L-serine is a promising building block biochemical with a high theoretical production yield from glucose.Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Other mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium.Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology. AbbreviationsAdaptive Laboratory Evolution (ALE); Mutiplex Genome Engineering (MAGE).
Escherichia coli strains are widely used in academic research and biotechnology. New technologies for quantifying strain-specific differences and their underlying contributing factors promise greater understanding of how these differences significantly impact physiology, synthetic biology, metabolic engineering, and process design. Here, we quantified strain-specific differences in seven widely used strains of E. coli (BL21, C, Crooks, DH5a, K-12 MG1655, K-12 W3110, and W) using genomics, phenomics, transcriptomics, and genome-scale modelling. Metabolic physiology and gene expression varied widely with downstream implications for productivity, product yield, and titre. These differences could be linked to differential regulatory structure. Analysing high-flux reactions and expression of encoding genes resulted in a correlated and quantitative link between these sets, with strain-specific caveats. Integrated modelling revealed that certain strains are better suited to produce given compounds or express desired constructs considering native expression states of pathways that enable high-production phenotypes. This study yields a framework for quantitatively comparing strains in a species with implications for strain selection.
We present an individual-based experimental framework to identify and estimate the main parameters governing bacterial conjugation at the individual cell scale. From this analysis, we have established that transient periods of unregulated plasmid transfer within newly formed transconjugant cells, together with contact mechanics arising from cellular growth and division, are the two main processes determining the emergent inability of the pWW0 TOL plasmid to fully invade spatially structured Pseudomonas putida populations. We have also shown that pWW0 conjugation occurs mainly at advanced stages of the growth cycle and that nongrowing cells, even when exposed to high nutrient concentrations, do not display conjugal activity. These results do not support previous hypotheses relating conjugation decay in the deeper cell layers of bacterial biofilms to nutrient depletion and low physiological activity. We observe, however, that transient periods of elevated plasmid transfer in newly formed transconjugant cells are offset by unfavorable cell-to-cell contact mechanics, which ultimately precludes the pWWO TOL plasmid from fully invading tightly packed multicellular P. putida populations such as microcolonies and biofilms.
Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor. By extending an individual-based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that parameters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual-based framework introduced in this work is a powerful tool that enables one to test additional hypotheses on the spread and role of plasmids in microbial biofilms.
Although uterus and cervix infiltration is a frequent finding in the later stages of lymphomatous disease, primary lymphoma of the cervix is very uncommon; however, this entity can occasionally be distinguished from cervical carcinoma by means of MRI. This is an important fact as treatment and prognosis differ between these neoplasms. We present a case of primary cervical lymphoma studied with high-field (1.5 T) MRI and we make an extensive review of the literature. The cervical mass was found in a routine pelvic examination in a patient with no previous history of gynecologic disorders. This is an uncommon way of presentation of this disease. T2-weighted turbo spin-echo (TSE) sequences in the axial, sagittal, and coronal planes, and T1-weighted SE pre- and post-contrast images, were obtained. The lack of involvement of the mucosa, as well as sparing of cervical stroma and uterine junctional zone, are the most important findings to differentiate cervical lymphoma from carcinoma, and are best evaluated with T2 TSE sequences. Post-contrast images help to delineate the extent of the disease.
Start-up phenomena in microbial biokinetic assays are not captured by the most commonly used growth-related equations. In this study we propose a new respirometric experimental design to estimate intrinsic growth parameters that allow us to avoid these limitations without data omission, separate mathematical treatment, or wake-up pulses prior to the analysis. Identifiability and sensitivity analysis were performed to confirm the robustness of the new approach for obtaining unique and accurate estimates of growth kinetic parameters. The new experimental design was applied to establish the metabolic burden caused by the carriage of a pWW0 TOL plasmid in the model organism Pseudomonas putida KT2440. The metabolic burden associated was manifested as a reduction in the yield and the specific growth rate of the host, with both plasmid maintenance and the over-expression of recombinant proteins from the plasmid contributing equally to the overall effect.
Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.
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