Showing papers in "ACS Synthetic Biology in 2021"

Transcription-Factor-based Biosensor Engineering for Applications in Synthetic Biology.

Abstract: Transcription-factor-based biosensors (TFBs) are often used for metabolite detection, adaptive evolution, and metabolic flux control. However, designing TFBs with superior performance for applications in synthetic biology remains challenging. Specifically, natural TFBs often do not meet real-time detection requirements owing to their slow response times and inappropriate dynamic ranges, detection ranges, sensitivity, and selectivity. Furthermore, designing and optimizing complex dynamic regulation networks is time-consuming and labor-intensive. This Review highlights TFB-based applications and recent engineering strategies ranging from traditional trial-and-error approaches to novel computer-model-based rational design approaches. The limitations of the applications and these engineering strategies are additionally reviewed.

Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput.

Abstract: Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme.

Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons.

Abstract: mRNA degradation is a central process that affects all gene expression levels, and yet, the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biol...

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria.

Abstract: Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

Statistical Design of Experiments for Synthetic Biology.

Abstract: The design and optimization of biological systems is an inherently complex undertaking that requires careful balancing of myriad synergistic and antagonistic variables. However, despite this complexity, much synthetic biology research is predicated on One Factor at A Time (OFAT) experimentation; the genetic and environmental variables affecting the activity of a system of interest are sequentially altered while all other variables are held constant. Beyond being time and resource intensive, OFAT experimentation crucially ignores the effect of interactions between factors. Given the ubiquity of interacting genetic and environmental factors in biology this failure to account for interaction effects in OFAT experimentation can result in the development of suboptimal systems. To address these limitations, an increasing number of studies have turned to Design of Experiments (DoE), a suite of methods that enable efficient, systematic exploration and exploitation of complex design spaces. This review provides an overview of DoE for synthetic biologists. Key concepts and commonly used experimental designs are introduced, and we discuss the advantages of DoE as compared to OFAT experimentation. We dissect the applicability of DoE in the context of synthetic biology and review studies which have successfully employed these methods, illustrating the potential of statistical experimental design to guide the design, characterization, and optimization of biological protocols, pathways, and processes.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

Abstract: Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP) These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600 We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin These circuits expand the applicability of optogenetics to metabolic engineering

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles.

Abstract: Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

Rational Promoter Engineering Enables Robust Terpene Production in Microalgae.

Abstract: Microalgal biotechnology promises sustainable light-driven production of valuable bioproducts and addresses urgent demands to attain a sustainable economy. However, to unfold its full potential as a platform for biotechnology, new and powerful tools for nuclear engineering need to be established. Chlamydomonas reinhardtii, the model for microalgal synthetic biology and genetic engineering has already been used to produce various bioproducts. Nevertheless, low transgene titers, the lack of potent expression elements, and sparse comparative evaluation prevents further development of C. reinhardtii as a biotechnological host. By systematically evaluating existing expression elements combined with rational promoter engineering, we established novel synthetic expression elements, improved the standardized application of synthetic biology tools, and unveiled an existing synergism between the PSAD 5' UTR and its corresponding chloroplast targeting peptide. Promoter engineering strategies, implemented in a newly designed synthetic algal promoter, increased the production of the sesquiterpene (E)-α-bisabolene by 18-fold compared to its native version and 4-fold to commonly used expression elements. Our results improve the application of synthetic biology in microalgae and display a significant step toward establishing C. reinhardtii as a sustainable green cell-factory.

CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research.

Abstract: Over the past decades, significant progress has been made in targeted cancer therapy. In precision oncology, molecular profiling of cancer patients enables the use of targeted cancer therapeutics. However, current diagnostic methods for molecular analysis of cancer are costly and require sophisticated equipment. Moreover, targeted cancer therapeutics such as monoclonal antibodies and small-molecule drugs may cause off-target effects and they are available for only a minority of cancer driver proteins. Therefore, there is still a need for versatile, efficient, and precise tools for cancer diagnostics and targeted cancer treatment. In recent years, the CRISPR-based genome and transcriptome engineering toolbox has expanded rapidly. Particularly, the RNA-targeting CRISPR-Cas13 system has unique biochemical properties, making Cas13 a promising tool for cancer diagnosis, therapy, and research. Cas13-based diagnostic methods allow early detection and monitoring of cancer markers from liquid biopsy samples without the need for complex instrumentation. In addition, Cas13 can be used for targeted cancer therapy through degrading and manipulating cancer-associated transcripts with high efficiency and specificity. Moreover, Cas13-mediated programmable RNA manipulation tools offer invaluable opportunities for cancer research, identification of drug-resistance mechanisms, and discovery of novel therapeutic targets. Here, we review and discuss the current use and potential applications of the CRISPR-Cas13 system in cancer diagnosis, therapy, and research. Thus, researchers will gain a deep understanding of CRISPR-Cas13 technologies, which have the potential to be used as next-generation cancer diagnostics and therapeutics.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Abstract: Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Morphological and Metabolic Engineering of Yarrowia lipolytica to Increase β-Carotene Production.

