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Pichia MoClo Toolkit (Lu Lab)
Pichia_MoClo_Toolkit_Lu_Lab_collection Version 1 (Collection)
Pichia MoClo Toolkit (Lu Lab)
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Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae
sb7b00466 Version 1 (Collection)
Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA and n-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in S. cerevisiae. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore S. cerevisiae as a heterologous production host for novel secondary metabolites.
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Engineering Mannitol Biosynthesis in Escherichia coli and Synechococcus sp. PCC 7002 Using a Green Algal Fusion Protein
sb8b00238 Version 1 (Collection)
The genetic engineering of microbial cell factories is a sustainable alternative to the chemical synthesis of organic compounds. Successful metabolic engineering often depends on manipulating several enzymes, requiring multiple transformation steps and selection markers, as well as protein assembly and efficient substrate channeling. Naturally occurring fusion genes encoding two or more enzymatic functions may offer an opportunity to simplify the engineering process and to generate ready-made protein modules, but their functionality in heterologous systems remains to be tested. Here we show that heterologous expression of a fusion enzyme from the marine alga Micromonas pusilla, comprising a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase, leads to synthesis of mannitol by Escherichia coli and by the cyanobacterium Synechococcus sp. PCC 7002. Neither of the heterologous systems naturally produce this sugar alcohol, which is widely used in food, pharmaceutical, medical, and chemical industries. While the mannitol production rates obtained by single-gene manipulation were lower than those previously achieved after pathway optimization with multiple genes, our findings show that naturally occurring fusion proteins can offer simple building blocks for the assembly and optimization of recombinant metabolic pathways.
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Parallel Integration and Chromosomal Expansion of Metabolic Pathways
sb8b00243 Version 1 (Collection)
Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.
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Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini
sb8b00525 Version 1 (Collection)
Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.
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Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables Development of a Multiplexed, Orthogonal Gene Activation and Repression System
sb8b00527 Version 1 (Collection)
The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.
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Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli
sb9b00078 Version 1 (Collection)
The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription–translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse α-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.
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Bacterial MbtH-like Proteins Stimulate Nonribosomal Peptide Synthetase-Derived Secondary Metabolism in Filamentous Fungi
sb9b00106 Version 1 (Collection)
Filamentous fungi are known producers of bioactive natural products, low molecular weight molecules that arise from secondary metabolism. MbtH-like proteins (MLPs) are small (∼10 kDa) proteins, which associate noncovalently with adenylation domains of some bacterial nonribosomal peptide synthetases (NRPS). MLPs promote the folding, stability, and activity of NRPS enzymes. MLPs are highly conserved among a wide range of bacteria; however, they are absent from all fungal species sequenced to date. We analyzed the interaction potential of bacterial MLPs with eukaryotic NRPS enzymes first using crystal structures, with results suggesting a conservation of the interaction surface. Subsequently, we transformed five MLPs into Penicillium chrysogenum strains and analyzed changes in NRPS-derived metabolite profiles. Three of the five transformed MLPs increased the rate of nonribosomal peptide formation and elevated the concentrations of intermediate and final products of the penicillin, roquefortine, chrysogine, and fungisporin biosynthetic pathways. Our results suggest that even though MLPs are not found in the fungal domain of life, they can be used in fungal hosts as a tool for natural product discovery and biotechnological production.
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Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae
sb9b00144 Version 1 (Collection)
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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Genome Engineering of Eubacterium limosum Using Expanded Genetic Tools and the CRISPR-Cas9 System
sb9b00150 Version 1 (Collection)
Eubacterium limosum is one of the important bacteria in C1 feedstock utilization as well as in human gut microbiota. Although E. limosum has recently garnered much attention and investigation on a genome-wide scale, a bottleneck for systematic engineering in E. limosum is the lack of available genetic tools and an efficient genome editing platform. To overcome this limitation, we here report expanded genetic tools and the CRISPR-Cas9 system. We have developed an inducible promoter system that enables implementation of the CRISPR-Cas9 system to precisely manipulate target genes of the Wood-Ljungdahl pathway with 100% efficiency. Furthermore, we exploited the effectiveness of CRISPR interference to reduce the expression of target genes, exhibiting substantial repression of several genes in the Wood-Ljungdahl pathway and fructose-PTS system. These expanded genetic tools and CRISPR-Cas9 system comprise powerful and widely applicable genetic tools to accelerate functional genomic study and genome engineering in E. limosum.
