High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space.

Coenzyme F420 is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F420-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F420 levels are produced in microorganisms, F420 availability is a serious bottleneck for research and application. Recent advances in our understanding of the F420 biosynthesis enabled heterologous overproduction of F420 in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F420 biosynthesis in E. coli. However, it still led to the production of low amounts of F420 and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module ("core") addressed parts of the riboflavin biosynthesis pathway and FO synthase for the conversion of GTP to the stable F420 intermediate FO. The enzymes of the second module ("decoration") were chosen to turn FO into F420. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F420-derived fluorescence. This approach yielded the highest titer of coenzyme F420 produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F420-dependent bioprocesses as well as applications of F420-dependent enzymes in biocatalysis.

Tight association of autophagy and cell cycle in leukemia cells.

BACKGROUND: Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined. RESULTS: In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis. CONCLUSION: This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.

Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds.

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.