Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to ...petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of
(CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of
(CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both
and
genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both
and
in C. acetobutylicum. As suggested by these data, overexpression of
and
led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars.
C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them.
Although
is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, ...the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene,
Although the literature has previously shown that inactivation of either
,
, or
had only moderate effects on the strain, this study shows that clean deletion of both
and
strongly impaired solvent production and that a triple mutant Δ
Δ
Δ
was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each
copy to fully restore efficient ABE fermentation in the triple deletion strain.
An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for
and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of
expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in
, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.
CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of ...Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.
•A two-plasmid inducible CRISPR/Cas9 genome editing tool has been developed•Editing efficiency of 100% was achieved in Clostridium acetobutylicum ATCC 824•Nucleotide substitution, deletion and integrations up to 3.6kb were performed
Abstract
Acquisition of differential signals from low voltage and high impedance sources is difficult when using commonly available laboratory test equipment such as a standard oscilloscope or single ...ended acquisition system. This paper presents a versatile differential preamplifier for universal signal conditioning. The probe offers a wide input voltage range and can measure signals ranging from a few hundred micro-volts up to 100 V with 1 MHz bandwidth. It also contains a programmable output filter for bandwidth limitation and noise reduction, user-accessible controls for offset adjustment and input coupling mode selection. The probe can be powered through an USB port or by using an internal battery. Input impedance can be made virtually infinite by the JFET input stage, which makes this differential preamplifier usable with a variety of high-impedance sources commonly encountered in the sensors research field and in electronic engineering. Experimental validation shows that the proposed device has better performance levels and interoperability than commercially available instrumentation at a reduced cost, even while using off-the-shelf components.
Abstract
The solventogenic
C
.
beijerinckii
DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the ...resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (
sigL
), which encodes the σ
54
sigma factor. Complementation with wild-type
sigL
fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ
54
in
C
.
beijerinckii
DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of
sigL
in the model strain
C
.
beijerinckii
NCIMB 8052.
There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol ...(ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol.
The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol.
We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
► We study the effect of pretreatment and enzymatic hydrolysis on wheat straw. ► We focus on wettability, lignin structure and adsorption by three fungi cellulases. ► Organosolv treatment and ...steam-explosion decreases the hydrophobicity of wheat straw. ► Wettability of pretreated hydrolyzed samples negatively correlates to lignin content. ► Cellulase adsorption decreases with increasing hydrolysis time and lignin content.
The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20–50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from
Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.
Through biomass valorization, sugars can become a major carbon resource for the production of fuels and chemicals by using catalysis. Hybrid catalysis, a direct combination of biocatalysis and ...chemocatalysis, may yield innovative solutions. 5‐Hydroxymethylfurfural (5‐HMF) is a platform molecule derived from glucose, for which fructose is a key intermediate. To overcome the thermodynamic enzymatic equilibrium between glucose and fructose, two methodologies based on hybrid catalysis are described herein. In the first method, the glucose isomerase and fructose‐to‐5‐HMF dehydration chemocatalysts are simultaneously implemented in a multiphasic way to imply the complexation and transport of fructose. The second method proposes a route through sorbitol, obtained by the hydrogenation of glucose. Sorbitol is then enzymatically converted into fructose, whereas regeneration of the cofactor is performed in situ with an organometallic complex as a chemocatalyst.
Mixing it up: Hybrid catalysis, which intimately combines bio‐ and chemocatalytic systems, is a promising strategy to convert bioderived molecules. We present two variations of this technology with the potential to unlocking the quantitative production of 5‐hydroxymethylfurfural from glucose.
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