Efficient Production of Triacetic Acid Lactone from Lignocellulose Hydrolysate by Metabolically Engineered Yarrowia lipolytica.

Lignocellulose is a promising renewable feedstock for the bioproduction of high-value biochemicals. The poorly expressed xylose catabolic pathway was the bottleneck in the efficient utilization of the lignocellulose feedstock in yeast. Herein, multiple genetic and process engineering strategies were explored to debottleneck the conversion of xylose to the platform chemical triacetic acid lactone (TAL) in Yarrowia lipolytica. We identified that xylose assimilation generating more cofactor NADPH was favorable for the TAL synthesis. pH control improved the expression of acetyl-CoA carboxylase and generated more precursor malonyl-CoA. Combined with the suppression of the lipid synthesis pathway, 5.03 and 4.18 g/L TAL were produced from pure xylose and xylose-rich wheat straw hydrolysate, respectively. Our work removed the bottleneck of the xylose assimilation pathway and effectively upgraded wheat straw hydrolysate to TAL, which enabled us to build a sustainable oleaginous yeast cell factory to cost-efficiently produce green chemicals from low-cost lignocellulose by Y. lipolytica.

DeepEdit: single-molecule detection and phasing of A-to-I RNA editing events using nanopore direct RNA sequencing.

Single-molecule detection and phasing of A-to-I RNA editing events remain an unresolved problem. Long-read and PCR-free nanopore native RNA sequencing offers a great opportunity for direct RNA editing detection. Here, we develop a neural network model, DeepEdit, that not only recognizes A-to-I editing events in single reads of Oxford Nanopore direct RNA sequencing, but also resolves the phasing of RNA editing events on transcripts. We illustrate the robustness of DeepEdit by applying it to Schizosaccharomyces pombe and Homo sapiens transcriptome data. We anticipate DeepEdit to be a powerful tool for the study of RNA editing from a new perspective.

Targeted inhibition of Zika virus infection in human cells by CRISPR-Cas13b.

Zika virus (ZIKV) outbreaks occurred in recent years on an unprecedented scale, which caused fever and severe complications like Guillain-Barré syndrome in adults and fetal abnormalities. No vaccines or other effective treatments against ZIKV are available to date. The CRISPR-Cas13 family has the unique ability to target single-strand RNA molecules and mediate RNA cleavage. In the present study, we sought to exploit CRISPR-Cas13b for developing an anti-ZIKV system in mammalian cells. We first generated a ZIKV infection and reporting system by: (1) fusing mCherry to the ZIKV capsid protein for reporting infection by fluorescence; and (2) deriving a 293T cell line (293T-DC-SIGN) stably expressing DC-SIGN (Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) that became highly susceptible to ZIKV infection. The CRISPR Cas13b expression was reported to be in the cytoplasm of 293T-DC-SIGN cells using a Cas13b-GFP fusion expression vector. Fourteen CRISPR RNAs (crRNAs) were designed to target the most conserved regions of the ZIKV genome through bioinformatics analysis of 1138 ZIKV genome sequences. Five crRNAs were found to have significant effects (p < 0.001; two-sided t test) for Cas13b-targeted inhibition on ZIKV infection in 293T-DC-SIGN cells. Our study demonstrated an exciting example of using the CRISPR-Cas13b system for the treatment and prevention of ZIKV infection, highlighting CRISPR-Cas13 as a promising therapeutic anti-RNA virus strategy.

Interfering with retrotransposition by two types of CRISPR effectors: Cas12a and Cas13a.