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De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast
sb9b00152 Version 1 (Collection)
Tropine and pseudotropine with opposite stereospecific configurations as platform compounds are central building blocks in both biosynthesis and chemical synthesis of pharmacologically important tropane and nortropane alkaloids. The supply of plant-derived tropine and pseudotropine still heavily depends on either plant extraction or chemical synthesis. Advances in synthetic biology prompt the microbial synthesis of various valuable chemicals. With the biosynthetic pathway elucidation of tropine and pseudotropine in several Solanaceae plants, the key genes were sequentially identified. Here, the enzymes responsible for converting N-methylpyrrolinium into tropine and pseudotropine from Anisodus acutangulus were characterized. Reconstruction of the six-step biosynthetic pathways into Saccharomyces cerevisiae provides cell chassis producing tropine and pseudotropine with 0.13 and 0.08 mg/L titers from simple feedstocks in a shake flask, respectively. The strains described not only offer alternative sources of these central intermediates and their derived alkaloids but also provide platforms for pathway enzyme discovery.
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Heterologous Microcompartment Assembly in Bacillaceae : Establishing the Components Necessary for Scaffold Formation
sb9b00155 Version 1 (Collection)
Bacterial microcompartments (BMCs) are organelles that host specific biochemical reactions for both anabolic and catabolic functions. Engineered morphologically diverse BMCs bearing heterologous enzymatic pathways have shown enhanced productivity for commodity chemicals, which makes BMCs an important focus for metabolic engineering. Gaining control of BMC assembly and incorporation of a heterologous enzymatic cargo has yet to be achieved in thermophiles. Herein, we address this by first conducting a detailed bioinformatic analysis of the propanediol utilization (pdu) operon in the thermophile Parageobacillus thermoglucosidasius. We then demonstrated, in vivo, the ability to assemble the native BMCs at an elevated temperature of 60 °C. Heterologous expression of Pdu shell proteins from P. thermoglucosidasius in Bacillus subtilis resulted in the assembly of a single tubular BMC with an average length of 1.4 μm; BMCs assembled after a 20 min induction of expression of the shell operons. Moreover, we show that it is possible to target the monomeric superfolder GFP (msfGFP) to the interior of the compartment by fusion of an N-terminal sequence of the propanediol utilization protein (PduP) of at least 24 amino acids. This study establishes the feasibility of constructing cell factories for small molecules in industrially important Bacillus and Geobacillus spp. by heterologous cargo-carrying BMC production and assembly. Additionally, the study provides experimental confirmation that BMCs are produced in thermophilic bacteria, which opens a path for future research on repurposing the native organelles to provide new functionality at elevated temperatures.
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Enhancing Light-Driven 1,3-Propanediol Production by Using Natural Compartmentalization of Differentiated Cells
sb8b00239 Version 1 (Collection)
Synthetic biology emerges as a powerful approach for unlocking the potential of cyanobacteria to produce various chemicals. However, the highly oxidative intracellular environment of cyanobacteria is incompatible to numerous introduced enzymes from anaerobes. In this study, we explore a strategy based on natural compartmentalization of cyanobacterial heterocysts to overcome the incompatibility. Hence, the oxygen-sensitive 1,3-propanediol (1,3-PDO) biosynthetic pathway was selected as a model and insulated in heterocysts to evaluate the proposed strategy. Thus, the genes from different sources for 1,3-PDO production were tandemly arrayed with promoter, resulting the assembled 1,3-PDO synthetic cassettes. Then the synthetic cassettes were integrated into the chromosome of Anabaena sp. strain PCC7120 by homologous recombination, respectively. The engineered strain P11 containing the genes from facultative anaerobe Klebsiella pneumoniae (cassette KP) accumulated 46.0 mg L–1 of 1,3-PDO when heterocysts were present, which is approximately 1.7-fold higher than that of no heterocysts. As for the strains (P12, P13, and P14) containing the genes from strictly anaerobic bacterium Clostridium butyricum (cassette CB), the product 1,3-PDO could only be detected when heterocysts were present. These results indicate that insulation of the oxygen-sensitive 1,3-PDO pathway with heterocysts is an effective way to protect these enzymes in cyanobacteria. The strategy may have the potential of serving as a universal strategy to overcome the incompatibility of oxygen-sensitivity in synthetic biology.