Abstract: The oleaginous yeast Yarrowia lipolytica represents an environmentally friendly platform cell factory for β-carotene production. However, Y. lipolytica is a dimorphic species that can undergo a yeast-to-mycelium transition when exposed to stress. The mycelial form is unfavorable for industrial fermentation. In this study, β-carotene-producing Y. lipolytica strains were constructed via the integration of multiple copies of 13 genes related to the β-carotene biosynthesis pathway. The β-carotene content increased by 11.7-fold compared with the start strain T1. As the β-carotene content increased, the oval-shaped yeast form was gradually replaced by hyphae, implying that the accumulation of β-carotene in Y. lipolytica induces a morphological transition. To relieve this metabolic stress, the strains were morphologically engineered by deleting CLA4 and MHY1 genes to convert the mycelium back to the yeast form, which further increased the β-carotene production by 139%. In fed-batch fermentation, the engineered strain produced 7.6 g/L and 159 mg/g DCW β-carotene, which is the highest titer and content reported to date. The morphological engineering strategy developed here may be useful for enhancing chemical synthesis in dimorphic yeasts.

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.

Abstract: Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.

Kinetics of RNA and RNA:DNA Hybrid Strand Displacement.

Abstract: In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields

Abstract: Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Metabolic Engineering of Escherichia coli for De Novo Production of 1,5-Pentanediol from Glucose.

Abstract: 1,5-Pentanediol (1,5-PDO) is an important C5 building block for the synthesis of different value-added polyurethanes and polyesters. However, no natural metabolic pathway exists for the biosynthesis of 1,5-PDO. Herein we designed and constructed a promising nonnatural pathway for de novo production of 1,5-PDO from cheap carbohydrates. This biosynthesis route expands natural lysine pathways and employs two artificial metabolic modules to sequentially convert lysine into 5-hydroxyvalerate (5-HV) and 1,5-PDO via 5-hydroxyvaleryl-CoA. Theoretically, the 5-hydroxyvaleryl-CoA-based pathway is more energy-efficient than a recently published carboxylic acid reductase-based pathway for 1,5-PDO production. By combining strategies of systematic enzyme screening, pathway balancing, and transporter engineering, we successfully constructed a minimally engineered Escherichia coli strain capable of producing 3.19 g/L of 5-HV and 0.35 g/L of 1,5-PDO in a medium containing 20 g/L of glucose and 5 g/L lysine. Introducing the synthetic modules into a lysine producer and enhancing NADPH supply enabled the strain to accumulate 1.04 g/L of 5-HV and 0.12 g/L of 1,5-PDO using glucose as the main carbon source. This work lays the basis for the development of a biological route for 1,5-PDO production from renewable bioresources.

Dual Regulation of Cytoplasm and Peroxisomes for Improved Α-Farnesene Production in Recombinant Pichia pastoris.

Abstract: Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii

Abstract: Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small mo...

High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae.

Abstract: Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons

Abstract: The construction of non-native biosynthetic pathways represents a powerful, modular strategy for the production of valuable synthons and fine chemicals. Accordingly, artificially affixing enzymes that catalyze sequential reactions onto DNAs, proteins, or synthetic scaffolds has proven to be an effective route for generating de novo metabolons with novel functionalities and superior efficiency. In recent years, nanoscale microbial compartments known as encapsulins have emerged as a class of robust and highly engineerable proteinaceous containers with myriad applications in biotechnology and synthetic biology. Herein we report the concurrent surface functionalization and internal packaging of encapsulins from Thermotoga maritima to generate a catalytically competent two-enzyme metabolon. Encapsulins were engineered to covalently sequester up to 60 copies of a dihydrofolate reductase (DHFR) enzyme variant on their exterior surfaces using the SpyCatcher bioconjugation system, while their lumens were packaged with a tetrahydrofolate-dependent demethylase enzyme using short peptide affinity tags abstracted from the encapsulin's native protein cargo. Successful cross-talk between the two colocalized enzymes was confirmed as tetrahydrofolate produced by externally tethered DHFR was capable of driving the demethylation of a lignin-derived aryl substrate by packaged demethylases, albeit slowly. The subsequent introduction of a previously reported pore-enlarging deletion in the encapsulin shell was shown to enhance metabolite exchange such that the encapsulin-based metabolon functioned at speeds equivalent to those of the two enzymes freely dispersed in solution. Our work thus further emphasizes the engineerability of encapsulins and their potential use as flexile scaffolds for biocatalytic applications.

Engineering an Optogenetic CRISPRi Platform for Improved Chemical Production.

Abstract: Microbial synthesis of chemicals typically requires the redistribution of metabolic flux toward the synthesis of targeted products. Dynamic control is emerging as an effective approach for solving the hurdles mentioned above. As light could control the cell behavior in a spatial and temporal manner, the optogenetic-CRISPR interference (opto-CRISPRi) technique that allocates the metabolic resources according to different optical signal frequencies will enable bacteria to be controlled between the growth phase and the production stage. In this study, we applied a blue light-sensitive protein EL222 to regulate the expression of the dCpf1-mediated CRISPRi system that turns off the competitive pathways and redirects the metabolic flux toward the heterologous muconic acid synthesis in Escherichia coli. We found that the opto-CRISPRi system dynamically regulating the suppression of the central metabolism and competitive pathways could increase the muconic acid production by 130%. These results demonstrated that the opto-CRISPRi platform is an effective method for enhancing chemical synthesis with broad utilities.

