este insecto ha sido motivo de numerosos estudios, sin embargo, muchos aspectos de su dispersión se desconocen o requieren ser determinados. La presente investigación tuvo el objetivo de estudiar los factores que influyen en la dispersión espacio-temporal de la broca del café. Dado que la dispersión es un proceso complejo, para ser estudiada se desarrollaron técnicas para la obtención del material biológico con condiciones adecuadas para el estudio, para facilitar el estudio de la morfología del complejo espermático de H. hampei y de marcaje-liberación-recaptura para estudiar la capacidad de vuelo y los factores que la afectan. Los resultados más sobresalientes indican que la espermateca de H. hampei está ubicada en la parte final del abdomen entre la glándula accesoria y el oviducto común y está compuesta por un ducto espermático, músculos espermáticos y una glándula espermática; además, posee músculos al final de su curvatura extrema y su cutícula está finamente reticulada. Los espermatozoides se observaron como filamentos minúsculos, endebles y homogéneos, congregados longitudinalmente dentro de la espermateca. La emergencia masiva de las CH se relacionó con las lluvias y se presentó entre febrero y junio, que corresponde al periodo intercosecha. Todas las CH examinadas tenían espermatozoides en la espermateca, lo q ue sugiere que se habían apareado antes de abandonar el fruto natal. La sobrevivencia de las hembras colonizadoras fue mayor en ambientes humedos y en sustratos diferentes a su hospedero. Durante el periodo de fructificación del café, las hembras se dispersaron mediante vuelo a los glomérulos de frutos adyacentes. Después de colonizar un fruto, la CH perdió su capacidad de vuelo de forma gradual en el transcurso de 5 a 6 días; durante este tiempo, las hembras evaluadas realizar más de un vuelo. Algunas CH marcadas y liberadas se recapturaron a 75 m de distancia del sitio de liberación a las 24 h después de haber sido liberadas. Finalmente, se discute la importancia de los hallazgos de la dispersión espacio-temporal de la broca del café, como información clave para mejorar la comprensión del complejo café-broca, y el manejo de la broca. (Résumé d'auteur

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

Wolf T, Droste J, Gren T, et al. The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110. BMC Genomics. 2017;18(1): 562.Background Acarbose is used in the treatment of diabetes mellitus type II and is produced by Actinoplanes sp. SE50/110. Although the biosynthesis of acarbose has been intensively studied, profound knowledge about transcription factors involved in acarbose biosynthesis and their binding sites has been missing until now. In contrast to acarbose biosynthetic gene clusters in Streptomyces spp., the corresponding gene cluster of Actinoplanes sp. SE50/110 lacks genes for transcriptional regulators. Results The acarbose regulator C (AcrC) was identified through an in silico approach by aligning the LacI family regulators of acarbose biosynthetic gene clusters in Streptomyces spp. with the Actinoplanes sp. SE50/110 genome. The gene for acrC, located in a head-to-head arrangement with the maltose/maltodextrin ABC transporter malEFG operon, was deleted by introducing PCR targeting for Actinoplanes sp. SE50/110. Characterization was carried out through cultivation experiments, genome-wide microarray hybridizations, and RT-qPCR as well as electrophoretic mobility shift assays for the elucidation of binding motifs. The results show that AcrC binds to the intergenic region between acbE and acbD in Actinoplanes sp. SE50/110 and acts as a transcriptional repressor on these genes. The transcriptomic profile of the wild type was reconstituted through a complementation of the deleted acrC gene. Additionally, regulatory sequence motifs for the binding of AcrC were identified in the intergenic region of acbE and acbD. It was shown that AcrC expression influences acarbose formation in the early growth phase. Interestingly, AcrC does not regulate the malEFG operon. Conclusions This study characterizes the first known transcription factor of the acarbose biosynthetic gene cluster in Actinoplanes sp. SE50/110. It therefore represents an important step for understanding the regulatory network of this organism. Based on this work, rational strain design for improving the biotechnological production of acarbose can now be implemented

The Transformers. From Carbon Dioxide to Biofuel

Fust A, Hollmann B, Pucker B, et al. The Transformers. From Carbon Dioxide to Biofuel. Presented at the International Genetically Engineered Machine Competition (IGEM) 2014, Boston, Mass., USA

Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications

Whitford CM, Dymek S, Kerkhoff D, et al. Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications. Journal of Biological Engineering. 2018;12(1): 13

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110

Schaffert L, März C, Burkhardt L, et al. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microbial Cell Factories. 2019;18(1): 114.Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives

Model-checking games for fixpoint logics with partial order models

In this paper, we introduce model-checking games that allow local second-order power on sets of independent transitions in the underlying partial order models where the games are played. Since the interleaving semantics of such models is not considered, some problems that may arise when using interleaving representations are avoided and new decidability results for partial order models of concurrency are achieved. The games are shown to be sound and complete, and therefore determined. While in the interleaving case they coincide with the local model-checking games for the μ-calculus, in a partial order setting they verify properties of a number of fixpoint modal logics that can specify, in concurrent systems with partial order semantics, several properties not expressible with the μ-calculus. The games underpin a novel decision procedure for model-checking all temporal properties of a class of infinite and regular event structures, thus improving, in terms of temporal expressive power, previous results in the literature