Development and application of genetic engineering methods for Actinoplanes sp. SE50/110

Gren T. Development and application of genetic engineering methods for Actinoplanes sp. SE50/110. Bielefeld: Universität Bielefeld; 2017.The alpha-glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type 2, and is manufactured industrially with overproducing derivatives of _Actinoplanes_ sp. SE50/110. This strain was reportedly optimized through step-by-step conventional mutagenesis procedures in the past, however this strategy seems to reach its limits by now. Despite of high industrial significance, only limited information exists regarding acarbose metabolism, function and regulation of these processes, due to the absence of proper genetic engineering methods and tools developed for this strain. In this work, a full toolkit and set of methods for genetic engineering of _Actinoplanes_ sp. SE50/110 were developed. A standardized protocol for a DNA transfer through _E. coli_ - _Actinoplanes_ conjugation was adjusted and applied for the transfer of phiC31, phiBT1 and VWB actinophage-based integrative vectors and pSG5-based replicative vector. Integration sites, occurring once per genome for all integrative vectors, were sequenced and characterized for the first time in _{Actinoplanes_ sp. SE50/110. Notably, the studied plasmids were proven to be stable and neutral with respect to strain morphology and acarbose production, enabling future use for genetic manipulations of _Actinoplanes_ sp. SE50/110. To further broaden the spectrum of available tools, a GUS reporter system, was established in _Actinoplanes_ sp. SE50/110. The set of different methods for gene knockouts was tested, which included integrative and replicative vector based knockouts, ReDirect system based knockouts and CRISPR-Cas9 genetic engineering. ReDirect system was further used to create a library of _Actinoplanes_ single knockout strains. Two of the strains, _Actinoplanes_ _acbD_ and _Actinoplanes_ _cadC_ knockout mutants were further characterized in detail regarding their phenotype. The developed gene cloning system offers multiple possibilities to solve fundamental questions regarding acarbose production, in particular, formulation and verification of the complete acarbose metabolism model, as well as the rational design of acarbose overproducing strains

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

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

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the β-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites

Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes

CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.</p

Targeted genome editing in the rare actinomycete Actinoplanes sp SE50/110 by using the CRISPR/Cas9 System

Wolf T, Gren T, Thieme E, et al. Targeted genome editing in the rare actinomycete Actinoplanes sp SE50/110 by using the CRISPR/Cas9 System. Journal of Biotechnology. 2016;231:122-128.The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the starless and antibiotic marker-free deletion of the gene encoding the tyrosinase Me1C, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated AmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants. (C) 2016 Elsevier B.V. All rights reserved

Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions

Yushchuk O, Ostash I, Vlasiuk I, et al. Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2018;102(19):8419-8428.Streptomyces cyanogenus S136 is the only known producer of landomycin A (LaA), one of the largest glycosylated angucycline antibiotics possessing strong antiproliferative properties. There is rising interest in elucidation of mechanisms of action of landomycins, which, in turn, requires access to large quantities of the pure compounds. Overproduction of LaA has been achieved in the past through manipulation of cluster-situated regulatory genes. However, other components of the LaA biosynthetic regulatory network remain unknown. To fill this gap, we elucidated the contribution of AdpA family pleiotropic regulators in landomycin production via expression of adpA genes of different origins in S. cyanogenus S136. Overexpression of the native S. cyanogenus S136 adpA ortholog had no effect on landomycin titers. In the same time, expression of several heterologous adpA genes led to significantly increased landomycin production under different cultivation conditions. Hence, heterologous adpA genes are a useful tool to enhance or activate landomycin production by S. cyanogenus. Our ongoing research effort is focused on identification of mutations that render S. cyanogenus AdpA nonfunctional

Complete genome sequence of Streptomyces sp. strain CA-256286

Here, we report the sequencing, assembly, and annotation of the genome of Streptomyces sp. strain CA-256286. The genome consists of a linear 7,726,360-nucleotide chromosome and a linear 466,817-nucleotide putative plasmid. This strain is predicted to produce a range of novel secondary metabolites