Modular Synthetic Biology Toolkit for Filamentous Fungi.

Abstract: Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.

A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens.

Abstract: Water contamination by pathogenic bacteria is a major public health concern globally Monitoring bacterial contamination in water is critically important to protect human health, but this remains a

Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis

Abstract: Human milk oligosaccharides (HMOs) are one of the important ingredients in human milk, which have attracted great interest due to their beneficial effect on the health of newborns. The large-scale production of HMOs has been researched using engineered microbial routes due to the availability, safety, and low cost of host strains. In addition, the development of molecular biology technology and metabolic engineering has promoted the effectiveness of HMOs production. According to current reports, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), and some fucosylated HMOs with complex structures have been produced via the engineered microbial route, with 2'-FL having been produced the most. However, due to the uncertainty of metabolic patterns, the selection of host strains has certain limitations. Aside from that, the expression of appropriate glycosyltransferase in microbes is key to the synthesis of different HMOs. Therefore, finding a safe and efficient glycosyltransferase has to be addressed when using engineered microbial pathways. In this review, the latest research on the production of HMOs using engineered microbial routes is reported. The selection of host strains and adapting different metabolic pathways helped researchers designing engineered microbial routes that are more conducive to HMOs production.

Optogenetic Control of Microbial Consortia Populations for Chemical Production.

Abstract: Microbial co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. In this study, we demonstrate that optogenetics is an effective strategy to dynamically control populations in microbial co-cultures. Using a new optogenetic circuit we call OptoTA, we regulate an endogenous toxin-antitoxin system, enabling tunability of Escherichia coli growth using only blue light. With this system we can control the population composition of co-cultures of E. coli and Saccharomyces cerevisiae. When introducing in each strain different metabolic modules of biosynthetic pathways for isobutyl acetate or naringenin, we found that the productivity of co-cultures increases by adjusting the population ratios with specific light duty cycles. This study shows the feasibility of using optogenetics to control microbial consortia populations and the advantages of using light to control their chemical production.

A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica.

Abstract: Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Systematically Engineered Fatty Acid Catabolite Pathway for the Production of (2S)-Naringenin in Saccharomyces cerevisiae.

Abstract: The (2S)-naringenin is an important natural flavonoid with several bioactive effects on human health. It is also a key precursor in the biosynthesis of other high value compounds. The production of (2S)-naringenin is significantly influenced by the acetyl-CoA available in the cytosol. In this study, we increased the acetyl-CoA supply via the β-oxidation of fatty acids in the peroxisomes of Saccharomyces cerevisiae. Several lipases from different sources and PEX11, FOX1, FOX2, and FOX3, the key genes of the fatty acid β-oxidation pathway, were overexpressed during the production of (2S)-naringenin in yeast. The level of acetyl-CoA was 0.205 nmol higher than that in the original strain and the production of (2S)-naringenin increased to 286.62 mg/g dry cell weight when PEX11 was overexpressed in S. cerevisiae strain L07. Remarkable (2S)-naringenin production (1129.44 mg/L) was achieved with fed-batch fermentation, with the highest titer reported in any microorganism. Our results demonstrated the use of fatty acid β-oxidation to increase the level of cytoplasmic acetyl-CoA and the production of its derivatives.

A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets.

Abstract: Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.

A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology.

Abstract: Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test tube. The key advantages of this approach are the reduced experimental time scales and controlled reaction conditions. To realize this potential, it is essential to develop specialized cell-free systems in organisms enriched for biosynthetic gene clusters. This requires strong protein production and well-characterized synthetic biology tools. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool, and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterize a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes, and demonstrate an initial proof of concept for combined transcription, translation, and biosynthesis of Streptomyces metabolic pathways in a single "one-pot" reaction.

Prime Editing Guide RNA Design Automation Using PINE-CONE

Abstract: CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.

Biotechnological Production of 2'-Fucosyllactose: A Prevalent Fucosylated Human Milk Oligosaccharide.

Abstract: Human milk oligosaccharide (HMO) is a key component of human milk carbohydrates and is closely related to the nutrition and health benefits of breastfeeding in infants. 2'-Fucosyllactose (2'-FL) is the most abundant fucosylated HMO, which has remarkable value in nutrition and medicine, such as suppressing pathogen infection, regulating intestinal flora, and boosting immunity. However, 2'-FL production via the method of extraction or chemical synthesis cannot meet its large demand, and as a result, environmentally friendly and efficient biotechnological approaches, including in vitro enzymatic synthesis and microbial cell factory production, have been developed and applied to its commercialized production. This review introduces, summarizes, and discusses the recent advances in the biotechnological production of 2'-FL. Furthermore, future research directions for the biotechnological production of 2'-FL as well as the strategies to further improve its concentration are highlighted and discussed.