A signal transmission strategy driven by gap-regulated exonuclease hydrolysis for hierarchical molecular networks.

Exonucleases serve as efficient tools for signal processing and play an important role in biochemical reactions. Here, we identify the mechanism of cooperative exonuclease hydrolysis, offering a method to regulate the cooperative hydrolysis driven by exonucleases through the modulation of the number of bases in gap region. A signal transmission strategy capable of producing amplified orthogonal DNA signal is proposed to resolve the polarity of signals and byproducts, which provides a solution to overcome the signal attenuation. The gap-regulated mechanism combined with DNA strand displacement (DSD) reduces the unpredictable secondary structures, allowing for the coexistence of similar structures in hierarchical molecular networks. For the application of the strategy, a molecular computing model is constructed to solve the maximum weight clique problems (MWCP). This work enhances for our knowledge of these important enzymes and promises application prospects in molecular computing, signal detection, and nanomachines.

Catalytic Gold Nanoparticle Assembly Programmed by DNAzyme Circuits.

Assembled gold nanoparticle (AuNP) superstructures can generate unique physicochemical characteristics and be used in various applications, thus becoming an attractive research field. Recently, several DNA-assisted gold nanoparticle assembly methods have been rigorously developed that typically require a non-catalytic equimolar molecular assembly to guarantee the designed assembly. Although efficient and accurate, exploring such non-catalytic nanoparticle assemblies in the complex cellular milieu under low trigger concentrations remains challenging. Therefore, developing a catalytic method that facilitates gold nanoparticle assemblies with relatively low DNA trigger concentrations is desirable. In this report, a catalytic method to program gold nanoparticle assemblies by DNAzyme circuits is presented, where only a small number of DNA triggers are able to induce the production of a large number of the desired nanoparticle assemblies. The feasibility of using logic DNAzyme circuits to control catalytic nanoparticle assemblies is experimentally verified. Additionally, catalytic AuNP assembly systems are established with cascading and feedback functions. The work provides an alternative research direction to enrich the tool library of nanoparticle assembly and their application in biosensing and nanomedicine.

[Multiplex gene editing and regulation techniques based on CRISPR/Cas system].

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.

Transcriptional Regulatory Networks Involved in C3-C4 Alcohol Stress Response and Tolerance in Yeast.

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. In this study, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3-C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3-C4 alcohols by perturbing the transcriptional regulatory network.

Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing.

CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.

Engineering regulatory networks for complex phenotypes in E. coli.

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli, with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts.

Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2-C6 Bioalcohol Production.

Biofuel production from renewable and sustainable resources is playing an increasingly important role within the fuel industry. Among biofuels, bioethanol has been most widely used as an additive for gasoline. Higher alcohols can be blended at a higher volume compared to ethanol and generate lower greenhouse gas (GHG) emissions without a need to change current fuel infrastructures. Thus, these fuels have the potential to replace fossil fuels in support of more environmentally friendly processes. This review summarizes the efforts to enhance bioalcohol production in engineered Escherichia coli over the last 5 years and analyzes the current challenges for increasing productivities for industrial applications.

Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform.

Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.

Genome engineering of E. coli for improved styrene production.

Microbial production of exogenous organic compounds is challenging as biosynthetic pathways are often complex and produce metabolites that are toxic to the hosts. Biogenic styrene is an example of this problem, which if addressed could result in a more sustainable supply of this important component of the plastics industry. In this study, we engineered Escherichia coli for the production of styrene. We systematically optimized the production capability by first screening different pathway expression levels in E. coli strains. We then further designed and constructed a transcription regulator library targeting 54 genes with 85,420 mutations, and tested this library for increased styrene resistance and production. A series of tolerant mutants not only exhibited improved styrene tolerance but also produced higher styrene concentrations compared to the parent strain. The best producing mutant, ST05 LexA_E45I, produced a 3.45-fold increase in styrene compared to the parent strain. The produced styrene was extracted via gas stripping into dodecane and used in a direct free radical synthesis of polystyrene.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

Rational Control of Calcium Carbonate Precipitation by Engineered Escherichia coli.

Ureolytic bacteria ( e.g., Sporosarcina pasteurii) can produce calcium carbonate (CaCO3). Tailoring the size and shape of biogenic CaCO3 may increase the range of useful applications for these crystals. However, wild type Sporosarcina pasteurii is difficult to genetically engineer, limiting control of the organism and its crystal precipitates. Therefore, we designed, constructed, and compared different urease operons and expression levels for CaCO3 production in engineered Escherichia coli strains. We quantified urease expression and calcium uptake and characterized CaCO3 crystal phase and morphology for 13 engineered strains. There was a weak relationship between urease expression and crystal size, suggesting that genes surrounding the urease gene cluster affect crystal size. However, when evaluating strains with a wider range of urease expression levels, there was a negative relationship between urease activity and polycrystal size (e.g., larger crystals with lower activity). The resulting range of crystal morphologies created by the rationally designed strains demonstrates the potential for controlling biogenic CaCO3 precipitation.

Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production.

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP). Specifically, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~ 162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~ 1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~ 60-fold higher than the wild-type strain.

Combinatorial pathway engineering using type I-E CRISPR interference.

Optimization of metabolic flux is a difficult and time-consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial-and-error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I-E CRISPR-Cas system and an iterative cloning strategy for construction of guide RNA arrays. This approach allowed us to build a combinatorial gene expression library three orders of magnitude larger than previous studies. In less than 1 month, we generated ∼12,000 combinatorial gene expression variants that target six different genes and screened these variants for increased malonyl-CoA flux and 3-hydroxypropionate (3HP) production. We were able to identify a set of variants that exhibited a significant increase in malonyl-CoA flux and up to a 98% increase in 3HP production. This approach provides a fast and easy-to-implement strategy for engineering metabolic pathway flux for development of industrially relevant strains, as well as investigation of fundamental biological questions.

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering.

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

Renewable acrylonitrile production.

Acrylonitrile (ACN) is a petroleum-derived compound used in resins, polymers, acrylics, and carbon fiber. We present a process for renewable ACN production using 3-hydroxypropionic acid (3-HP), which can be produced microbially from sugars. The process achieves ACN molar yields exceeding 90% from ethyl 3-hydroxypropanoate (ethyl 3-HP) via dehydration and nitrilation with ammonia over an inexpensive titanium dioxide solid acid catalyst. We further describe an integrated process modeled at scale that is based on this chemistry and achieves near-quantitative ACN yields (98 ± 2%) from ethyl acrylate. This endothermic approach eliminates runaway reaction hazards and achieves higher yields than the standard propylene ammoxidation process. Avoidance of hydrogen cyanide as a by-product also improves process safety and mitigates product handling requirements.

The Prevalence of Ocular Allergy and Comorbidities in Chinese School Children in Shanghai.

OBJECTIVE: To investigate the prevalence and features of ocular allergy (OA) and comorbidities among school children in Shanghai, China. METHODS: This was a population-based cross-sectional study. Each participant completed an ISAAC-based questionnaire. The prevalence of OA symptoms, allergic rhinitis (AR) asthma, atopic dermatitis (AD), and sensitization to mites, pollen, and food was analyzed. RESULTS: A total of 724 and 942 completed questionnaires from the 7-9-year-old (young group) and the 12-14-year-old (teen group) groups were analyzed, respectively. The overall prevalence of OA symptoms was 28%. However, more young students (10.6%) reported mild to severe daily life interference caused by OA than the teens (5.7%). The young group had higher prevalence of diagnosed allergic conjunctivitis (10.2%). The overall prevalence of AR symptom, diagnosed asthma, and diagnosed AD was 40.4%, 11.6%, and 16.7%, respectively. Young children had higher prevalence of diagnosed AR and AD than the teens. There were gender associated differences in the prevalence of AR and asthma among young children, but not among the teens. The comorbidities associated with OA was also analyzed. Sensitization to mites, food, and pollen was associated with higher prevalence of allergic conditions. CONCLUSIONS: OA together with other allergic conditions affected a significant number of children in Shanghai.

Progress of succinic acid production from renewable resources: Metabolic and fermentative strategies.

Succinic acid is a four-carbon dicarboxylic acid, which has attracted much interest due to its abroad usage as a precursor of many industrially important chemicals in the food, chemicals, and pharmaceutical industries. Facing the shortage of crude oil supply and demand of sustainable development, biological production of succinic acid from renewable resources has become a topic of worldwide interest. In recent decades, robust producing strain selection, metabolic engineering of model strains, and process optimization for succinic acid production have been developed. This review provides an overview of succinic acid producers and cultivation technology, highlight some of the successful metabolic engineering approaches.

CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli.

Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1g/L at 24h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62g/L/h (which was 0.22g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

Directed evolution and synthetic biology applications to microbial systems.

Biotechnology applications require engineering complex multi-genic traits. The lack of knowledge on the genetic basis of complex phenotypes restricts our ability to rationally engineer them. However, complex phenotypes can be engineered at the systems level, utilizing directed evolution strategies that drive whole biological systems toward desired phenotypes without requiring prior knowledge of the genetic basis of the targeted trait. Recent developments in the synthetic biology field accelerates the directed evolution cycle, facilitating engineering of increasingly complex traits in biological systems. In this review, we summarize some of the most recent advances in directed evolution and synthetic biology that allows engineering of complex traits in microbial systems. Then, we discuss applications that can be achieved through engineering at the systems level.