Effect of ozone treatment on phenylpropanoid metabolism in harvested cantaloupes.

Phenylpropanoid metabolism plays an important role in cantaloupe ripening and senescence, but the mechanism of ozone regulation on phenylpropanoid metabolism remains unclear. This study investigated how ozone treatment modulates the levels of secondary metabolites associated with phenylpropanoid metabolism, the related enzyme activities, and gene expression in cantaloupe. Treating cantaloupes with 15 mg/m3 of ozone after precooling can help maintain postharvest hardness. This treatment also enhances the production and accumulation of secondary metabolites, such as total phenols, flavonoids, and lignin. These metabolites are essential components of the phenylpropanoid metabolic pathway, activating enzymes like phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4CL, chalcone synthase, and chalcone isomerase. The results of the transcriptional expression patterns showed that differential gene expression related to phenylpropanoid metabolism in the peel of ozone-treated cantaloupes was primarily observed during the middle and late storage stages. In contrast, the pulp exhibited significant differential gene expression mainly during the early storage stage. Furthermore, it was observed that the level of gene expression in the peel was generally higher than that in the pulp. The correlation between the relative amount of gene changes in cantaloupe, activity of selected enzymes, and concentration of secondary metabolites could be accompanied by positive regulation of the phenylpropanoid metabolic pathway. Therefore, ozone stress induction positively enhances the biosynthesis of flavonoids in cantaloupes, leading to an increased accumulation of secondary metabolites. Additionally, it also improves the postharvest storage quality of cantaloupes.

Extraction of flavonoids from black mulberry wine residues and their antioxidant and anticancer activity in vitro.

Enhancing the valorization of fruit processing by-products is pivotal for advancing the industry. Black mulberry wine residues, a by-product, contains some bioactive compounds, yet its antioxidant and anticancer potentials remain unverified. In this study, ultrasound-assisted enzymatic extraction was optimized by response surface methodology to obtain the flavonoids extracts from black mulberry wine residues, whose antioxidant capacity and anti-cancer activity in vitro was investigated. The results showed that under the optimal extraction conditions (enzyme ratio at pectinase:cellulose = 2:1, mixed enzyme concentration 0.31 mg/mL, enzymatic hydrolysis temperature 55.35 °C, enzymatic hydrolysis time 79.03 min, and ultrasonic time 22.71 min), the extracts from black mulberry wine residues (BMWR-E) reached 5.672 mg/g. At a concentration of 1.2 mg/mL, BMWR-E exhibited strong DPPH and hydroxyl radical scavenging activities. At a concentration of 2.5 mg/mL, BMWR-E showed a strong superoxide anion radical scavenging capacity, with no significant distinction compared to the positive control group (Vitamin C) (p > 0.05). Cell viability assay results showed that BMWR-E was non-toxic to normal BRL-3A cells when applied at concentrations of 0.1-0.3 mg/mL for an incubation period of 24 h, but BMWR-E exhibited the ability to inhibit the proliferation of HepG2 cells. At concentrations of 0.2 mg/mL and above, BMWR-E could induce late apoptosis of HepG2 cells by increasing the protein expression levels of Bax, caspase-3, and caspase-12, reducing the protein expression levels of Bcl-2, inducing cell cycle arrest at G0/G1 phase, thereby inhibiting the proliferation of HepG2 cells. The bioactive properties make BMWR-E possess potential in developing new antioxidants and anti-cancer agents, which would significantly enhance the economic worth of agricultural by-products in product processing. This research can improve the utilization rate of agricultural product processing by-products and protect the environment.

Rapid Cooling Delays the Occurring of Core Browning in Postharvest 'Yali' Pear at Advanced Maturity by Inhibiting Ethylene Metabolism.

During the storage and transportation processes, the occurrence of core browning in 'Yali' pear fruit due to adversity injury can be easily mitigated by implementing different cooling methods, especially in advanced maturity fruits. In this study, 'Yali' pears at an advanced maturity stage were subjected to slow cooling and rapid cooling treatment. The quality-related physiological percentage and severity, and the rate of good fruits were determined, and RNA-seq was used to explore the effects of different cooling methods on pathways related to core browning in advanced-maturity pears at the transcriptional level. The results indicated that, compared with slow cooling treatment, rapid cooling significantly inhibited core browning in advanced-maturity 'Yali' pears. Measurements of quality-related physiological indexes suggested that rapid cooling treatment led to higher SSC content, firmness, L* value, and b* value, indicating better brightness, coloration, and higher soluble solid content, which are desirable for commercial sale. Rapid cooling effectively suppressed the physiological metabolism of 'Yali' pears, delaying fruit senescence compared with slow-cooling treatment. Furthermore, the RNA-Seq sequencing results revealed that pathways related to browning are involved in hormone signal transduction pathways, which are associated with resistance and aging processes of pear fruit. In summary, rapid cooling treatment delayed the core browning of advanced maturity of 'Yali' pears, indicating that the core browning of 'Yali' pears is related to the cooling method, and the mechanism of rapid cooling in reducing the core browning of advanced maturity of 'Yali' pears was by delaying the aging process of the fruit. This provides a new perspective for alleviating the core browning of advanced-maturity 'Yali' pears during storage and transportation, and provides a theoretical reference for studying the mechanism of core browning of 'Yali' pears.

