Machine Learning to Predict Enzyme-Substrate Interactions in Elucidation of Synthesis Pathways: A Review.

Enzyme-substrate interactions play a fundamental role in elucidating synthesis pathways and synthetic biology, as they allow for the understanding of important aspects of a reaction. Establishing the interaction experimentally is a slow and costly process, which is why this problem has been addressed using computational methods such as molecular dynamics, molecular docking, and Monte Carlo simulations. Nevertheless, this type of method tends to be computationally slow when dealing with a large search space. Therefore, in recent years, methods based on artificial intelligence, such as support vector machines, neural networks, or decision trees, have been implemented, significantly reducing the computing time and covering vast search spaces. These methods significantly reduce the computation time and cover broad search spaces, rapidly reducing the number of interacting candidates, as they allow repetitive processes to be automated and patterns to be extracted, are adaptable, and have the capacity to handle large amounts of data. This article analyzes these artificial intelligence-based approaches, presenting their common structure, advantages, disadvantages, limitations, challenges, and future perspectives.

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-mediated Isothermal Amplification.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Persistent and fatal severe acute respiratory syndrome coronavirus 2 infection in a patient with severe hypogammaglobulinemia: a case report.

BACKGROUND: Viruses are constantly changing as a result of mutations, and new viral variants are expected to appear over time. The virus that causes coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2, is not excluded from this condition. Patients with some types of immunodeficiency have been reported to experience symptoms that vary from mild to severe, or even death, after being infected with severe acute respiratory syndrome coronavirus 2. We report a case of a woman with severe hypogammaglobulinemia who developed a prolonged and fatal severe acute respiratory syndrome coronavirus 2 infection. CASE PRESENTATION: A 60-year-old mestizo female with a previous history of severe hypogammaglobulinemia manifested by recurrent pulmonary infections and follicular bronchiolitis. She received a monthly treatment of intravenous immunoglobulins and was admitted after report of a neurological manifestation related to a left thalamic inflammatory lesion, for a duration of 2 weeks of hospitalization, indicated for the study of her neurological condition, including brain biopsy. Both on admission and 1 week later, nasopharyngeal polymerase chain reaction tests for severe acute respiratory syndrome coronavirus 2 were performed and reported negative. In the third week of hospitalization, she developed pulmonary symptoms, and a positive test result for severe acute respiratory syndrome coronavirus 2 was evidenced. On Day 3, the patients' condition worsened as the infection progressed to respiratory failure and required mechanical ventilation. On Day 8 after the coronavirus disease 2019 diagnosis, the polymerase chain reaction test for severe acute respiratory syndrome coronavirus 2 showed persistent detection of the virus. Various bacterial coinfections, including Klebsiella pneumoniae and Enterobacter cloacae, were diagnosed and treated. On Day 35, her pulmonary symptoms worsened, and the results of the severe acute respiratory syndrome coronavirus 2 polymerase chain reaction test remained positive. On Day 36, despite all the respiratory support, the patient died. The severe acute respiratory syndrome coronavirus 2 virus was sequenced at the beginning and 8 days after the onset of the disease, and the strain, without obvious mutations in the gene that encodes spike protein, was identified. CONCLUSIONS: This clinical case showed persistent severe acute respiratory syndrome coronavirus 2 detection after 35 days of infection in a patient with severe hypogammaglobulinemia. The sequencing of the virus showed no mutations on the spike protein at 8 days, indicating that, in this case, the persistence of the viral detection was associated with immunodeficiency instead of changes in the viral components.

Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach.

Chiral amino alcohols are compounds of pharmaceutical interest as they are building blocks of sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Due to the challenges of chemical synthesis we recently developed two TK-TAm reaction cascades using natural and low cost feedstocks as substrates: a recycling cascade comprising of 2 enzymes and a sequential 3-step enzyme cascade yielding 30% and 1% conversion, respectively. In order to improve the conversion yield and aid the future host strain engineering for whole cell biocatalysis, we used a combination of microscale experiments and statistical experimental design. For this we implemented a full factorial design to optimise pH, temperature and buffer type, followed by the application of Response Surface Methodology for the optimisation of substrates and enzymes concentrations. Using purified enzymes we achieved 60% conversion for the recycling cascade and 3-fold improvement using the sequential pathway. Based on the results, limiting steps and individual requirements for host cell metabolic integration were identified expanding the understanding of the cascades without implementing extensive optimisation modelling. Therefore, the approach described here is well suited for optimising reaction conditions as well as defining the relative enzyme expression levels required for construction of microbial cell factories.

Multi-step biocatalytic strategies for chiral amino alcohol synthesis.

Chiral amino alcohols are structural motifs present in sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Their chemical synthesis presents several challenges in establishing at least two chiral centres. Here a de novo metabolic pathway using a transketolase enzyme coupled with a transaminase enzyme has been assembled. To synthesise this motif one of the strategies to obtain high conversions from the transaminase/transketolase cascade is the use of hydroxypyruvate (HPA) as a two-carbon donor for the transketolase reaction; although commercially available it is relatively expensive limiting application of the pathway on an industrial scale. Alternately, HPA can be synthesised but this introduces a further synthetic step. In this study two different biocatalytic strategies were developed for the synthesis of (2S,3R)-2-amino-1,3,4-butanetriol (ABT) without adding HPA into the reaction. Firstly, a sequential cascade of three enzymatic steps (two transaminases and one transketolase) for the synthesis of ABT from serine, pyruvate and glycolaldehyde as substrates. Secondly, a two-step recycling cascade where serine is used as donor to aminate erythrulose (catalysed by a transketolase) for the simultaneous synthesis of ABT and HPA. In order to test the novel pathways, three new transaminases are described, two ω-transaminases able to accept a broad range of amine acceptors with serine as amine donor; and an α-transaminase, which showed high affinity towards serine (KM: 18mM) using pyruvate as amine acceptor. After implementation of the above enzymes in the biocatalytic pathways proposed in this paper, the two-step recycling pathway was found to be the most promising for its integration with E. coli metabolism. It was more efficient (10-fold higher conversion), more sustainable and cost-effective (use of low cost natural substrates and only two enzymes), and the reaction could be performed in a one-pot system.