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1.	Arevalo, Sergio, et al. (författare) Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120 2024 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 13:3, s. 901-912 Tidskriftsartikel (refereegranskat)abstract In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities. [GRAPHICS] .
2.	Babaei, Mahsa, et al. (författare) Metabolic Engineering of Saccharomyces cerevisiae for Rosmarinic Acid Production 2020 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:8, s. 1978-1988 Tidskriftsartikel (refereegranskat)abstract Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast Saccharomyces cerevisiae. Multiple biosynthetic pathway variants, carrying only plant genes or a combination of plant and Escherichia coli genes, were implemented using a full factorial design of experiment. Through analysis of variances, the effect of each enzyme variant (factors), together with possible interactions between these factors, was assessed. The best pathway variant produced 2.95 ± 0.08 mg/L rosmarinic acid in mineral medium with glucose as the sole carbon source. Increasing the copy number of rosmarinic acid biosynthetic genes increased the titer to 5.93 ± 0.06 mg/L. The study shows the feasibility of producing rosmarinic acid by yeast fermentation.
3.	Behle, Anna, et al. (författare) Comparative Dose-Response Analysis of Inducible Promoters in Cyanobacteria 2020 Ingår i: ACS Synthetic Biology. - : AMER CHEMICAL SOC. - 2161-5063. ; 9:4, s. 843-855 Tidskriftsartikel (refereegranskat)abstract Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter P-vanCC was compared to the previously described rhamnose-inducible P-rha, the anhydrotetracycline-inducible P-L03, and the Co2+-inducible P-coaT. We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.
4.	Cengic, Ivana, et al. (författare) Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803 2022 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:9, s. 3100-3113 Tidskriftsartikel (refereegranskat)abstract Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system, contained on a single replicative vector, for the model cyanobacterium Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector intoSynechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits performed better, reaching, e.g., 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel in Synechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction. Lastly, a vector curing system based on the nickel-inducible expression of the toxic mazF (from Escherichia coli) was added to the CRISPR/Cas9 vector. This inducible system allowed for curing of the vector in 25-75% of screened colonies, enabling edited mutants to become markerless.
5.	Claassens, Nico J., et al. (författare) Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coil 2019 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:7, s. 1685-1690 Tidskriftsartikel (refereegranskat)abstract Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.
6.	Cumming, Alister James, et al. (författare) Antibiotic-Efficient Genetic Cassette for the TEM-1 β-Lactamase That Improves Plasmid Performance 2022 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:1, s. 241-253 Tidskriftsartikel (refereegranskat)abstract Antibiotic resistance cassettes are indispensable tools in recombinant DNA technology, synthetic biology, and metabolic engineering. The genetic cassette encoding the TEM-1 β-lactamase (denoted Tn3.1) is one of the most commonly used and can be found in more than 120 commercially available bacterial expression plasmids (e.g., the pET, pUC, pGEM, pQE, pGEX, pBAD, and pSEVA series). A widely acknowledged problem with the cassette is that it produces excessively high titers of β-lactamase that rapidly degrade β-lactam antibiotics in the culture media, leading to loss of selective pressure, and eventually a large percentage of cells that do not have a plasmid. To address these shortcomings, we have engineered a next-generation version that expresses minimal levels of β-lactamase (denoted Tn3.1MIN). We have also engineered a version that is compatible with the Standard European Vector Architecture (SEVA) (denoted Ap (pSEVA#1MIN--)). Expression plasmids containing either Tn3.1MIN or Ap (pSEVA#1MIN--) can be selected using a 5-fold lower concentration of β-lactam antibiotics and benefit from the increased half-life of the β-lactam antibiotics in the culture medium (3- to 10-fold). Moreover, more cells in the culture retain the plasmid. In summary, we present two antibiotic-efficient genetic cassettes encoding the TEM-1 β-lactamase that reduce antibiotic consumption (an integral part of antibiotic stewardship), reduce production costs, and improve plasmid performance in bacterial cell factories.