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Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l ‑Methionine Biosynthesis in Escherichia coli
sb8b00249 Version 1 (Collection)
To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E. coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise improve the l-methionine titer. The metabolomics data further revealed a more evenly distributed metabolic flux in l-methionine biosynthesis pathway of the optimal strain and provided valuable suggestions for further optimization. The optimal strain produced 16.86 g/L of l-methionine in 48 h by fed-batch fermentation. This work is the first to our knowledge to systematically elucidate the molecular fundamentals of multilevel regulation of l-methionine biosynthesis. It also demonstrated that the systematic analysis strategy can boost our ability to identify the potential bottlenecks and optimize the metabolic flux in multibranched and multilevel regulated pathways for the production of corresponding chemicals.
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Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens
sb8b00252 Version 1 (Collection)
A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries (e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 ± 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E. coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V. natriegens-derived CFPS platform is versatile and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.
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A System for Gene Expression Noise Control in Yeast
sb8b00279 Version 1 (Collection)
Gene expression noise arises from stochastic variation in the synthesis and degradation of mRNA and protein molecules and creates differences in protein numbers across populations of genetically identical cells. Such variability can lead to imprecision and reduced performance of both native and synthetic networks. In principle, gene expression noise can be controlled through the rates of transcription, translation and degradation, such that different combinations of those rates lead to the same protein concentrations but at different noise levels. Here, we present a “noise tuner” which allows orthogonal control over the transcription and the mRNA degradation rates by two different inducer molecules. Combining experiments with theoretical analysis, we show that in this system the noise is largely determined by the transcription rate, whereas the mean expression is determined by both the transcription rate and mRNA stability and can thus be decoupled from the noise. This noise tuner enables 2-fold changes in gene expression noise over a 5-fold range of mean protein levels. We demonstrated the efficacy of the noise tuner in a complex regulatory network by varying gene expression noise in the mating pathway of Saccharomyces cerevisiae, which allowed us to control the output noise and the mutual information transduced through the pathway. The noise tuner thus represents an effective tool of gene expression noise control, both to interrogate noise sensitivity of natural networks and enhance performance of synthetic circuits.
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Enhanced Isoprene Production by Reconstruction of Metabolic Balance between Strengthened Precursor Supply and Improved Isoprene Synthase in Saccharomyces cerevisiae
sb8b00289 Version 1 (Collection)
Isoprene, as a versatile bulk chemical, has wide industrial applications. Here, we attempted to improve isoprene biosynthesis in Saccharomyces cerevisiae by simultaneous strengthening of precursor supply and conversion via a combination of pathway compartmentation and protein engineering. At first, a superior isoprene synthase mutant ISPSLN was created by saturation mutagenesis, leading to almost 4-fold improvement in isoprene production. Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP) pool. To reconstruct metabolic balance between the upstream and downstream flux, additional copies of diphosphomevalonate decarboxylase gene (MVD1) and isopentenyl-diphosphate delta-isomerase gene (IDI1) were introduced into the cytoplasmic and mitochondrial engineered strains. Finally, the diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene, the highest ever reported in eukaryotic cells.
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Constructing Yeast Chimeric Pathways To Boost Lipophilic Terpene Synthesis
sb8b00360 Version 1 (Collection)
Synthetic chimeric biological system offers opportunities to illuminate principles of designing life, and a primary step is constructing synthetic chimeric pathways. Here, we constructed yeast chimeric pathways by transferring the genes from Saccharomyces cerevisiae pathways into another budding yeast Yarrowia lipolytica for in vivo assembly. We efficiently diversified gene option, combination, localization order, and copy number as expected. Convergence of two yeast pathways, especially mevalonic acid (MVA) pathways, remarkably enhanced synthesis of a lipophilic terpene, lycopene. In the selected champion strain with 50-fold of enhanced lycopene production, the chimeric MVA pathway gathered three S. cerevisiae genes with particular copies and locations. Amazingly, therein we discovered distinct transcriptional up-regulation of three significant pathways correlated with acetyl-CoA supply and tuning of cellular lipid amounts and composition. Modulating these pathways further improved lycopene production to 150-fold, a final 259 mg/L (approximately 80 mg/g DCW). We primarily showed the capacity of boosting the synthesis of lipophilic products with yeast chimeric pathways.
