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Tuesday, April 21, 2020

Rational development of transformation in Clostridium thermocellum ATCC 27405 via complete methylome analysis and evasion of native restriction-modification systems.

A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction-modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction-modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases…

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Tuesday, April 21, 2020

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7a-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens, the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens…

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Tuesday, April 21, 2020

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting…

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Tuesday, April 21, 2020

FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift…

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Tuesday, April 21, 2020

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development…

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Tuesday, April 21, 2020

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively…

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Tuesday, April 21, 2020

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039…

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Tuesday, April 21, 2020

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two…

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Tuesday, April 21, 2020

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with…

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Tuesday, April 21, 2020

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (>?5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of…

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