Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum.

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Recent and emerging trends of metal-organic frameworks (MOFs)-based sensors for detecting food contaminants: A critical and comprehensive review.

Interest in the use of sensors based on metal-organic frameworks (MOFs) to detect food pollutants has been growing recently due to the desirable characteristics of MOFs, including uniform structures, large surface area, ultrahigh porosity and easy-to-functionalize surface. Fundamentally, this review offers an excellent solution using MOFs-based sensors (e.g., fluorescent, electrochemical, electrochemiluminescence, surface-enhanced Raman spectroscopy, and colorimetric sensors) to detect food contaminants such as pesticide residues, mycotoxins, antibiotics, food additives, and other hazardous candidates. More importantly, their application scenarios and advantages in food detection are also introduced in more detail. Therefore, this systematic review analyzes detection limits, linear ranges, the role of functionalities, and immobilized nanoparticles utilized in preparing MOFs-based sensors. Additionally, the main limitations of each sensing type, along with the enhancement mechanisms of MOFs in addressing efficient sensing are discussed. Finally, the limitations and potential trends of MOFs-based materials in food contaminant detection are also highlighted.

Magnetic nanoparticle-based immunosensors and aptasensors for mycotoxin detection in foodstuffs: An update.

Mycotoxin contamination of food crops is a global challenge due to their unpredictable occurrence and severe adverse health effects on humans. Therefore, it is of great importance to develop effective tools to prevent the accumulation of mycotoxins through the food chain. The use of magnetic nanoparticle (MNP)-assisted biosensors for detecting mycotoxin in complex foodstuffs has garnered great interest due to the significantly enhanced sensitivity and accuracy. Within such a context, this review includes the fundamentals and recent advances (2020-2023) in the area of mycotoxin monitoring in food matrices using MNP-based aptasensors and immunosensors. In this review, we start by providing a comprehensive introduction to the design of immunosensors (natural antibody or nanobody, random or site-oriented immobilization) and aptasensors (techniques for aptamer selection, characterization, and truncation). Meanwhile, special attention is paid to the multifunctionalities of MNPs (recoverable adsorbent, versatile carrier, and signal indicator) in preparing mycotoxin-specific biosensors. Further, the contribution of MNPs to the multiplexing determination of various mycotoxins is summarized. Finally, challenges and future perspectives for the practical applications of MNP-assisted biosensors are also discussed. The progress and updates of MNP-based biosensors shown in this review are expected to offer readers valuable insights about the design of MNP-based tools for the effective detection of mycotoxins in practical applications.

Recent advances in electrochemical cell-based biosensors for food analysis: Strategies for sensor construction.

Owing to their advantages such as great specificity, sensitivity, rapidity, and possibility of noninvasive and real-time monitoring, electrochemical cell-based biosensors (ECBBs) have been a powerful tool for food analysis encompassing the areas of nutrition, flavor, and safety. Notably, the distinctive biological relevance of ECBBs enables them to mimic physiological environments and reflect cellular behaviors, leading to valuable insights into the biological function of target components in food. Compared with previous reviews, this review fills the current gap in the narrative of ECBB construction strategies. The review commences by providing an overview of the materials and configuration of ECBBs, including cell types, cell immobilization strategies, electrode modification materials, and electrochemical sensing types. Subsequently, a detailed discussion is presented on the fabrication strategies of ECBBs in food analysis applications, which are categorized based on distinct signal sources. Lastly, we summarize the merits, drawbacks, and application scope of these diverse strategies, and discuss the current challenges and future perspectives of ECBBs. Consequently, this review provides guidance for the design of ECBBs with specific functions and promotes the application of ECBBs in food analysis.

A film-like SERS aptasensor for sensitive detection of patulin based on GO@Au nanosheets.

Patulin (PAT) commonly contaminates fruits, posing a significant risk to human health. Therefore, a highly effective and sensitive approach in identifying PAT is warranted. Herein, a SERS aptasensor was constructed based on a two-dimensional film-like structure. GO@Au nanosheets modified with SH-cDNA were employed as capture probes, while core-shell Au@Ag nanoparticles modified with 4-MBA and SH-Apt were utilized as signal probes. Through the interaction between capture probes and signal probes, adjustable hotspots were formed, yielding a significant Raman signal. During sensing, the GO@Au-cDNA competitively attached to Au@AgNPs@MBA-Apt, resulting in an inverse relationship between PAT levels and SERS intensity. The acquired results exhibited linear responses to PAT within the range of 1-70 ng/mL, with a calculated limit of detection of 0.46 ng/mL. In addition, the SERS aptasensor exhibited satisfactory recoveries in apple samples, which aligned closely with HPLC. With high sensitivity and specificity, this method holds significant potential for PAT detection.

Significance of the antibody orientation for the lateral flow immunoassays: A mini-review.

Lateral flow immunoassays (LFIAs) are well-established and broadly commercialized tools in the field of point-of-care testing due to their simplicity, rapidity, cost-effectiveness, and low requirements for users and equipment. However, the insensitivity and the possibility of producing inaccurate results associated with conventional LFIAs have impeded their wide-ranging implementation, especially for monitoring ultra-trace level of analytes. Moreover, the heterogeneous distribution of amino acids on the surface of antibody (Ab) results in a lack of precise control over their orientation, which ultimately leads to unsatisfactory detection performance. To address those concerns, herein we provide an overview of the emerging efforts to prepare well-established LFIAs from the perspective of orientation manipulation of immobilized Abs on the nanoprobes or membranes. The preparation of excellent nanoprobes with Abs being oriented immobilized, consisting of the nanoprobe types, Ab types, and their conjugation chemistries, are reviewed. Followed by the introduction of efforts highlight the importance of directionally immobilized Ab on the membrane. The effects of Ab orientation on the analytical performance of LFIA platforms in terms of sensitivity, specificity, rapidity, reliability, cost-effectiveness, and stability are also summarized. Finally, the future development and challenges of Ab-oriented immobilization-assisted LFIAs are also discussed.

Dextran-coated nanoparticles as immunosensing platforms: Consideration of polyaldehyde density, nanoparticle size and functionality.

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle's colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 µmol analyte per µmol antibody, corresponding to 139 µmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL-1, while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL-1. In addition, polyaldehyde-dextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors.

Oriented immobilization of antibodies onto sensing platforms - A critical review.

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.

Oriented immobilization of antibodies through different surface regions containing amino groups: Selective immobilization through the bottom of the Fc region.

Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (ß-galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples.

Endoglucanase enzymatic modification of kraft pulp during recycling.

OBJECTIVE: To investigate the cellulose modification process on kraft pulp during recycling by mono-endoglucanase. RESULTS: Pichia pastoris expressing endoglucanase, EG1, was grown in a 10 l fermenter yielding a high carboxymethyl cellulase (CMCase) activity of 340 U mg(-1). EG1-mediated modification of kraft pulp resulted in a paper sheet with the tensile index and burst index increased by 10 and 6.5 %, respectively. The kink index (indicating abrupt bends in fibres) of the enzyme-treated group decreased sharply by 45 % after the first recycling, compared with a reduction of only 1 % in the control group. Furthermore, EG1 treatment decreased the growth of crystallinity from 73.5 to 73.2 % and crystal size from 7.45 to 7.21 nm, which alleviated paper aging. CONCLUSION: Endoglucanase EG1 modifies the interfacial properties of fibers, which affects fibre morphology during the recycling process and improves the technical properties of the resulting pulp and paper.