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

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