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Building Microbial Hosts for Heterologous Production of N ‑Methylpyrrolinium
sb8b00483 Version 1 (Collection)
N-Methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and in Saccharomyces cerevisiae by employing the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we produced N-methylpyrrolinium in vitro from l-ornithine via a combination of the three cascade enzymes, ornithine decarboxylase from Erythroxylum coca, putrescine N-methyltransferase from Anisodus tanguticus, and DAOs from A. acutangulus. Construction of the plant biosynthetic pathway in E. coli and S. cerevisiae resulted in de novo bioproduction of N-methylpyrrolinium with titers of 3.02 and 2.07 mg/L, respectively. Metabolic engineering of the yeast strain to produce N-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving the cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of N-methylpyrrolinium but also lays the foundation for heterologous biosynthesis of N-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed herein can serve as important alternative tools for identifying undiscovered pathway enzymes with a synthetic biology strategy.
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Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae
sb9b00060 Version 1 (Collection)
Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.
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CRISPRi-Based Downregulation of Transcriptional Feedback Improves Growth and Metabolism of Arginine Overproducing E. coli
sb9b00183 Version 1 (Collection)
Removing transcriptional feedback regulation of metabolic pathways is a classical approach to enhance overproduction of chemicals in microbes. However, disrupting transcriptional regulation can have broad physiological consequences that decrease cellular growth and productivity. Here, we compared downregulation and deletion of the transcriptional repressor ArgR in arginine overproducing Escherichia coli. Different levels of ArgR downregulation were achieved with CRISPR interference (CRISPRi) and resulted in 2-times higher growth rates compared to deletion of ArgR, while specific arginine production was similar (∼2 mmol gDW –1 h–1). Metabolomics and proteomics data revealed that poor growth of the ArgR deletion strain was caused by a limitation of pyrimidine nucleotide biosynthesis, because a 17-fold overexpression of ornithine carbamoyltransferase (ArgI) perturbed the arginine–pyrimidine branch point. These results demonstrate that overexpression of enzymes in an engineered pathway can impair metabolism of the host, especially in the case of branch-point enzymes. Thus, balancing enzyme levels is important to optimize industrial microbes, and CRISPRi of a transcription factor is a versatile tool for this purpose.
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Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis
sb9b00193 Version 1 (Collection)
Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon–nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature’s biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.
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CIDAR MoClo Extension Kit Volume I (Murray Lab)
CIDAR_MoClo_Extension_Kit_Volume_I_Murray_Lab_collection Version 1 (Collection)
CIDAR MoClo Extension Kit Volume I (Murray Lab)
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CIDAR MoClo Toolkit (Densmore Lab)
CIDAR_MoClo_Toolkit_Densmore_Lab_collection Version 1 (Collection)
CIDAR MoClo Toolkit (Densmore Lab)
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Natural and Synthetic Terminators (Voigt Lab)
Natural_and_Synthetic_Terminators_Voigt_Lab_collection Version 1 (Collection)
Natural and Synthetic Terminators (Voigt Lab)
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Engineered Networks of Synthetic and Natural Proteins To Control Cell Migration
sb3000172 Version 1 (Collection)
Mammalian cells reprogrammed with engineered transgenes have the potential to be useful therapeutic platforms because they can support large genetic networks, can be taken from a host or patient, and perform useful functions such as migration and secretion. Successful engineering of mammalian cells will require the development of modules that can perform well-defined, reliable functions, such as directed cell migration toward a chemical or physical signal. One inherently modular cellular pathway is the Ca2+ signaling pathway: protein modules that mobilize and respond to Ca2+ are combined across cell types to create complexity. We have designed a chimera of Rac1, a GTPase that controls cell morphology and migration, and calmodulin (CaM), a Ca2+-responsive protein, to control cell migration. The Rac1-CaM chimera (named RACer) controlled lamellipodia growth in response to Ca2+. RACer was combined with LOVS1K (a previously engineered light-sensitive Ca2+-mobilizing module) and cytokine receptors to create protein networks where blue light and growth factors regulated cell morphology and, thereby, cell migration. To show the generalizability of our design, we created a Cdc42-CaM chimera that controls filopodia growth in response to Ca2+. The insights that have been gained into Ca2+ signaling and cell migration will allow future work to combine engineered protein systems to enable reprogrammed cell sensing of relevant therapeutic targets in vivo.
