Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Possible intraindividual evolution of SARS-CoV-2 in nasopharyngeal and anal swab in an octogenarian: a case report.

Introduction: COVID-19 is an emerging infectious disease that remains to be further investigated. Case report: Here, we describe a case of COVID-19 in an octogenarian woman with comorbidities who slowly recovered during hospitalization, but died due to sudden cardiac death after 2 weeks of hospitalization. Her nasopharyngeal and anal swabs returned positive for SARS-CoV-2 by RT-PCR on day 7 of hospitalization. The NGS showed possible intraindividual evolution of virus. The sample from the nasopharyngeal swab yielded a B.1470 variant classified as clade GH. This variant showed mutation in the spike gene D614G; N gene; NS3 gene; NSP2 gene and NSP12 gene. The sample from the anal swab showed similar mutation but with additional point mutation in spike gene S12F and was classified as B.1.465 variant. Conclusions: The possibility of the gastrointestinal tract that served as reservoir for virus mutation accumulation should also be considered and the potential impact of viral fecal transmission in the environment should be further investigated.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia.

Background: Indonesia is one of the Southeast Asian countries with high case numbers of COVID-19 with up to 4.2 million confirmed cases by 29 October 2021. Understanding the genome of SARS-CoV-2 is crucial for delivering public health intervention as certain variants may have different attributes that can potentially affect their transmissibility, as well as the performance of diagnostics, vaccines, and therapeutics. Objectives: We aimed to investigate the dynamics of circulating SARS-CoV-2 variants over a 15-month period in Bogor and its surrounding areas in correlation with the first and second wave of COVID-19 in Indonesia. Methods: Nasopharyngeal and oropharyngeal swab samples collected from suspected patients from Bogor, Jakarta and Tangerang were confirmed for SARS-CoV-2 infection with RT-PCR. RNA samples of those confirmed patients were subjected to whole genome sequencing using the ARTIC Network protocol and sequencer platform from Oxford Nanopore Technologies (ONT). Results: We successfully identified 16 lineages and six clades out of 202 samples (male n = 116, female n = 86). Genome analysis revealed that Indonesian lineage B.1.466.2 dominated during the first wave (n = 48, 23.8%) while Delta variants (AY.23, AY.24, AY.39, AY.42, AY.43 dan AY.79) were dominant during the second wave (n = 53, 26.2%) following the highest number of confirmed cases in Indonesia. In the spike protein gene, S_D614G and S_P681R changes were dominant in both B.1.466.2 and Delta variants, while N439K was only observed in B.1.466.2 (n = 44) and B.1.470 (n = 1). Additionally, the S_T19R, S_E156G, S_F157del, S_R158del, S_L452R, S_T478K, S_D950N and S_V1264L changes were only detected in Delta variants, consistent with those changes being characteristic of Delta variants in general. Conclusions: We demonstrated a shift in SARS-CoV-2 variants from the first wave of COVID-19 to Delta variants in the second wave, during which the number of confirmed cases surpassed those in the first wave of COVID-19 pandemic. Higher proportion of unique mutations detected in Delta variants compared to the first wave variants indicated potential mutational effects on viral transmissibility that correlated with a higher incidence of confirmed cases. Genomic surveillance of circulating variants, especially those with higher transmissibility, should be continuously conducted to rapidly inform decision making and support outbreak preparedness, prevention, and public health response.

From mannan to bioethanol: cell surface co-display of ß-mannanase and ß-mannosidase on yeast Saccharomyces cerevisiae.

BACKGROUND: Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays ß-mannanase and ß-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. RESULTS: Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered ß-mannanase and ß-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-ß-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-ß-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. CONCLUSIONS: We successfully displayed ß-mannanase and ß-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying ß-mannanase and ß-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering ß-mannanase and ß-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

Effects of saline-alkaline stress on benzo[a]pyrene biotransformation and ligninolytic enzyme expression by Bjerkandera adusta SM46.

Benzo[a]pyrene (BaP) accumulates in marine organisms and contaminated coastal areas. The biotreatment of waste water using saline-alkaline-tolerant white rot fungi (WRF) represents a promising method for removing BaP under saline-alkaline conditions based on WRF's ability to produce ligninolytic enzymes. In a pre-screening for degradation of polycyclic aromatic hydrocarbons of 82 fungal strains using Remazol brilliant blue R, Bjerkandera adusta SM46 exhibited the highest tolerance to saline-alkaline stress. Moreover, a B. adusta culture grown in BaP-containing liquid medium exhibited resistance to salinities up to 20 g l(-1). These conditions did not inhibit fungal growth or the expression of manganese peroxidase (MnP) or lignin peroxidase (LiP). The degradation rate also became higher as salinity increased to 20 g l(-1). Fungal growth and enzyme expression were inhibited at a salinity of 35 g l(-1). These inhibitory effects directly decreased the degradation rate (>24%). The presence of MnSO4 as an inducer improved the degradation rate and enzyme expression. MnP and LiP activity also increased by seven- and fivefold, respectively. SM46 degraded BaP (38-89% over 30 days) in an acidic environment (pH 4.5) and under saline-alkaline stress conditions (pH 8.2). Investigating the metabolites produced revealed BaP-1,6-dione as the main product, indicating the important role of ligninolytic enzymes in initializing BaP cleavage. The other metabolites detected, naphthalene acetic acid, hydroxybenzoic acid, benzoic acid, and catechol, may have been ring fission products. The wide range of activities observed suggests that B. adusta SM46 is a potential agent for biodegrading BaP under saline conditions.