7.	Cumming, Alister James, et al. (författare) Biosensor that Detects Stress Caused by Periplasmic Proteins 2024 Ingår i: ACS Synthetic Biology. - 2161-5063. ; 13:5, s. 1477-1491 Tidskriftsartikel (refereegranskat)abstract Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.
8.	Dabirian, Yasaman, 1992, et al. (författare) Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae 2019 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:9, s. 1968-1975 Tidskriftsartikel (refereegranskat)abstract Metabolite biosensors are useful tools for high-throughput screening approaches and pathway regulation approaches. An important feature of biosensors is the dynamic range. To expand the maximum dynamic range of a transcription factor-based biosensor in Saccharomyces cerevisiae, using the fapO/FapR system from Bacillus subtilis as an example case, five native promoters, including constitutive and glucose-regulated ones, were modified. By evaluating different binding site (BS) positions in the core promoters, we identified locations that resulted in a high maximum dynamic range with low expression under repressed conditions. We further identified BS positions in the upstream element region of the TEF1 promoter that did not influence the native promoter strength but resulted in repression in the presence of a chimeric repressor consisting of FapR and the yeast repressor Mig1. These modified promoters with broad dynamic ranges will provide useful information for the engineering of future biosensors and their use in complex genetic circuits.
9.	Dabirian, Yasaman, 1992, et al. (författare) FadR-Based Biosensor-Assisted Screening for Genes Enhancing Fatty Acyl-CoA Pools in Saccharomyces cerevisiae 2019 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:8, s. 1788-1800 Tidskriftsartikel (refereegranskat)abstract Fatty acid-derived compounds have a range of industrial applications, from chemical building blocks to biofuels. Due to the highly dynamic nature of fatty acid metabolism, it is difficult to identify genes modulating fatty acyl-CoA levels using a rational approach. Metabolite biosensors can be used to screen genes from large-scale libraries in vivo in a high throughput manner. Here, a fatty acyl-CoA sensor based on the transcription factor FadR from Escherichia coli was established in Saccharomyces cerevisiae and combined with a gene overexpression library to screen for genes increasing the fatty acyl-CoA pool. Fluorescence-activated cell sorting, followed by data analysis, identified genes enhancing acyl-CoA levels. From these, overexpression of RTC3, GGA2, and LPP1 resulted in about 80% increased fatty alcohol levels. Changes in fatty acid saturation and chain length distribution could also be observed. These results indicate that the use of this acyl-CoA biosensor combined with a gene overexpression library allows for identification of gene targets improving production of fatty acids and derived products.
10.	David, Florian, 1981, et al. (författare) Flux Control at the Malonyl-CoA Node through Hierarchical Dynamic Pathway Regulation in Saccharomyces cerevisiae 2016 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 5:3, s. 224-233 Tidskriftsartikel (refereegranskat)abstract The establishment of a heterologous pathway in a microbial host for the production of industrially relevant chemicals at high titers and yields requires efficient adjustment of the central carbon metabolism to ensure that flux is directed toward the product of interest. This can be achieved through regulation at key branch points in the metabolic networks, and here we present a novel approach for dynamic modulation of pathway flux and enzyme expression levels. The approach is based on a hierarchical dynamic control system around the key pathway intermediate malonyl-CoA. The upper level of the control system ensures downregulation of endogenous use of malonyl-CoA for fatty acid biosynthesis, which results in accumulation of this pathway intermediate. The lower level of the control system is based on use of a novel biosensor for malonyl-CoA to activate expression of a heterologous pathway using this metabolite for production of 3-hydroxypropionic acid (3-HP). The malonyl-CoA sensor was developed based on the FapR transcription factor of Bacillus subtilis, and it demonstrates one of the first applications of a bacterial metabolite sensor in yeast. Introduction of the dual pathway control increased the production of 3-HP by 10-fold and can also be applied for production of other malonyl-CoA-derived products.
