Acarbose, a pseudo-tetrasaccharide produced by Actinoplanes sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in Actinoplanes sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into Actinoplanes sp. These vectors were validated by silencing the known acarbose biosynthesis genes acbB and acbV, as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein–DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production.

Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL; Wendler et al., 2015a), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of the CRISPR/Cas9 technology. By growth experiments and functional assays of ΔamlE, we could show that AmlE is essential for the maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ΔamlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ΔamlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.

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.

Fusing We designed and synthesized a novel ncAA called CBT-asparagine. It enables a highly specific covalent fusing reaction to another ncAA, cysteinyl-lysine. We confirmed the fusion in vitro via MS and successfully modeled several aaRS sequences for the translational incorporation of CBT-asparagine into proteins. Protein design is usually limited to 20 amino acids. Since the chemical abilities of these amino acids are limited, a lot of interesting functions are not applicable in protein design. We aim to enable the translational incorporation of non-canonical amino acids (ncAAs) through an expanded genetic code.

We worked in many different scientific fields to find suitable ways for the translational incorporate of non-canonical amino acids into proteins. Repurposing existing codons or incorporating new bases are two possible ways. We realized both ways to expand the genetic code of Escherichia coli. The repurposing of a codon for the incorporation of a non-canonical amino acid (ncAA) is possible using the rarely used amber stop codon UAG or other rarely used codons like the leucine codon CUA. To incorporate a non-canonical amino acid using these codons, an orthogonal tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is necessary, which can charge the ncAA to the tRNA. We designed and synthetized the novel ncAA Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT-asparagine). This ncAA has the chemical ability of perform a highly specific covalent binding reaction, which we wanted to incorporate into our target protein. Therefore, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that specifically incorporates the ncAA. In parallel to the libary and selection based approach, we modeled the aaRS which could incorporate our new amino acid CBT-asparagine. We demonstrated that both ways are suitable for the evolution of aaRS. Although incorporation of ncAAs through the amber codon works, there are challenges associated with this approach. The repurposing of codons leads to the decrease of the growth rate of E. coli and it is only feasible to incorporate up to two different ncAAs. Therefore, we took a new way to incorporate ncAAs. The incorporation of an unnatural base pair into the DNA generates 64 new codons. Our first challenge was the uptake of the unnatural base from the media, because E.coli has no nucleoside triphosphate transporter and is not able to synthetize the bases itself. We cloned a nucleoside triphosphate transporter that enables the uptake of both bases from the media. Furthermore, we analyzed the transcriptome of the plant Croton tiglium, which produces the unnatural base isoG. The transcriptome revealed an enzyme for the biosynthesis, which was cloned and characterized for the biosynthesis of isoG in E. coli. To detect the unnatural base we developed two orthogonal systems. A restriction experiment based on the software tool M.A.X. and an adaption of the Oxford Nanopore sequencing, which were combined into one software suite. To demonstrate the possibilities offered by the incorporation of ncAAs, we developed a toolbox containing five different tools. We chose seven different ncAAs for these five tools and demonstrated interesting applications for them. These ncAAs can be used for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted parts, every iGEM team can incorporate these ncAAs into their target proteins. Regarding our project, two of the ncAAs that are part of our toolbox perform an autocatalytic reaction upon irradiation with ultraviolet light. Therefore, we decided to build our own LED panel that allows us to perform experiments with these non‑canonical amino acids under reproducible irradiation conditions.