Prospects for utilizing microbial consortia for lignin conversion

Naturally occurring microbial communities are able to decompose lignocellulosic biomass through the concerted production of a myriad of enzymes that degrade its polymeric components and assimilate the resulting breakdown compounds by members of the community. This process includes the conversion of lignin, the most recalcitrant component of lignocellulosic biomass and historically the most difficult to valorize in the context of a biorefinery. Although several fundamental questions on microbial conversion of lignin remain unanswered, it is known that some fungi and bacteria produce enzymes to break, internalize, and assimilate lignin-derived molecules. The interest in developing efficient biological lignin conversion approaches has led to a better understanding of the types of enzymes and organisms that can act on different types of lignin structures, the depolymerized compounds that can be released, and the products that can be generated through microbial biosynthetic pathways. It has become clear that the discovery and implementation of native or engineered microbial consortia could be a powerful tool to facilitate conversion and valorization of this underutilized polymer. Here we review recent approaches that employ isolated or synthetic microbial communities for lignin conversion to bioproducts, including the development of methods for tracking and predicting the behavior of these consortia, the most significant challenges that have been identified, and the possibilities that remain to be explored in this field

Improvement of the overall quality of amplification by ddMDA.

<p>(A) The fraction of reads correctly mapped to <i>E</i>. <i>coli</i> genome depending on the DNA concentration and the MDA reaction volume. Blue and red columns show ddMDA and tube MDA results, respectively. The green line shows the fold change of the % mapping from tube MDA to ddMDA (= ddMDA/tubeMDA). It stayed around 1 for high concentrations (10–100 pg/μL) while it considerably increased for low concentrations (0.1–1 pg/μL). (B) The fraction of an <i>E</i>. <i>coli</i> genome covered by one or more sequencing reads depending on the DNA concentration and the MDA reaction volume. Tube MDA and ddMDA showed little difference at high concentrations but the fold change significantly increased at low concentrations. (C) The fraction of an <i>E</i>. <i>coli</i> genome covered by contigs during de novo assembly. While the advantage of ddMDA over tube MDA was still limited at high concentrations but the fold change significantly increased at low concentrations. (D) Lorenz curves depict the amplification bias in read coverage across the <i>E</i>. <i>coli</i> genome. Each curve was calculated by evaluating the read depth for each base and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bases (x-axis). The ideal Lorentz curve (black dotted line) for a distribution in which all of the bases have the same coverage and a Lorenz curve for gDNA were plotted for comparison. Other solid curves show ddMDA curves while dotted curves indicate tube MDA. (Upper Left) For 100 pg/μL, ddMDA in dark purple and tube MDA in bright purple. (Bottom Left) For 10 pg/μL, ddMDA in dark brown and tube MDA in bright brown. (Upper Right) For 1 pg/μL, ddMDA in dark red and tube MDA in bright red. (Bottom Right) For 0.1 pg/μL, ddMDA in dark blue and tube MDA in bright blue.</p

Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples

<div><p>Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an <i>E</i>. <i>coli</i> genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.</p></div

Statistics of sequence mapping and assembly of <i>E</i>. <i>coli</i> K12 MG1655 samples prepared with ddMDA and conventional tube MDA at different initial copy numbers.

<p>Total number of reads included all sequencing reads mapped and unmapped to <i>E</i>. <i>coli</i> genome. % Reads mapped to genome corresponds to the percentage of sequencing reads that are specifically aligned to <i>E</i>. <i>coli</i> genome. % Genome covered by reads refers to the percentage of <i>E</i>. <i>coli</i> genome covered by one or more sequencing reads. % Genome covered by assembly indicated the percentage of <i>E</i>. <i>coli</i> genome covered by contigs. Gini indices were calculated based on the cumulative distribution of sequencing reads.</p

The working principles of ddMDA.

<p>(A) The ddMDA procedures as a high quality alternative to the conventional tube MDA. The MDA ready <i>E</i>. <i>coli</i> samples were partitioned into millions of picoliter droplets using a microfluidic droplet generator. Upon collection, droplets were tightly sealed for isothermal incubation at 30°C for 18 hours. DNA amplicons were then purified, cleaned, and prepared for the following sequencing. (B) Denatured and fragmented whole genomes consist of highly amplifiable (yellow) and weakly amplifiable (red) sequences. During tube MDA, yellow fragments are preferred and repeatedly amplified with a high gain until it reaches a concentration plateau, whereas red fragments are less preferred and barely amplify. For ddMDA, DNA fragments are randomly partitioned into picoliter droplets, resulting in different subsets of the template DNA. When a droplet contains yellow fragments, the amplification kinetics favor the yellow fragments, ending up with significant biases on amplification. The enzyme will amplify red fragments at a slower rate only in the absence of yellow fragments. The overall gain of ddMDA is always lower than tube MDA because of the volume constraint. Every droplet is uniquely composed of fragments and ends up with a different amplification gain after MDA. (C) A fluorescence micrograph showing ddMDA endpoint with the initial template DNA concentration of 100 pg/μL. Having started with different parts of the <i>E</i>. <i>coli</i> genome, individual droplets expressed discrete levels of amplification by showing different sizes of DNA amplicon aggregates and different fluorescent signals. The scale bar shows 100μm.</p

Comparison of whole genome coverage of assembled contigs mapped onto <i>E</i>. <i>coli</i> K12 genome sequences for ddMDA and tube MDA.

<p>(A) From the outermost circle, ddMDA for 100 pg/μL (dark purple), tube MDA for 100 pg/μL (bright purple), 10 pg/μL (dark brown), and 10 pg/μL (bright brown), respectively. GC contents across the genome were depicted in black in the innermost circle and reads from genomic DNA were illustrated in green as a reference. (B) From the outermost circle, ddMDA for 1 pg/μL (dark red), tube MDA for 1 pg/μL (bright red), 0.1 pg/μL (dark blue), and 0.1 pg/μL (bright blue), respectively. (C) GC contents (top) and amplification read depths over the entire E. coli genome for ddMDA (middle) and tube MDA (bottom) at the DNA concentration of 10 pg/μL. The right panels show zoomed-in plots of the dotted-line box region of the genome for close-up visualization.</p

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with <i>S. pyogenes</i> (Spy), <i>S. aureus</i> (Sau), and <i>C. jejuni</i> (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants

Phenothiazines Rapidly Induce Laccase Expression and Lignin-Degrading Properties in the White-Rot Fungus Phlebia radiata

Phlebia radiata is a widespread white-rot basidiomycete fungus with significance in diverse biotechnological applications due to its ability to degrade aromatic compounds, xenobiotics, and lignin using an assortment of oxidative enzymes including laccase. In this work, a chemical screen with 480 conditions was conducted to identify chemical inducers of laccase expression in P. radiata. Among the chemicals tested, phenothiazines were observed to induce laccase activity in P. radiata, with promethazine being the strongest laccase inducer of the phenothiazine-derived compounds examined. Secretomes produced by promethazine-treated P. radiata exhibited increased laccase protein abundance, increased enzymatic activity, and an enhanced ability to degrade phenolic model lignin compounds. Transcriptomics analyses revealed that promethazine rapidly induced the expression of genes encoding lignin-degrading enzymes, including laccase and various oxidoreductases, showing that the increased laccase activity was due to increased laccase gene expression. Finally, the generality of promethazine as an inducer of laccases in fungi was demonstrated by showing that promethazine treatment also increased laccase activity in other relevant fungal species with known lignin conversion capabilities including Trametes versicolor and Pleurotus ostreatus

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA