Showing papers in "ACS Synthetic Biology in 2016"
TL;DR: The performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system, which is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, are reported on.
Abstract: We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free t...
342 citations
TL;DR: In this article, a CRISPR/Cas-based method for generating random-sized deletions around at least one target nucleic acid sequence, or for generating precise indels around one target DNA sequence, was presented.
Abstract: The present invention relates to CRISPR/Cas-based methods for generating random-sized deletions around at least one target nucleic acid sequence, or for generating precise indels around at least one target nucleic acid sequence, or for modulating transcription of at least one target nucleic acid sequence. Also disclosed is a clonal library comprising clones with random-sized deletions, as well as polynucleotides, polypeptides, cells and kits useful for performing the present methods. The present methods can be performed in organisms where gene editing is typically considered as difficult, such as actinomycetes, in particular streptomycetes.
272 citations
TL;DR: The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica, a valuable microbial host for chemical production.
Abstract: The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR–Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III–tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1′–tRNAGly promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8–TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor pla...
265 citations
TL;DR: In this paper, the authors developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory.
Abstract: CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.
252 citations
TL;DR: This approach for C. glutamicum metabolic engineering indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.
Abstract: Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.
213 citations
TL;DR: The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels and it is demonstrated that tightly repressed promoters allow for inducible and reversible CRISpri in cyanobacteria.
Abstract: We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50–95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.
204 citations
TL;DR: Communication systems for microbial consortia are developed in an effort to allow for complex social behavior across different members of a community to outpace monocultures in their ability to perform complicated tasks if communication among and between members of the community is well regulated.
Abstract: The power of a single engineered organism is limited by its capacity for genetic modification. To circumvent the constraints of any singular microbe, a new frontier in synthetic biology is emerging: synthetic ecology, or the engineering of microbial consortia. Here we develop communication systems for such consortia in an effort to allow for complex social behavior across different members of a community. We posit that such communities will outpace monocultures in their ability to perform complicated tasks if communication among and between members of the community is well regulated. Quorum sensing was identified as the most promising candidate for precise control of engineered microbial ecosystems, due to its large diversity and established utility in synthetic biology. Through promoter and protein modification, we engineered two quorum sensing systems (rpa and tra) to add to the extensively used lux and las systems. By testing the cross-talk between all systems, we thoroughly characterized many new indu...
155 citations
TL;DR: The development of a highly efficient CRISPR/Cas9 system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the commercial production of ethanol from synthesis gas, overcomes the deficiencies of currently available tools and will find utility in other acetogens, including the pathogen Clostridgeium difficile.
Abstract: Acetogenic bacteria have the potential to convert single carbon gases (CO and CO2) into a range of bulk chemicals and fuels. Realization of their full potential is being impeded by the absence of effective genetic tools for high throughput genome modification. Here we report the development of a highly efficient CRISPR/Cas9 system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the commercial production of ethanol from synthesis gas. Following the experimental selection of two promoters (Pthl and ParaE) for expression of cas9 and the requisite single guide RNA (sgRNA), the efficiency of system was tested by making precise deletions of four genes, pta, adhE1, ctf and pyrE. Deletion efficiencies were 100%, >75%, 100% and >50%, respectively. The system overcomes the deficiencies of currently available tools (more rapid, no added antibiotic resistance gene, scarless and minimal polar effects) and will find utility in other acetogens, including the pathogen Clostridium difficile.
154 citations
TL;DR: This work has developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli.
Abstract: Multipart and modular DNA part libraries and assembly standards have become common tools in synthetic biology since the publication of the Gibson and Golden Gate assembly methods, yet no multipart modular library exists for use in bacterial systems. Building upon the existing MoClo assembly framework, we have developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli. The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from that of previously published protocols.
148 citations
TL;DR: In this paper, the authors explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer).
Abstract: CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for th...
145 citations
TL;DR: The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.
Abstract: Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.
TL;DR: The ATLAS of Biochemistry is constructed, a database of all theoretical biochemical reactions based on known biochemical principles and compounds and the approach of introducing novel biochemistry into pathway design and associated databases will be important for synthetic biology and metabolic engineering.
