Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli.

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen.

One of the key challenges of the recent COVID-19 pandemic is the ability to accurately estimate the number of infected individuals, particularly asymptomatic and/or early-stage patients. We herewith report the proof-of-concept development of a biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus. The biosensor is based on membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody. We demonstrate that the attachment of the protein to the membrane-bound antibodies resulted in a selective and considerable change in the cellular bioelectric properties measured by means of a Bioelectric Recognition Assay. The novel biosensor provided results in an ultra-rapid manner (3 min), with a detection limit of 1 fg/mL and a semi-linear range of response between 10 fg and 1 µg/mL. In addition, no cross-reactivity was observed against the SARS-CoV-2 nucleocapsid protein. Furthermore, the biosensor was configured as a ready-to-use platform, including a portable read-out device operated via smartphone/tablet. In this way, we demonstrate that the novel biosensor can be potentially applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing, therefore offering a possible solution for the timely monitoring and eventual control of the global coronavirus pandemic.

Clinical Application of the Novel Cell-Based Biosensor for the Ultra-Rapid Detection of the SARS-CoV-2 S1 Spike Protein Antigen: A Practical Approach.

The availability of antigen tests for SARS-CoV-2 represents a major step for the mass surveillance of the incidence of infection, especially regarding COVID-19 asymptomatic and/or early-stage patients. Recently, we reported the development of a Bioelectric Recognition Assay-based biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus in just three minutes, with high sensitivity and selectivity. The working principle was established by measuring the change of the electric potential of membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody after attachment of the respective viral protein. In the present study, we applied the novel biosensor to patient-derived nasopharyngeal samples in a clinical set-up, with absolutely no sample pretreatment. More importantly, membrane-engineered cells were pre-immobilized in a proprietary biomatrix, thus enabling their long-term preservation prior to use as well as significantly increasing their ease-of-handle as test consumables. The plug-and-apply novel biosensor was able to detect the virus in positive samples with a 92.8% success rate compared to RT-PCR. No false negative results were recorded. These findings demonstrate the potential applicability of the biosensor for the early, routine mass screening of SARS-CoV-2 on a scale not yet realized.