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Modular Construction of a Functional Artificial Epothilone Polyketide Pathway
sb300080t Version 1 (Collection)
Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.
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Rewiring the Wax Ester Production Pathway of Acinetobacter baylyi ADP1
sb4000788 Version 1 (Collection)
Wax esters are industrially relevant high-value molecules. For sustainable production of wax esters, bacterial cell factories are suggested to replace the chemical processes exploiting expensive starting materials. However, it is well recognized that new sophisticated solutions employing synthetic biology toolbox are required to improve and tune the cellular production platform to meet the product requirements. For example, saturated wax esters with alkanol chain lengths C12 or C14 that are convenient for industrial uses are rare among bacteria. Acinetobacter baylyi ADP1, a natural producer of wax esters, is a convenient model organism for studying the potentiality and modifiability of wax esters in a natural host by means of synthetic biology. In order to establish a controllable production platform exploiting well-characterized biocomponents, and to modify the wax ester synthesis pathway of A. baylyi ADP1 in terms product quality, a fatty acid reductase complex LuxCDE with an inducible arabinose promoter was employed to replace the natural fatty acyl-CoA reductase acr1 in ADP1. The engineered strain was able to produce wax esters by the introduced synthetic pathway. Moreover, the fatty alkanol chain length profile of wax esters was found to shift toward shorter and more saturated carbon chains, C16:0 accounting for most of the alkanols. The study demonstrates the potentiality of recircuiting a biosynthesis pathway in a natural producer, enabling a regulated production of a customized bioproduct. Furthermore, the LuxCDE complex can be potentially used as a well-characterized biopart in a variety of synthetic biology applications involving the production of long-chain hydrocarbons.
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Optimization of a Yeast RNA Interference System for Controlling Gene Expression and Enabling Rapid Metabolic Engineering
sb4001432 Version 1 (Collection)
Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design–build–test cycle in yeast.
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Cloning and Optimization of a Nisin Biosynthesis Pathway for Bacteriocin Harvest
sb500225r Version 1 (Collection)
Nisin is an important antimicrobial peptide that has enormous applications in biotechnology. Despite many encouraging efforts, its overproduction has been a long-standing challenge due to the complexity of the underlying pathway and the difficulty in genetic modification of lactic acid bacteria. Here, we cloned an entire nisin biosynthesis pathway from a nisin-producing strain (Lactococcus lactis K29) into a plasmid and transplanted the plasmid into a nisin deficient strain Lactococcus lactis MG1363, resulting in successful heterologous expression of bioactive recombinant nisin. To increase nisin harvest, we also overexpressed nisA, a gene responsible for nisin precursor production, with a set of constitutive promoters. To further optimize nisin yield, we minimized the metabolic cost of the engineered strains by integrating nisA overexpression cassettes and the recombinant pathway into a single circuit. With our rational construction and optimization, our engineered optimized strain is able to produce bioactive nisin with a yield of 1098 IU/mL, which is more than six times higher than that of the original strain.
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Homology-Integrated CRISPR–Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae
sb500255k Version 1 (Collection)
One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.
