An elevated OmpA expression during the production of a recombinant protein in Escherichia coli.

Escherichia coli cells rapidly respond to changes in the environment. Such response must be anticipated upon development of fermentation strategy for commercial purposes. The response may signal changes in cell physiology, which is critical for the cell growth and the level of the target protein production. One of the responses is the elevated expression of membrane proteins to tightly control the trafficking of molecules into and out from the cells. Normally, the expression level of the membrane protein is basal as the fermentation is carried out in physiological conditions. Here, we reported an elevated expression of the outer membrane protein A (OmpA) during a series of fermentation conduct, starting from the shake flask, 1-L to finally 10-L fermentor. The incidence led to a lower expression of the target protein and thereby resulting in lower process efficiency. OmpA expression was concomitant to the bacterial growth and already observed in the early exponential phase. Despite the drawback, this phenomenon actually inspires the observation of OmpA expression as one of the indicators for the E. coli cells response to the fermentation conditions. This auxiliary check would prevent the higher OmpA expression that led to the low expression of the target protein.

Update on the Development of Toehold Switch-Based Approach for Molecular Diagnostic Tests of COVID-19.

A high volume of diagnostic tests is needed during the coronavirus disease 2019 (COVID-19) pandemic to obtain representative results. These results can help to design and implement effective policies to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis using current gold standard methods, i.e., real-time quantitative PCR (RT-qPCR), is challenging, especially in areas with limited trained personnel and health-related infrastructure. The toehold switch-based diagnostic system is a promising alternative method for detecting SARS-CoV-2 that has advantages such as inexpensive cost per testing, rapid, and highly sensitive and specific analysis. Moreover, the system can be applied to paper-based platforms, simplifying the distribution and utilization in low-resource settings. This review provides insight into the development of toehold switch-based diagnostic devices as the most recent methods for detecting SARS-CoV-2.

Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms.

Microbes have been the preferred hosts for producing high-value chemicals from cheap raw materials. However, metabolic flux imbalance, the presence of competing pathways, and toxic intermediates often lead to low production efficiency. The spatial organization of the substrates, intermediates, and enzymes is critical to ensuring efficient metabolic activity by microorganisms. One of the most common approaches for bringing the key components of biosynthetic pathways together is through molecular scaffolds, which involves the clustering of pathway enzymes on engineered molecules via different interacting mechanisms. In particular, synthetic scaffold systems have been applied to improve the efficiency of various heterologous and synthetic pathways in Escherichia coli and Saccharomyces cerevisiae, with varying degrees of success. Herein, we review the recent developments and applications of protein-based and nucleic acid-based scaffold systems and discuss current challenges and future directions in the use of such approaches.

Screening, gene sequencing and characterising of lipase for methanolysis of crude palm oil.

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/ß hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K(M) of 0.75 mM and a V(max) of 0.33 mMmin(-1) on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.