| 1. |
- Arevalo, Sergio, et al.
(författare)
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Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120
- 2024
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 13:3, s. 901-912
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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] .
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| 2. |
- Babaei, Mahsa, et al.
(författare)
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Metabolic Engineering of Saccharomyces cerevisiae for Rosmarinic Acid Production
- 2020
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:8, s. 1978-1988
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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.
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| 3. |
- Behle, Anna, et al.
(författare)
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Comparative Dose-Response Analysis of Inducible Promoters in Cyanobacteria
- 2020
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Ingår i: ACS Synthetic Biology. - : AMER CHEMICAL SOC. - 2161-5063. ; 9:4, s. 843-855
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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.
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| 4. |
- Cengic, Ivana, et al.
(författare)
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Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803
- 2022
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:9, s. 3100-3113
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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.
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| 5. |
- Claassens, Nico J., et al.
(författare)
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Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coil
- 2019
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:7, s. 1685-1690
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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.
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| 6. |
- Cumming, Alister James, et al.
(författare)
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Antibiotic-Efficient Genetic Cassette for the TEM-1 β-Lactamase That Improves Plasmid Performance
- 2022
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:1, s. 241-253
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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.
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| 7. |
- Cumming, Alister James, et al.
(författare)
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Biosensor that Detects Stress Caused by Periplasmic Proteins
- 2024
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Ingår i: ACS Synthetic Biology. - 2161-5063. ; 13:5, s. 1477-1491
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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.
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| 8. |
- Dabirian, Yasaman, 1992, et al.
(författare)
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Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae
- 2019
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:9, s. 1968-1975
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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.
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| 9. |
- Dabirian, Yasaman, 1992, et al.
(författare)
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FadR-Based Biosensor-Assisted Screening for Genes Enhancing Fatty Acyl-CoA Pools in Saccharomyces cerevisiae
- 2019
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:8, s. 1788-1800
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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.
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| 10. |
- David, Florian, 1981, et al.
(författare)
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Flux Control at the Malonyl-CoA Node through Hierarchical Dynamic Pathway Regulation in Saccharomyces cerevisiae
- 2016
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 5:3, s. 224-233
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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.
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