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Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems
sb500273n Version 1 (Collection)
Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms. Previously, we have engineered red/far-red and green/red photoreversible two-component signal transduction systems (TCSs) with transcriptional outputs in E. coli and used them to characterize and control synthetic gene circuits with exceptional quantitative, temporal, and spatial precision. However, the broad utility of these light sensors is limited by bulky DNA encoding, incompatibility with commonly used ligand-responsive transcription factors, leaky output in deactivating light, and less than 10-fold dynamic range. Here, we compress the four genes required for each TCS onto two streamlined plasmids and replace all chemically inducible and evolved promoters with constitutive, engineered versions. Additionally, we systematically optimize the expression of each sensor histidine kinase and response regulator, and redesign both pathway output promoters, resulting in low leakiness and 72- and 117-fold dynamic range, respectively. These second-generation light sensors can be used to program the expression of more genes over a wider range and can be more easily combined with additional plasmids or moved to different host strains. This work demonstrates that bacterial TCSs can be optimized to function as high-performance sensors for scientific and engineering applications.
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Systematically Engineering Escherichia coli for Enhanced Production of 1,2-Propanediol and 1‑Propanol
sb500345t Version 1 (Collection)
The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.
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Evolution of Synthetic Signaling Scaffolds by Recombination of Modular Protein Domains
sb5003482 Version 1 (Collection)
Signaling scaffolds are proteins that interact via modular domains with multiple partners, regulating signaling networks in space and time and providing an ideal platform from which to alter signaling functions. However, to better exploit scaffolds for signaling engineering, it is necessary to understand the full extent of their modularity. We used a directed evolution approach to identify, from a large library of randomly shuffled protein interaction domains, variants capable of rescuing the signaling defect of a yeast strain in which Ste5, the scaffold in the mating pathway, had been deleted. After a single round of selection, we identified multiple synthetic scaffold variants with diverse domain architectures, able to mediate mating pathway activation in a pheromone-dependent manner. The facility with which this signaling network accommodates changes in scaffold architecture suggests that the mating signaling complex does not possess a single, precisely defined geometry into which the scaffold has to fit. These relaxed geometric constraints may facilitate the evolution of signaling networks, as well as their engineering for applications in synthetic biology.
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High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System
sb500351f Version 1 (Collection)
Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.
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Orthogonal Fatty Acid Biosynthetic Pathway Improves Fatty Acid Ethyl Ester Production in Saccharomyces cerevisiae
sb500319p Version 1 (Collection)
Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms.
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Expansion of Bisindole Biosynthetic Pathways by Combinatorial Construction
sb5003218 Version 1 (Collection)
Cladoniamides are indolotryptoline natural products that derive from indolocarbazole precursors. Here, we present a microbial platform to artificially redirect the cladoniamide pathway to generate unnatural bisindoles for drug discovery. Specifically, we target glycosyltransferase, halogenase, and oxidoreductase genes from the phylogenetically related indolocarbazole rebeccamycin and staurosporine pathways. We generate a series of novel compounds, reveal details about the substrate specificities of a number of enzymes, and set the stage for future efforts to develop new catalysts and compounds by engineering of bisindole genes. The strategy for structural diversification we use here could furthermore be applied to other natural product families with known biosynthetic genes.
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Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli
sb5b00092 Version 1 (Collection)
Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, Mentha spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)­synthesis of (1R,2S,5R)-(−)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an “ene”-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.
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Development of an Unnatural Amino Acid Incorporation System in the Actinobacterial Natural Product Producer Streptomyces venezuelae ATCC 15439
sb5b00209 Version 1 (Collection)
Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439. We observed notable differences in the fidelity and efficiency of these systems between S. venezuelae and previously used hosts. Our findings serve as a foundation for using an expanded genetic code in Streptomyces to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.
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Flux Control at the Malonyl-CoA Node through Hierarchical Dynamic Pathway Regulation in Saccharomyces cerevisiae
sb5b00161 Version 1 (Collection)
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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Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae
sb5b00164 Version 1 (Collection)
Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae . In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.
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Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p ‑Coumaric Acid Production in Pseudomonas putida KT2440
sb6b00081 Version 1 (Collection)
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 useful tools for optimizing gene expression in a variety of bacteria.
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Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening
sb6b00083 Version 1 (Collection)
As a product of a multistep enzymatic reaction, accumulation of poly­(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%–92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (M w ) of 2.7–6.8 × 106 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.
