Enhancing Micropropagation of Adenophora liliifolia: Insights from PGRs, Natural Extracts, and pH Optimization.

The endangered plant species Adenophora liliifolia faces threats to its survival in the wild, necessitating the development of effective micropropagation techniques for potential reintroduction efforts. This study demonstrates that Adenophora liliifolia effectively reproduces on MS synthetic medium with diverse plant growth regulators (PGR) and natural extracts, facilitating swift micropropagation for potential future reintroduction endeavors. It highlights the substantial impact of PGR composition and natural extracts on the growth and development of A. liliifolia. The ideal growth medium for A. liliifolia was determined to be ½ MS with specific treatments. Additionally, incorporating silver nitrate (AgNO3) at 5 mg L-1 into the medium led to enhanced root formation and shoot length, albeit excessive concentrations adversely affected root development. Varying concentrations of NAA significantly affected different plant growth parameters, with the 0.1 mg L-1 treatment yielding comparable plant height to the control. Moreover, 50 mL L-1 of coconut water bolstered root formation, while 200 mL L-1 increased shoot formation during in vitro propagation. However, elevated doses of coconut water (CW) impeded root development but stimulated shoot growth. Experiments measuring chlorophyll a + b and carotenoid content indicated higher concentrations in the control group than differing levels of applied coconut water. Optimizing pH levels from 6.8-7 to 7.8-8.0 notably enhanced plant height and root formation, with significant carotenoid accumulation observed at pH 6.8-7. Soil samples from A. liliifolia's natural habitat exhibited a pH of 6.65. Ultimately, the refined in vitro propagation protocol effectively propagated A. liliifolia, representing a pioneering effort and setting the stage for future restoration initiatives and conservation endeavors.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the ß-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.