The non-mevalonate pathway requires a delicate balance of intermediates to maximize terpene production.

Terpenes are valuable industrial chemicals whose demands are increasingly being met by bioengineering microbes such as E. coli. Although the bioengineering efforts commonly involve installing the mevalonate (MVA) pathway in E. coli for terpene production, the less studied methylerythritol phosphate (MEP) pathway is a more attractive target due to its higher energy efficiency and theoretical yield, despite its tight regulation. In this study, we integrated an additional copy of the entire MEP pathway into the E. coli genome for stable, marker-free terpene production. The genomically integrated strain produced more monoterpene geraniol than a plasmid-based system. The pathway genes' transcription was modulated using different promoters to produce geraniol as the reporter of the pathway flux. Pathway genes, including dxs, idi, and ispDF, expressed from a medium-strength promoter, led to the highest geraniol production. Quantifying the MEP pathway intermediates revealed that the highest geraniol producers had high levels of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), but moderate levels of the pathway intermediates upstream of these two building blocks. A principal component analysis demonstrated that 1-deoxy-D-xylulose 5-phosphate (DXP), the product of the first enzyme of the pathway, was critical for determining the geraniol titer, whereas MEP, the product of DXP reductoisomerase (Dxr or IspC), was the least essential. This work shows that an intricate balance of the MEP pathway intermediates determines the terpene yield in engineered E. coli. The genetically stable and intermediate-balanced strains created in this study will serve as a chassis for producing various terpenes. KEY POINTS: • Genome-integrated MEP pathway afforded higher strain stability • Genome-integrated MEP pathway produced more terpene than the plasmid-based system • High monoterpene production requires a fine balance of MEP pathway intermediates.

Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5ß-reductases (P5ßR and P5ßR2) did not correlate with the cardenolide increase. The expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

A cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis.

Digoxin extracted from the foxglove plant is a widely prescribed natural product for treating heart failure. It is listed as an essential medicine by the World Health Organization. However, how the foxglove plant synthesizes digoxin is mostly unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450scc), which catalyzes the first and rate-limiting step. Here we identify the long-speculated foxglove P450scc through differential transcriptomic analysis. This enzyme converts cholesterol and campesterol to pregnenolone, suggesting that digoxin biosynthesis starts from both sterols, unlike previously reported. Phylogenetic analysis indicates that this enzyme arises from a duplicated cytochrome P450 CYP87A gene and is distinct from the well-characterized mammalian P450scc. Protein structural analysis reveals two amino acids in the active site critical for the foxglove P450scc's sterol cleavage ability. Identifying the foxglove P450scc is a crucial step toward completely elucidating digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Evaluation of Antimicrobial Efficacy and Penetration Depth of Various Irrigants into the Dentinal Tubules with and without Lasers: A Stereomicroscopic Study.

AIM: To evaluate and compare the antibacterial efficacy and horizontal depth of penetration of various irrigants into the dentinal tubules when used alone and when combined with lasers. MATERIALS AND METHODS: An experimental study was done on 42 single-rooted teeth. Access cavity preparation was done and the canals were enlarged up to a ProTaper file size F2 of length 25 mm. They were inoculated with 0.1 mL of Enterococcus faecalis and the samples were randomly assigned into six different groups of seven teeth each. The following irrigation systems were used individually and in combinations-normal saline, sodium hypochlorite, chlorhexidine gluconate, diode laser and erbium, chromium: ytrrium scandium gallium garnet laser (Er,Cr:YSGG laser). The colony-forming units (CFU) of bacteria before and after disinfection and the penetration depth of different groups were determined. Statistical analysis was done by an ANOVA test. RESULTS: The highest number of CFU of bacteria was shown by the group where saline was used and it also showed the least penetration depth compared to that of the Er,Cr:YSGG laser group. CONCLUSION: Er,Cr:YSGG laser when used along with sodium hypochlorite and chlorhexidine gluconate showed the highest reduction in the CFU of bacteria and the greatest penetration depth when observed under a stereomicroscope. CLINICAL SIGNIFICANCE: Laser-assisted irrigation regimes have a high antibacterial efficacy and more penetration depth into the dentinal tubules. HOW TO CITE THIS ARTICLE: Subramani SM, Anjana G, et al. Evaluation of Antimicrobial Efficacy and Penetration Depth of Various Irrigants into the Dentinal Tubules with and without Lasers: A Stereomicroscopic Study. Int J Clin Pediatr Dent 2019;12(4):273-279.