11.	Dorado-Morales, P, et al. (författare) Engineering bacteria to form a biofilm and induce clumping in Caenorhabditis elegans 2014 Ingår i: ACS synthetic biology. - : American Chemical Society (ACS). - 2161-5063. ; 3:12, s. 941-943 Tidskriftsartikel (refereegranskat)
12.	Eisenhauer, Klara, et al. (författare) Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor 2023 Ingår i: ACS Synthetic Biology. - 2161-5063. ; 13:4, s. 1382-1392 Tidskriftsartikel (refereegranskat)abstract The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focusing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors and now provides a capacity to assess the specificity of a nanopore or ion channel. Finally, a comparison to high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.
13.	Englund, Elias, et al. (författare) Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide 2015 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:12, s. 1270-1278 Tidskriftsartikel (refereegranskat)abstract Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.
14.	Engqvist, Martin, 1983, et al. (författare) ANT: Software for Generating and Evaluating Degenerate Codons for Natural and Expanded Genetic Codes 2015 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:8, s. 935-938 Tidskriftsartikel (refereegranskat)abstract The Ambiguous Nucleotide Tool (ANT) is a desktop application that generates and evaluates degenerate codons. Degenerate codons are used to represent DNA positions that have multiple possible nucleotide alternatives. This is useful for protein engineering and directed evolution, where primers specified with degenerate codons are used as a basis for generating libraries of protein sequences. ANT is intuitive and can be used in a graphical user interface or by interacting with the code through a defined application programming interface. ANT comes with full support for nonstandard, user-defined, or expanded genetic codes (translation tables), which is important because synthetic biology is being applied to an ever widening range of natural and engineered organisms. The Python source code for ANT is freely distributed so that it may be used without restriction, modified, and incorporated in other software or custom data pipelines.
15.	Ferreira, Raphael, 1990, et al. (författare) Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation 2019 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:11, s. 2457-2463 Tidskriftsartikel (refereegranskat)abstract Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.
16.	Ferreira, Raphael, 1990, et al. (författare) Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae 2018 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 7:1, s. 10-15 Tidskriftsartikel (refereegranskat)abstract Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the three genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4.
17.	Gong, Guiping, et al. (författare) GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae 2021 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:6, s. 1328-1337 Tidskriftsartikel (refereegranskat)abstract Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.
18.	Gossing, Michael, et al. (författare) Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae 2023 Ingår i: ACS Synthetic Biology. - 2161-5063. ; 12:8, s. 2271-2277 Tidskriftsartikel (refereegranskat)abstract Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9technology, with its ability to target a specific DNA locus usingguide RNAs (gRNAs), is particularly suited for targeted mutagenesis.The targeted diversification of nucleotides in Saccharomycescerevisiae using a CRISPR-guided error-prone DNA polymerase calledyEvolvR was recently reported. Here, we investigate the effectof multiplexed expression of gRNAs flanking a short stretch of DNAon reversion and mutation frequencies using yEvolvR. Phenotypic assaysdemonstrate that higher reversion frequencies are observed when expressingmultiple gRNAs simultaneously. Next generation sequencing revealsa synergistic effect of multiple gRNAs on mutation frequencies, whichis more pronounced in a mutant with a partially defective DNA mismatchrepair system. Additionally, we characterize a galactose-inducibleyEvolvR, which enables temporal control of mutagenesis. This studydemonstrates that multiplex expression of gRNAs and induction of mutagenesisgreatly improves the capabilities of yEvolvR for generation of geneticlibraries in vivo.
19.	Govindarajan, Sridhar, et al. (författare) Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase 2015 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:3, s. 221-227 Tidskriftsartikel (refereegranskat)abstract We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.