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Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink
sb6b00100 Version 1 (Collection)
Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme Rubisco resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant, we engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450 CYP1A1 to serve as an artificial electron sink for excess electrons derived from light-catalyzed water-splitting. This improved photosynthetic efficiency by increasing the maximum rate of photosynthetic electron flow by 31.3%. A simple fluorescent assay for CYP1A1 activity demonstrated that the P450 was functional in the absence of its native reductase, that activity was light-dependent and scaled with irradiance. We show for the first time in live cells that photosynthetic reductant can be redirected to power a heterologous cytochrome P450. Furthermore, Synechococcus PCC 7002 expressing CYP1A1 degraded the herbicide atrazine, which is a widespread environmental pollutant.
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Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate
sb6b00206 Version 1 (Collection)
Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5α-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors.
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Improving Metabolic Pathway Efficiency by Statistical Model-Based Multivariate Regulatory Metabolic Engineering
sb6b00187 Version 1 (Collection)
Metabolic engineering entails target modification of cell metabolism to maximize the production of a specific compound. For empowering combinatorial optimization in strain engineering, tools and algorithms are needed to efficiently sample the multidimensional gene expression space and locate the desirable overproduction phenotype. We addressed this challenge by employing design of experiment (DoE) models to quantitatively correlate gene expression with strain performance. By fractionally sampling the gene expression landscape, we statistically screened the dominant enzyme targets that determine metabolic pathway efficiency. An empirical quadratic regression model was subsequently used to identify the optimal gene expression patterns of the investigated pathway. As a proof of concept, our approach yielded the natural product violacein at 525.4 mg/L in shake flasks, a 3.2-fold increase from the baseline strain. Violacein production was further increased to 1.31 g/L in a controlled benchtop bioreactor. We found that formulating discretized gene expression levels into logarithmic variables (Linlog transformation) was essential for implementing this DoE-based optimization procedure. The reported methodology can aid multivariate combinatorial pathway engineering and may be generalized as a standard procedure for accelerating strain engineering and improving metabolic pathway efficiency.
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Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants
sb6b00295 Version 1 (Collection)
Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. Meeting these challenges will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5′UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repress transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Using a bioinformatics approach, we identified 54 orthologous systems from various bacteria, and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.
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Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII
sb6b00341 Version 1 (Collection)
During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by “click” chemistry.
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Designing a New Entry Point into Isoprenoid Metabolism by Exploiting Fructose-6-Phosphate Aldolase Side Reactivity of Escherichia coli
sb7b00072 Version 1 (Collection)
The 2C-methyl-d-erythritol-4-phosphate (MEP) pathway in Escherichia coli has been highlighted for its potential to provide access to myriad isoprenoid chemicals of industrial and therapeutic relevance and discover antibiotic targets to treat microbial human pathogens. Here, we describe a metabolic engineering strategy for the de novo construction of a biosynthetic pathway that produces 1-dexoxy-d-xylulose-5-phosphate (DXP), the precursor metabolite of the MEP pathway, from the simple and renewable starting materials d-arabinose and hydroxyacetone. Unlike most metabolic engineering efforts in which cell metabolism is reprogrammed with enzymes that are highly specific to their desired reaction, we highlight the promiscuous activity of the native E. coli fructose-6-phosphate aldolase as central to the metabolic rerouting of carbon to DXP. We use mass spectrometric isotopomer analysis of intracellular metabolites to show that the engineered pathway is able to support in vivo DXP biosynthesis in E. coli. The engineered DXP synthesis is further able to rescue cells that were chemically inhibited in their ability to produce DXP and to increase terpene titers in strains harboring the non-native lycopene pathway. In addition to providing an alternative metabolic pathway to produce isoprenoids, the results here highlight the potential role of pathway evolution to circumvent metabolic inhibitors in the development of microbial antibiotic resistance.
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Improvement of Squalene Production from CO 2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor
sb7b00083 Version 1 (Collection)
The push-and-pull strategy for metabolic engineering was successfully demonstrated in Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate synthase) of the methylerythritol phosphate pathway or the β-subunit of phycocyanin (CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold compared to that of the control strain expressing SQS alone. To increase squalene production further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization, which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell weight). Further optimization for photobioprocessing and strain development will promote the construction of a solar-to-chemical platform.
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