CRISPRs are a promising tool being explored in combating exogenous retroviral pathogens and in disabling endogenous retroviruses for organ transplantation. The Cas12a and Cas13a systems offer novel mechanisms of CRISPR actions that have not been evaluated for retrovirus interference. Particularly, a latest study revealed that the activated Cas13a provided bacterial hosts with a "passive protection" mechanism to defend against DNA phage infection by inducing cell growth arrest in infected cells, which is especially significant as it endows Cas13a, a RNA-targeting CRISPR effector, with mount defense against both RNA and DNA invaders. Here, by refitting long terminal repeat retrotransposon Tf1 as a model system, which shares common features with retrovirus regarding their replication mechanism and life cycle, we repurposed CRISPR-Cas12a and -Cas13a to interfere with Tf1 retrotransposition, and evaluated their different mechanisms of action. Cas12a exhibited strong inhibition on retrotransposition, allowing marginal Tf1 transposition that was likely the result of a lasting pool of Tf1 RNA/cDNA intermediates protected within virus-like particles. The residual activities, however, were completely eliminated with new constructs for persistent crRNA targeting. On the other hand, targeting Cas13a to Tf1 RNA intermediates significantly inhibited Tf1 retrotransposition. However, unlike in bacterial hosts, the sustained activation of Cas13a by Tf1 transcripts did not cause cell growth arrest in S. pombe, indicating that virus-activated Cas13a likely acted differently in eukaryotic cells. The study gained insight into the actions of novel CRISPR mechanisms in combating retroviral pathogens, and established system parameters for developing new strategies in treatment of retrovirus-related diseases.

Modelling BioNano optical data and simulation study of genome map assembly.

Motivation: The launch of the BioNano next-generation mapping system has greatly enhanced the performance of physical map construction, thus rapidly expanding the application of optical mapping in genome research. Data biases have profound implications for downstream applications. However, very little is known about the properties and biases of BioNano data, and the very factors that contribute to whole-genome optical map assembly. Results: We generated BioNano molecule data from eight organisms with diverse base compositions. We first characterized the properties/biases of BioNano molecule data, i.e. molecule length distribution, false labelling signal, variation of optical resolution and coverage distribution bias, and their inducing factors such as chimeric molecules, fragile sites and DNA molecule stretching. Second, we developed the BioNano Molecule SIMulator (BMSIM), a novel computer simulation program for optical data. BMSIM, is of great use for future genome mapping projects. Third, we evaluated the experimental variables that impact whole-genome optical map assembly. Specifically, the effects of coverage depth, molecule length, false-positive and false-negative labelling signals, chimeric molecules and nicking enzyme and nick site density were investigated. Our simulation study provides the empirical findings on how to control experimental variables and gauge analytical parameters to maximize benefit and minimize cost on whole-genome optical map assembly. Availability and implementation: BMSIM is freely available on: https://github.com/pingchen09990102/BMSIM. Supplementary information: Supplementary data are available at Bioinformatics online.

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.

Global transcriptome analysis reveals extensive gene remodeling, alternative splicing and differential transcription profiles in non-seed vascular plant Selaginella moellendorffii.

BACKGROUND: Selaginella moellendorffii, a lycophyte, is a model plant to study the early evolution and development of vascular plants. As the first and only sequenced lycophyte to date, the genome of S. moellendorffii revealed many conserved genes and pathways, as well as specialized genes different from flowering plants. Despite the progress made, little is known about long noncoding RNAs (lncRNA) and the alternative splicing (AS) of coding genes in S. moellendorffii. Its coding gene models have not been fully validated with transcriptome data. Furthermore, it remains important to understand whether the regulatory mechanisms similar to flowering plants are used, and how they operate in a non-seed primitive vascular plant. RESULTS: RNA-sequencing (RNA-seq) was performed for three S. moellendorffii tissues, root, stem, and leaf, by constructing strand-specific RNA-seq libraries from RNA purified using RiboMinus isolation protocol. A total of 176 million reads (44 Gbp) were obtained from three tissue types, and were mapped to S. moellendorffii genome. By comparing with 22,285 existing gene models of S. moellendorffii, we identified 7930 high-confidence novel coding genes (a 35.6% increase), and for the first time reported 4422 lncRNAs in a lycophyte. Further, we refined 2461 (11.0%) of existing gene models, and identified 11,030 AS events (for 5957 coding genes) revealed for the first time for lycophytes. Tissue-specific gene expression with functional implication was analyzed, and 1031, 554, and 269 coding genes, and 174, 39, and 17 lncRNAs were identified in root, stem, and leaf tissues, respectively. The expression of critical genes for vascular development stages, i.e. formation of provascular cells, xylem specification and differentiation, and phloem specification and differentiation, was compared in S. moellendorffii tissues, indicating a less complex regulatory mechanism in lycophytes than in flowering plants. The results were further strengthened by the evolutionary trend of seven transcription factor families related to vascular development, which was observed among four representative species of seed and non-seed vascular plants, and nonvascular land and aquatic plants. CONCLUSIONS: The deep RNA-seq study of S. moellendorffii discovered extensive new gene contents, including novel coding genes, lncRNAs, AS events, and refined gene models. Compared to flowering vascular plants, S. moellendorffii displayed a less complexity in both gene structure, alternative splicing, and regulatory elements of vascular development. The study offered important insight into the evolution of vascular plants, and the regulation mechanism of vascular development in a non-seed plant.

