In silico characterization of microbial electrosynthesis for metabolic engineering of biochemicals

<p>Abstract</p> <p>Background</p> <p>A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates such as NADH. Bioelectrochemical techniques offer a novel and promising method to alleviate redox imbalances during the synthesis of biochemicals and biofuels. Broadly, these techniques reduce intracellular NAD⁺to NADH and therefore manipulate the cell's redox balance. The cellular response to such redox changes and the additional reducing power available to the cell can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can be used to improve product yields and/or productivity.</p> <p>Results</p> <p>We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of <it>E. coli</it>. The results in this paper elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.</p> <p>Conclusions</p> <p>The results in this paper provide a systematic understanding of the benefits and limitations of bioelectrochemical techniques for biochemical production and highlight how electrical enhancement can impact cellular metabolism and biochemical production.</p

Ribosomes slide on lysine-encoding homopolymeric A stretches.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.00

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality

Recent experimental findings have substantially advanced the notion that the codon-dependent rate of translation elongation is a major determinant of mRNA stability. While the role of the ribosome in identification and decay of aberrant mRNAs has been well established, how the process of translation elongation and mRNA decay communicate is less well understood. Here, we report that the yeast DEAD-box protein Dhh1, long implicated in regulation of translation and activation of mRNA decay, acts as a sensor of codon optimality that targets an mRNA for decay. First, we find that Dhh1 specifically associates with and degrades mRNAs of low codon optimality. We show that messages with greater numbers of slowly translating ribosomes are preferential targets for Dhh1 mediated decay. Moreover, we note that these mRNAs are degraded by Dhh1-specific mechanism separate from the standard cellular ribosome quality control apparatus. We find that overexpression of Dhh1 leads to accumulation of ribosomes specifically on mRNAs of poor optimality. We supplement this with high-throughput sequencing analysis to show that Dhh1 over-expression leads to ribosomal stalling on specific non-optimal codons. Taken together with the finding that Dhh1 is found to associate with ribosomes in vivo, these data suggest that Dhh1 acts a sensor for translation elongation, efficiently coupling codon optimality with mRNA decay

The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality

Recent experimental findings have substantially advanced the notion that the codon-dependent rate of translation elongation is a major determinant of mRNA stability. While the role of the ribosome in identification and decay of aberrant mRNAs has been well established, how the process of translation elongation and mRNA decay communicate is less well understood. Here, we report that the yeast DEAD-box protein Dhh1, long implicated in regulation of translation and activation of mRNA decay, acts as a sensor of codon optimality that targets an mRNA for decay. First, we find that Dhh1 specifically associates with and degrades mRNAs of low codon optimality. We show that messages with greater numbers of slowly translating ribosomes are preferential targets for Dhh1 mediated decay. Moreover, we note that these mRNAs are degraded by Dhh1-specific mechanism separate from the standard cellular ribosome quality control apparatus. We find that overexpression of Dhh1 leads to accumulation of ribosomes specifically on mRNAs of poor optimality. We supplement this with high-throughput sequencing analysis to show that Dhh1 over-expression leads to ribosomal stalling on specific non-optimal codons. Taken together with the finding that Dhh1 is found to associate with ribosomes in vivo, these data suggest that Dhh1 acts a sensor for translation elongation, efficiently coupling codon optimality with mRNA decay

Engineering Escherichia coli for the utilization of ethylene glycol

Abstract Background A considerable challenge in the development of bioprocesses for producing chemicals and fuels has been the high cost of feedstocks relative to oil prices, making it difficult for these processes to compete with their conventional petrochemical counterparts. Hence, in the absence of high oil prices in the near future, there has been a shift in the industry to produce higher value compounds such as fragrances for cosmetics. Yet, there is still a need to address climate change and develop biotechnological approaches for producing large market, lower value chemicals and fuels. Results In this work, we study ethylene glycol (EG), a novel feedstock that we believe has promise to address this challenge. We engineer Escherichia coli (E. coli) to consume EG and examine glycolate production as a case study for chemical production. Using a combination of modeling and experimental studies, we identify oxygen concentration as an important metabolic valve in the assimilation and use of EG as a substrate. Two oxygen-based strategies are thus developed and tested in fed-batch bioreactors. Ultimately, the best glycolate production strategy employed a target respiratory quotient leading to the highest observed fermentation performance. With this strategy, a glycolate titer of 10.4 g/L was reached after 112 h of production time in a fed-batch bioreactor. Correspondingly, a yield of 0.8 g/g from EG and productivity of 0.1 g/L h were measured during the production stage. Our modeling and experimental results clearly suggest that oxygen concentration is an important factor in the assimilation and use of EG as a substrate. Finally, our use of metabolic modeling also sheds light on the intracellular distribution through central metabolism, implicating flux to 2-phosphoglycerate as the primary route for EG assimilation. Conclusion Overall, our work suggests that EG could provide a renewable starting material for commercial biosynthesis of fuels and chemicals that may achieve economic parity with petrochemical feedstocks while sequestering carbon dioxide

Scikit-ribo Enables Accurate Estimation and Robust Modeling of Translation Dynamics at Codon Resolution

Summary Ribosome profiling (Ribo-seq) is a powerful technique for measuring protein translation; however, sampling errors and biological biases are prevalent and poorly understood. Addressing these issues, we present Scikit-ribo (https://github.com/schatzlab/scikit-ribo), an open-source analysis package for accurate genome-wide A-site prediction and translation efficiency (TE) estimation from Ribo-seq and RNA sequencing data. Scikit-ribo accurately identifies A-site locations and reproduces codon elongation rates using several digestion protocols (r = 0.99). Next, we show that the commonly used reads per kilobase of transcript per million mapped reads-derived TE estimation is prone to biases, especially for low-abundance genes. Scikit-ribo introduces a codon-level generalized linear model with ridge penalty that correctly estimates TE, while accommodating variable codon elongation rates and mRNA secondary structure. This corrects the TE errors for over 2,000 genes in S. cerevisiae, which we validate using mass spectrometry of protein abundances (r = 0.81), and allows us to determine the Kozak-like sequence directly from Ribo-seq. We conclude with an analysis of coverage requirements needed for robust codon-level analysis and quantify the artifacts that can occur from cycloheximide treatment