A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid

The Mode of Action of Cyclic Monoterpenes (−)-Limonene and (+)-α-Pinene on Bacterial Cells

A broad spectrum of volatile organic compounds’ (VOCs’) biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs—the cyclic monoterpenes (−)-limonene and (+)-α-pinene—on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (−)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (−)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (−)-limonene completely inhibits the DnaKJE–ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ΔibpB strains. (+)-α-Pinene partially inhibits refolding only in ΔibpB mutant strain

Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM)

The N-Terminal Domain of Aliivibrio fischeri LuxR Is a Target of the GroEL Chaperonin▿

Here we show that the C-terminal domain of LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in Escherichia coli SKB178 gro+ and E. coli OFB1111 groEL673 strains to the same level. Using affinity chromatography, we showed that GroEL binds to the N-terminal domain of LuxR, pointing to a GroEL/GroES requirement for the folding of the N-terminal domain of LuxR

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts

A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering

Abstract CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid

HYPOLIPIDEMIC EFFECT AND LESKOL TOLERANCE IN HYPERTENSIVE PATIENTS WITH HYPERCHOLESTEROLEMIA - RESULTS OF MULTICENTER TRIAL

Hypertensive patients with hyperlipidemia are at high risk to develop coronary heart disease (CHD). Chemotherapeutic correction of hyperlipidemia seems most reliable modality to prevent CHD. Hypolipidemic effect and tolerance of leskol (fluvastatin) in dietotherapy-resistant hypercholesterolemia were studied in 74 patients with essential hypertension treated with hypotensive drugs. The patients were included in a multicenter trial. A 12-week course reduced total cholesterol level under 6.2 mmol/l in 59% of the patients, under 5.2 mmol/l in 29% of them. LDLP cholesterol lowered to 3.5% in 34% of the patients. Mean apo B diminished by 23%. There was a 27% decrease in the proportion of atherogenic fraction apo B to antiatherogenic fraction to transport proteins apo A-I. Leskol is well tolerated and effective against hypercholesterolemia, it is safe in relation to side effects and blood biochemistry

ABDOMINAL OBESITY IN ARTERIAL HYPERTENSION: ATHEROGENIC ALTERATIONS IN LIPID TRANSPORT SYSTEMS AND CARBOHYDRATES METABOLISM

The comparative analysis of CHD risk factors which are known metabolic syndrome components was conducted in groups of patients with arterial hypertension (AH) combined with abdominal obesity (AO), in hypertensive subjects with normal body mass index, and in those with AH, AO and glucose intolerance. The combination of AH with AO was coupled with higher SBP and DBP, and with more pronounced atherogenic shifts in lipoprotein profile: elevated levels of triglycerides, total cholesterol, apo B and apoB/AI ratio. The magnitude of these parameters was closed to that in group with AH, AO and glucose intolerance; hyperlipidemia in the last group was aggravated by decreased HDL cholesterol level. Patients with AH and AO had significantly higher blood insulin level and lower glucose/insulin ratio than subjects with AH only. These results might indicate the existence of latent insulin resistance in hypertensive patients with abdominal (visceral) obesity. Global CHD risk for these patients appeared to be higher than in subjects with AH only

Hypolipidemic effect and leskol tolerance in hypertensive patients with hypercholesterolemia: Results of multicenter trial

Hypertensive patients with hyperlipidemia are at high risk to develop coronary heart disease (CHD). Chemotherapeutic correction of hyperlipidemia seems most reliable modality to prevent CHD. Hypolipidemic effect and tolerance of leskol (fluvastatin) in dietotherapy-resistant hypercholesterolemia were studied in 74 patients with essential hypertension treated with hypotensive drugs. The patients were included in a multicenter trial. A 12-week course reduced total cholesterol level under 6.2 mmol/l in 59% of the patients, under 5.2 mmol/l in 29% of them. LDLP cholesterol lowered to 3.5% in 34% of the patients. Mean apo B diminished by 23%. There was a 27% decrease in the proportion of atherogenic fraction apo B to antiatherogenic fraction of transport proteins apo A-I. Leskol is well tolerated and effective against hypercholesterolemia, it is safe in relation to side effects and blood biochemistry