BackgroundBiodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth-inhibiting byproducts which reduce feedstock consumption and solvent production.ResultsTo obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild-type C. pasteurianum (ATCC 6013) culture was chemically mutagenized, and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80–150 g/L). The best-performing mutant (M150B) showed a 91 % increase in butanol production in 100 g/L crude glycerol compared to the wild-type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol). Wild-type and M150B strains were sequenced via Single Molecule Real-Time (SMRT) sequencing. Mutations introduced to the M150B genome were identified by sequence comparison to the wild-type and published closed sequences. A major mutation (a deletion) in the gene of the master transcriptional regulator of sporulation, Spo0A, was identified. A spo0A single gene knockout strain was constructed using a double--crossover genome-editing method. The Spo0A-deficient strain showed similar tolerance to crude glycerol as the evolved mutant strain M150B. Methylation patterns on genomic DNA identified by SMRT sequencing were used to transform plasmid DNA to overcome the native C. pasteurianum restriction endonuclease.ConclusionsSolvent production in the absence of Spo0A shows C. pasteurianum differs in solvent-production regulation compared to other solventogenic Clostridium. Growth-associated butanol production shows C. pasteurianum to be an attractive option for further engineering as it may prove a better candidate for butanol production through continuous fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0408-7) contains supplementary material, which is available to authorized users.

Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including: i. glycosyltransferases that add saccharides, ii. glycosidases that trim glycans, iii. Conserved Oligomeric Golgi complex (COG) members that regulate intracellular transport, iv. enzymes aiding the biosynthesis of sugar-nucleotides, and v. sulfotransferases. This manuscript describes a pooled ‘glycoGene CRISPR’ lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P−/E-selectin, anti-sialyl Lewis-X mAb HECA-452 and selected lectins (PHA-L, VVA, PNA) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand, PSGL-1 in leukocytes. ~ 80–90% of the sgRNA used in this study displayed high editing efficiency and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation related genes and pathways that control lectin recognition in a variety of contexts.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on-and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycyclinedependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genomewide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA‐seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)‐hepatic differentiation, siRNA knockdown demonstrated de‐differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.