Decipher the ancestry of the plant-specific LBD gene family.

BACKGROUND: Lateral Organ Boundaries Domain (LBD) genes arise from charophyte algae and evolve essential functions in land plants in regulating organ development and secondary metabolism. Although diverse plant species have been investigated to construct the phylogeny of LBD gene family, a detailed and reliable ancestry that characterizes their evolutionary patterns has not been revealed. RESULTS: We develop an improved bioinformatic method that allows robust detection of 431 LBD genes in 11 high-quality land plant genomes. Phylogenetic analysis classifies the LBD genes into six subfamilies which support the existence of 7 ancient gene lineages. Phylogenetic relationship and gene collinearity are combined to retrace 11 ancestor genes for seed plants and 18 ancestor genes for angiosperms, which improves the resolution of LBD gene ancestry. The ancient gene lineages are strictly preserved in current plant genomes, including the previously controversial class IB gene in Selaginella moellendorphii, suggesting extreme reluctance of LBD genes to be lost during evolution. Meanwhile, whole-genome and dispersed gene duplications substantially expand LBD gene family in angiosperms, and elaborate functions of LBD genes through frequent expression pattern change and protein sequence variation. CONCLUSIONS: Through phylogenetic and gene collinearity analyses, we retrace the landscape of LBD gene ancestry which lays foundation for elucidating evolutionary diversification of LBD genes in land plants.

Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor.

Streptomyces coelicolor, with its 8 667 507-bp linear chromosome, is the genetically most studied Streptomyces species and is an excellent model for studying antibiotic production and cell differentiation. Here, we report construction of S. coelicolor derivatives containing sequential deletions of all the 10 polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters and a 900-kb subtelomeric sequence (total c. 1.22 Mb, 14% of the genome). No obvious differences in growth rates and sporulation of the strains were found. An artificially circularized S. coelicolor genome with deletions of total c. 1.6 Mb segments (840-kb for the left and 761-kb for the right arm of the linear chromosome) was obtained. The actinorhodin biosynthetic gene cluster could be overexpressed in some of the constructed strains.

Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis.

This study examined the inhibition performance by the major lignocellulose degradation products, including organic acids, furan derivatives, lignin derivatives, and ethanol, on a broadly used commercial cellulase enzyme Spezyme CP (Genencor International, Rochester, NY, USA) to cellulose hydrolysis at both the well-mixing state (shaking flask) and the static state (test tube). The cellulase activity in the cellulase complex of Spezyme CP was assumed to be one single "cellulase", and the apparent kinetic parameters of this cellulase enzyme were measured as an approximate index of the inhibitory effect to the industrial cellulase enzyme. The inhibition performance of these degradation products was compared and analyzed using the determined apparent kinetic parameters. All the degradation products strongly inhibit the cellulose hydrolysis by cellulase enzyme, and the inhibitions on cellulase were all competitive type. The order of the inhibition strength by the lignocellulose degradation products to cellulase is lignin derivatives > furan derivatives > organic acids > ethanol. This study gave a quantitative view to the enzymatic hydrolysis of lignocellulose under the inhibition performance of the lignocellulose degradation products and will help to understand the lignocellulose recalcitrance to enzyme hydrolysis.