[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.

Effects of Atmospheric Cold Plasma Treatment on the Storage Quality and Chlorophyll Metabolism of Postharvest Tomato.

Atmospheric cold plasma (ACP) is a potential green preservation technology, but its preservation mechanism is still unclear, and the effects of different plasma intensities on postharvest tomatoes are little studied. In this study, the effects of different ACP treatments (0 kV, 40 kV, 60 kV, and 80 kV) on the sensory quality, physiological indexes, key enzyme activities, and gene expression related to the chlorophyll metabolism of postharvest tomatoes were investigated during the storage time. The results showed that compared with the control group, the tomatoes in the plasma treatment group had a higher hardness and total soluble solid (TSS) and titratable acid (TA) contents, a lower respiratory intensity and weight loss rate, a higher brightness, and a lower red transformation rate, especially in the 60 kV treatment group. In addition, chlorophyll degradation, carotenoid accumulation, and chlorophyllase and pheophorbide a mono-oxygenase (PAO) enzyme activities in the postharvest tomatoes were inhibited in the 60 kV treatment group, and the expressions of three key genes related to chlorophyll metabolism, chlorophyll (CLH1), pheophytinase (PPH), and red chlorophyll catabolic reductase (RCCR) were down-regulated. The results of the correlation analysis also confirmed that the enzyme activity and gene expression of the chlorophyll metabolism were regulated by the ACP treatment, aiming to maintain the greenness of postharvest tomatoes.

Environmentally relevant concentrations of F-53B induce eye development disorders-mediated locomotor behavior in zebrafish larvae.

The perfluorooctane sulfonate alternative, F-53B, induces multiple physiological defects but whether it can disrupt eye development is unknown. We exposed zebrafish to F-53B at four different concentrations (0, 0.15, 1.5, and 15 µg/L) for 120 h post-fertilization (hpf). Locomotor behavior, neurotransmitters content, histopathological alterations, morphological changes, cell apoptosis, and retinoic acid signaling were studied. Histology and morphological analyses showed that F-53B induced pathological changes in lens and retina of larvae and eye size were significantly reduced as compared to control. Acridine orange (AO) staining revealed a dose-dependent increase in early apoptosis, accompanied by upregulation of p53, casp-9 and casp-3 genes. Genes related to retinoic acid signaling (aldh1a2), lens developmental (cryaa, crybb, crygn, and mipa) and retinal development (pax6, rx1, gant1, rho, opn1sw and opn1lw) were significantly downregulated. In addition, behavioral responses (swimming speed) were significantly increased, while no significant changes in the neurotransmitters (dopamine and acetylcholine) level were observed. Therefore, in this study we observed that exposure to F-53B inflicted histological and morphological changes in zebrafish larvae eye, induced visual motor dysfunctions, perturbed retinoid signaling and retinal development and ultimately triggering apoptosis.

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.

Engineered Ureolytic Microorganisms Can Tailor the Morphology and Nanomechanical Properties of Microbial-Precipitated Calcium Carbonate.

We demonstrate for the first time that the morphology and nanomechanical properties of calcium carbonate (CaCO3) can be tailored by modulating the precipitation kinetics of ureolytic microorganisms through genetic engineering. Many engineering applications employ microorganisms to produce CaCO3. However, control over bacterial calcite morphology and material properties has not been demonstrated. We hypothesized that microorganisms genetically engineered for low urease activity would achieve larger calcite crystals with higher moduli. We compared precipitation kinetics, morphology, and nanomechanical properties for biogenic CaCO3 produced by two Escherichia coli (E. coli) strains that were engineered to display either high or low urease activity and the native producer Sporosarcina pasteurii. While all three microorganisms produced calcite, lower urease activity was associated with both slower initial calcium depletion rate and increased average calcite crystal size. Both calcite crystal size and nanoindentation moduli were also significantly higher for the low-urease activity E. coli compared with the high-urease activity E. coli. The relative resistance to inelastic deformation, measured via the ratio of nanoindentation hardness to modulus, was similar across microorganisms. These findings may enable design of novel advanced engineering materials where modulus is tailored to the application while resistance to irreversible deformation is not compromised.

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.

Rapid and Simple Determination of Sarafloxacin and Difloxacin in Beef by Capillary Electrophoresis Coupled with Solid-Phase Extraction.

A simple and rapid capillary electrophoresis method with diode array detector was developed for determination of sarafloxacin and difloxacin in beef. In this study, the experimental parameters affecting the determination were systematically optimized, including wavelength, buffer system, pH and concentration, and separation temperature and voltage. Under the optimal conditions, sarafloxacin and difloxacin could be quantified within 4 min using H3BO3/Na2B4O7 buffer (35 mmol/L, pH 8.8) as background electrolyte, 25 kV as separation voltage, and 22°C as the column temperature. The linear range of the method was 1-20 µg/mL with LOD 0.8 µg/mL for sarafloxacin and 0.5-20 µg/mL with LOD 0.3 µg/mL for difloxacin. The RSDs for the peak area of 8 µg/mL sarafloxacin were 4.8% (intraday) and 7.8% (interday), respectively. The proposed method has been applied to determine the residue of sarafloxacin and difloxacin in beef samples with the satisfactory recovery.

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.

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.