20.	Hedin, Karl Alex, et al. (författare) Cold Exposure and Oral Delivery of GLP-1R Agonists by an Engineered Probiotic Yeast Strain Have Antiobesity Effects in Mice 2023 Ingår i: ACS Synthetic Biology. - 2161-5063. ; 12:11, s. 3433-3442 Tidskriftsartikel (refereegranskat)abstract Advanced microbiome therapeutics (AMTs) holds promise in utilizing engineered microbes such as bacteria or yeasts for innovative therapeutic applications, including the in situ delivery of therapeutic peptides. Glucagon-like peptide-1 receptor agonists, such as Exendin-4, have emerged as potential treatments for type 2 diabetes and obesity. However, current administration methods face challenges with patient adherence and low oral bioavailability. To address these limitations, researchers are exploring improved oral delivery methods for Exendin-4, including utilizing AMTs. This study engineered the probiotic yeast Saccharomyces boulardii to produce Exendin-4 (Sb-Exe4) in the gastrointestinal tract of male C57BL/6 mice to combat diet-induced obesity. The biological efficiency of Exendin-4 secreted by S. boulardii was analyzed ex vivo on isolated pancreatic islets, demonstrating induced insulin secretion. The in vivo characterization of Sb-Exe4 revealed that when combined with cold exposure (8 °C), the Sb-Exe4 yeast strain successfully suppressed appetite by 25% and promoted a 4-fold higher weight loss. This proof of concept highlights the potential of AMTs to genetically modify S. boulardii for delivering active therapeutic peptides in a precise and targeted manner. Although challenges in efficacy and regulatory approval persist, AMTs may provide a transformative platform for personalized medicine. Further research in AMTs, particularly focusing on probiotic yeasts such as S. boulardii, holds great potential for novel therapeutic possibilities and enhancing treatment outcomes in diverse metabolic disorders.
21.	Hernández-Rollán, Cristina, et al. (författare) LyGo : A Platform for Rapid Screening of Lytic Polysaccharide Monooxygenase Production 2021 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:4, s. 897-906 Tidskriftsartikel (refereegranskat)abstract Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with 14 different expression vectors in a simple 15 min reaction, thus enabling rapid exploration of several gene contexts, hosts, and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration of its utility, we explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.
22.	Hjelm, Anna, et al. (författare) Tailoring Escherichia coli for the L-Rhamnose P-BAD Promoter-Based Production of Membrane and Secretory Proteins 2017 Ingår i: ACS Photonics. - : American Chemical Society (ACS). - 2330-4022. ; 6:6, s. 985-994 Tidskriftsartikel (refereegranskat)abstract Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli L-rhamnose PBAD promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an L-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an L-rhamnose catabolism deficient mutant, we demonstrate that the L-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to L-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated L-rhamnose transport and L-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an L-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an L-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.
23.	Jin, Haojie, et al. (författare) Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803 2019 Ingår i: ACS Synthetic Biology. - : AMER CHEMICAL SOC. - 2161-5063. ; 8:4, s. 655-660 Tidskriftsartikel (refereegranskat)abstract To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high, GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc10 and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.
24.	Kipper, Kalle, et al. (författare) Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli 2017 Ingår i: ACS Photonics. - : AMER CHEMICAL SOC. - 2330-4022. ; 6:2, s. 233-255 Tidskriftsartikel (refereegranskat)abstract Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.
25.	Li, Gang, 1991, et al. (författare) Machine Learning Applied to Predicting Microorganism Growth Temperatures and Enzyme Catalytic Optima 2019 Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:6, s. 1411-1420 Tidskriftsartikel (refereegranskat)abstract Enzymes that catalyze chemical reactions at high temperatures are used for industrial biocatalysis, applications in molecular biology, and as highly evolvable starting points for protein engineering. The optimal growth temperature (OGT) of organisms is commonly used to estimate the stability of enzymes encoded in their genomes, but the number of experimentally determined OGT values are limited, particularly for thermophilic organisms. Here, we report on the development of a machine learning model that can accurately predict OGT for bacteria, archaea, and microbial eukaryotes directly from their proteome-wide 2-mer amino acid composition. The trained model is made freely available for reuse. In a subsequent step we use OGT data in combination with amino acid composition of individual enzymes to develop a second machine learning model-for prediction of enzyme catalytic temperature optima (T-opt). The resulting model generates enzyme T-opt estimates that are far superior to using OGT alone. Finally, we predict T-opt for 6.5 million enzymes, covering 4447 enzyme classes, and make the resulting data set available to researchers. This work enables simple and rapid identification of enzymes that are potentially functional at extreme temperatures.

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