Engineering Bacillus subtilis for production of 3-hydroxypropanoic acid.

3-Hydroxypropionic acid (3-HP) is a valuable platform chemical that is used as a precursor for several higher value-added chemical products. There is an increased interest in development of cell factories as a means for the synthesis of 3-HP and various other platform chemicals. For more than a decade, concentrated effort has been invested by the scientific community towards developing bio-based approaches for the production of 3-HP using primarily Escherichia coli and Klebsiella pneumoniae as production hosts. These hosts however might not be optimal for applications in e.g., food industry due primarily to endotoxin production and the pathogenic origin of particularly the K. pneumoniae. We have previously demonstrated that the generally recognized as safe organism Bacillus subtilis can be engineered to produce 3-HP using glycerol, an abundant by-product of the biodiesel industry, as substrate. For commercial exploitation, there is a need to substantially increase the titer. In the present study, we optimized the bioprocess conditions and further engineered the B. subtilis 3-HP production strain. Thereby, using glycerol as substrate, we were able to improve 3-HP production in a 1-L bioreactor to a final titer of 22.9 g/L 3-HP.

An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.

Erratum: An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

[This corrects the article DOI: 10.1016/j.crmeth.2021.100062.].

Chemokine Receptors CCR1 and CCR2 on Peripheral Blood Mononuclear Cells of Newly Diagnosed Patients with the CD38-Positive Chronic Lymphocytic Leukemia.

Chemokines and their receptors direct migration and infiltration of immune cells. CCR1 and CCR2 maintain sequence similarity and respond to a number of the same chemokines secreted in lymphoid organs. Expression of CD38 on leukemic cells has been associated with poor clinical outcomes in patients with chronic lymphocytic leukemia (CLL) and is considered as the negative predictor of progression. In our study of newly diagnosed CLL patients, which included 39 CD38-positive and 22 CD38-negative patients, CCR1 and/or CCR2 were always detected, using flow cytometry, on the peripheral blood (PB) CD19+CD5+ lymphocytes in patients with >30% of the CD38+ CD19+CD5+ lymphocytes (n = 16). Spearman's rank correlation analysis determined correlations between the frequency of the CCR1- and CCR2-expressing PB CD19+CD5+ lymphocytes and the frequency of the CD38-positive CD19+CD5+ lymphocytes (rs = 0.50 and rs = 0.38, respectively). No significant correlations were observed between ZAP70 mRNA expression levels in PB mononuclear cells and the frequency of the circulating CCR1+ or CCR2+ CD19+CD5+ lymphocytes. Further association studies are needed to verify prognostic relevance of the CCR1/CCR2 expression on leukemic cells in CLL patients at diagnosis. We suggest that CCR1/CCR2 signaling pathways could represent attractive targets for development of CLL anti-progression therapeutics.