Abstract: Because the complexity of metabolism cannot be intuitively understood or analyzed, computational methods are indispensable for studying biochemistry and deepening our understanding of cellular metabolism to promote new discoveries. We used the computational framework BNICE.ch along with cheminformatic tools to assemble the whole theoretical reactome from the known metabolome through expansion of the known biochemistry presented in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We constructed the ATLAS of Biochemistry, a database of all theoretical biochemical reactions based on known biochemical principles and compounds. ATLAS includes more than 130 000 hypothetical enzymatic reactions that connect two or more KEGG metabolites through novel enzymatic reactions that have never been reported to occur in living organisms. Moreover, ATLAS reactions integrate 42% of KEGG metabolites that are not currently present in any KEGG reaction into one or more novel enzymatic reactions. The generated repos...
TL;DR: EcoFlex is introduced, a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard.
Abstract: Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluores...
TL;DR: A novel approach for dynamic modulation of pathway flux and enzyme expression levels around the key pathway intermediate malonyl-CoA is presented, which increased the production of 3-HP by 10-fold and can be applied for production of other malony l-coA-derived products.
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.
TL;DR: With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.
Abstract: The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the ...
TL;DR: A new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts, is reported and demonstrates the ability to rapidly prototype and debug enzymatic pathways in vitro for compelling metabolic engineering and synthetic biology applications.
Abstract: Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redir...
TL;DR: A minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology is developed.
Abstract: Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exp...
TL;DR: In this article, the authors present a framework for the development of a universal toolkit that can be ported into any E.coli chassis, which can be used to create robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies.
Abstract: Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This vi...
TL;DR: A motif is proposed that ensures tracking and robust perfect adaptation for the controlled reaction network through integral feedback and its metabolic load on the host is fully tunable and can be made arbitrarily close to the constitutive limit, the universal minimal metabolic load of all possible controllers
Abstract: The design and implementation of regulation motifs ensuring robust perfect adaptation are challenging problems in synthetic biology. Indeed, the design of high-yield robust metabolic pathways producing, for instance, drug precursors and biofuels, could be easily imagined to rely on such a control strategy in order to optimize production levels and reduce production costs, despite the presence of environmental disturbance and model uncertainty. We propose here a motif that ensures tracking and robust perfect adaptation for the controlled reaction network through integral feedback. Its metabolic load on the host is fully tunable and can be made arbitrarily close to the constitutive limit, the universal minimal metabolic load of all possible controllers. A DNA implementation of the controller network is finally provided. Computer simulations using realistic parameters demonstrate the good agreement between the DNA implementation and the ideal controller dynamics.
TL;DR: This work proposes an architecture for the systematic construction of DNA circuits for analog computation based on DNA strand displacement, and describes how DNA circuits to compute polynomial functions of inputs can be built.
Abstract: DNA circuits have been widely used to develop biological computing devices because of their high programmability and versatility. Here, we propose an architecture for the systematic construction of DNA circuits for analog computation based on DNA strand displacement. The elementary gates in our architecture include addition, subtraction, and multiplication gates. The input and output of these gates are analog, which means that they are directly represented by the concentrations of the input and output DNA strands, respectively, without requiring a threshold for converting to Boolean signals. We provide detailed domain designs and kinetic simulations of the gates to demonstrate their expected performance. On the basis of these gates, we describe how DNA circuits to compute polynomial functions of inputs can be built. Using Taylor Series and Newton Iteration methods, functions beyond the scope of polynomials can also be computed by DNA circuits built upon our architecture.
TL;DR: A software tool named FlowCal, which should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond, is reported.
Abstract: Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality...
TL;DR: The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.
Abstract: Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.
TL;DR: The MPC controller can track fast reference signals better than ZAD but with a higher actuation effort due to the large number of input switches it requires, and the PI controller's performance is comparable to that achieved by the MPC and the ZAD controllers only for the set-point regulation.
