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

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

Expanding The Genetic Code

Karsten L, Bergen D, Drake C, et al. Expanding The Genetic Code. Bielefeld University; 2017.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

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Role of MalQ Enzyme in a Reconstructed Maltose/Maltodextrin Pathway in <i>Actinoplanes</i> sp. SE50/110

The pseudotetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a relevant secondary metabolite used in diabetes type II medication. Although maltose plays a crucial role in acarbose biosynthesis, the understanding of the maltose/maltodextrin metabolism and its involvement in acarbose production is at an early stage. Here, we reconstructed the predicted maltose–maltodextrin pathway that involves four enzymes AmlE, MalZ, MalP, and MalQ. An investigation of enzyme activities was conducted through in vitro assays, leading to an expansion of previously postulated substrate spectra. The maltose-induced α-glucosidase AmlE is noteworthy for its high hydrolysis rate of linear α-1,4-glucans, and its capability to hydrolyze various glycosidic bonds. The predicted maltodextrin glucosidase MalZ showed slow hydrolysis activity on linear α-glucans, but it was resistant to acarbose and capable of releasing glucose from acarbose. AmlE compensates for the low activity of MalZ to ensure glucose supply. We determined the enzyme activity of MalP and its dual function as maltodextrin and glycogen phosphorylase. The 4-α-glucanotransferase MalQ plays a central role in the maltose/maltodextrin metabolism, alongside MalP. This study confirmed the simultaneous degradation and synthesis of long-chain α-glucans. The product distribution showed that with an increasing number of glycosidic bonds, less glucose is formed. We found that MalQ, like its sequence homolog AcbQ from the acarbose biosynthetic gene cluster, is involved in the formation of elongated acarviosyl metabolites. However, MalQ does not participate in the elongation of acarbose 7-phosphate, which is likely the more readily available acceptor molecule in vivo. Accordingly, MalQ is not involved in the formation of acarviosyl impurities in Actinoplanes sp. SE50/110

The 4-alpha-Glucanotransferase AcbQ Is Involved in Acarbose Modification in Actinoplanes sp. SE50/110

Nölting S, März C, Jacob L, Persicke M, Schneiker-Bekel S, Kalinowski J. The 4-alpha-Glucanotransferase AcbQ Is Involved in Acarbose Modification in Actinoplanes sp. SE50/110. Microorganisms. 2023;11(4): 848.The pseudo-tetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a alpha-glucosidase inhibitor used for treatment of type 2 diabetes patients. In industrial production of acarbose, by-products play a relevant role that complicates the purification of the product and reduce yields. Here, we report that the acarbose 4-alpha-glucanotransferase AcbQ modifies acarbose and the phosphorylated version acarbose 7-phosphate. Elongated acarviosyl metabolites (alpha-acarviosyl-(1,4)-maltooligosaccharides) with one to four additional glucose molecules were identified performing in vitro assays with acarbose or acarbose 7-phosphate and short alpha-1,4-glucans (maltose, maltotriose and maltotetraose). High functional similarities to the 4-alpha-glucanotransferase MalQ, which is essential in the maltodextrin pathway, are revealed. However, maltotriose is a preferred donor and acarbose and acarbose 7-phosphate, respectively, serve as specific acceptors for AcbQ. This study displays the specific intracellular assembly of longer acarviosyl metabolites catalyzed by AcbQ, indicating that AcbQ is directly involved in the formation of acarbose by-products of Actinoplanes sp. SE50/110

pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110

Schaffert L, Jacob L, Schneiker-Bekel S, et al. pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110. Microbiology Resource Announcements. 2020;9(39): e00596-20.ABSTRACT The pSETT4 vector integrates into the Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.</jats:p

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

Abstract 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

Functional Assessment of Vitamin D Status by a Novel Metabolic Approach: The Low Vitamin D Profile Concept.

peer reviewed[en] BACKGROUND: Determining serum 25-hydroxyvitamin D [25(OH)D], 24,25-dihydroxyvitamin D [24,25(OH)2D] and the vitamin D metabolite ratio (VMR) allows the identification of individuals with a low vitamin D metabolite profile. Here, we evaluated if such a functional approach provides superior diagnostic information to serum 25(OH)D alone. METHODS: 25(OH)D, 24,25(OH)2D, and the VMR were determined in participants of the DESIRE (Desirable Vitamin D Concentrations, n = 2010) and the LURIC (Ludwigshafen Risk and Cardiovascular Health, n = 2456) studies. A low vitamin D metabolite profile (vitamin D insufficiency) was defined by a 24,25(OH)2D concentration <1.2 ng/mL (<3 nmol/L) and a VMR <4%. Parathyroid hormone (PTH) and bone turnover markers were measured in both cohorts, whereas 10-year mortality data was recorded in LURIC only. RESULTS: The median age in DESIRE and LURIC was 43.3 and 63.8 years, respectively. Median 25(OH)D concentrations were 27.2 ng/mL (68.0 nmol/L) and 15.5 ng/mL (38.8 nmol/L), respectively. Serum 25(OH)D deficiency, defined as <20.2 ng/mL (<50 nmol/L), was present in 483 (24.0%) and 1701 (69.3%) participants of DESIRE and LURIC, respectively. In contrast, only 77 (3.8%) and 521 (21.2%) participants had a low vitamin D metabolite profile. Regardless of the serum 25(OH)D concentration, a low vitamin D metabolite profile was associated with a significantly higher PTH, accelerated bone metabolism, and higher all-cause mortality than an unremarkable vitamin D metabolite profile. CONCLUSIONS: The personalized assessment of vitamin D status using a functional approach better identifies patients with accelerated bone metabolism and increased mortality than the use of a fixed 25(OH)D cutoff of 20 ng/mL (50 nmol/L)