Abstract: Real-time automatic regulation of gene expression is a key technology for synthetic biology enabling, for example, synthetic circuit's components to operate in an optimal range. Computer-guided control of gene expression from a variety of inducible promoters has been only recently successfully demonstrated. Here we compared, in silico and in vivo, three different control algorithms: the Proportional-Integral (PI) and Model Predictive Control (MPC) controllers, which have already been used to control gene expression, and the Zero Average Dynamics (ZAD), a control technique used to regulate electrical power systems. We chose as an experimental testbed the most commonly used inducible promoter in yeast: the galactose-responsive GAL1 promoter. We set two control tasks: either force cells to express a desired constant fluorescence level of a reporter protein downstream of the GAL1 promoter (set-point) or a time-varying fluorescence (tracking). Using a microfluidics-based experimental platform, in which either glucose or galactose can be provided to the cells, we demonstrated that both the MPC and ZAD control strategies can successfully regulate gene expression from the GAL1 promoter in living cells for thousands of minutes. The MPC controller can track fast reference signals better than ZAD but with a higher actuation effort due to the large number of input switches it requires. Conversely, the PI controller's performance is comparable to that achieved by the MPC and the ZAD controllers only for the set-point regulation.
TL;DR: This work investigates how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway, and elucidates critical factors for engineeringextracellular electron transfer in heterologous organisms.
Abstract: Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. Here we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits, the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe2O3 (s) reducing conditions. Overall, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.
TL;DR: Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid and malonic acid, through the β-alanine route, and this platform strain was engineered by introducing E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP.
Abstract: Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the β-alanine (BA) route. First, various combinations of downstream enzymes were screened and BA pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate malonic semialdehyde (MSA) from BA. This platform strain was engineered by introducing E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP. Replacement of native promoter of the sdhC gene with the strong trc promoter in the genome increased 3-HP production to 3.69 g/L in flask culture. Introduction of E. coli semialdehyde dehydrogenase (encoded by yneI) into the platform strain resulted in the production of MA, and additional deletion of the ydfG gene increased MA production to 0.450 g/L in flask culture. Fed-batch cultures of final engineered strains resulted in the production of 31.1 g/L 3-HP or 3.60 g/L MA from glucose.
TL;DR: Variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription-translation (TX-TL) system for responsiveness to a new targeted effector, yielding two mutants that respond to vanillin both in vitro and in vivo.
Abstract: The pursuit of circuits and metabolic pathways of increasing complexity and robustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription–translation (TX-TL) system for responsiveness to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. Promising candidates from the in vitro screen were further characterized in vitro and in vivo in a gene circuit. The screen yielded two qacR mutants that respond to vanillin both in vitro and in vivo. While the mutants exhibit some toxicity to cells, presumably due to off-target effects, they are prime starting points...
TL;DR: This work demonstrates and discusses how a set of SBOL-enabled software tools can form an integrated, cross-organizational workflow to recapitulate the design of one of the largest published genetic circuits to date, a 4-input AND sensor.
Abstract: The Synthetic Biology Open Language (SBOL) is a standard that enables collaborative engineering of biological systems across different institutions and tools. SBOL is developed through careful consideration of recent synthetic biology trends, real use cases, and consensus among leading researchers in the field and members of commercial biotechnology enterprises. We demonstrate and discuss how a set of SBOL-enabled software tools can form an integrated, cross-organizational workflow to recapitulate the design of one of the largest published genetic circuits to date, a 4-input AND sensor. This design encompasses the structural components of the system, such as its DNA, RNA, small molecules, and proteins, as well as the interactions between these components that determine the system’s behavior/function. The demonstrated workflow and resulting circuit design illustrate the utility of SBOL 2.0 in automating the exchange of structural and functional specifications for genetic parts, devices, and the biological ...
TL;DR: ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning and serve as useful tools for optimizing gene expression in a variety of bacteria are generated.
Abstract: Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as...
TL;DR: These findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences, and identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activates sequences, which illustrates the complexity of fungal promoters.
Abstract: Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.
TL;DR: mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects.
Abstract: The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a s