Polylactic acid is a plastic polymer widely used in different applications from printing filaments for 3D printer to mulching films in agriculture, packaging materials, etc. Here, we report the production of poly-D-lactic acid (PDLA) in an engineered yeast strain of Yarrowia lipolytica. Firstly, the pathway for lactic acid consumption in this yeast was identified and interrupted. Then, the heterologous pathway for PDLA production, which contains a propionyl-CoA transferase (PCT) converting lactic acid into lactyl-CoA, and an evolved polyhydroxyalkanoic acid (PHA) synthase polymerizing lactyl-CoA, was introduced into the engineered strain. Among the different PCT proteins that were expressed in Y. lipolytica, the Clostridium propionicum PCT exhibited the highest efficiency in conversion of D-lactic acid to D-lactyl-CoA. We further evaluated the lactyl-CoA and PDLA productions by expressing this PCT and a variant of Pseudomonas aeruginosa PHA synthase at different subcellular localizations. The best PDLA production was obtained by expressing the PCT in the cytosol and the variant of PHA synthase in peroxisome. PDLA homopolymer accumulation in the cell reached 26 mg/g-DCW, and the molecular weights of the polymer (Mw = 50.5 × 10 3 g/mol and Mn = 12.5 × 10 3 g/mol) were among the highest reported for an in vivo production.
Inhalation
as a route for administering drugs and dietary
supplements
has garnered significant attention over the past decade. We performed
real-time analyses of aerosols using secondary electrospray ionization
(SESI) technology interfaced with high-resolution mass spectrometry
(HRMS), primarily developed for exhaled breath analysis with the goal
to detect the main aerosol constituents. Several commercially available
inhalation devices containing caffeine, melatonin, cannabidiol, and
vitamin B12 were tested. Chemical characterization of the aerosols
produced by these devices enabled detection of the main constituents
and screening for potential contaminants, byproducts, and impurities
in the aerosol. In addition, a programmable syringe pump was connected
to the SESI–HRMS system to monitor aerosolized active pharmaceutical
ingredients (APIs) such as chloroquine, hydroxychloroquine, and azithromycin.
This setup allowed us to detect caffeine, melatonin, hydroxychloroquine,
chloroquine, and cannabidiol in the produced aerosols. Azithromycin
and vitamin B12 in the aerosols could not be detected; however, our
instrument setup enabled the detection of vitamin B12 breakdown products
that were generated during the aerosolization process. Positive control
was realized by liquid chromatography-HRMS analyses. The compounds
detected in the aerosol were confirmed by exact mass measurements
of the protonated and/or deprotonated species, as well as their respective
collision-induced dissociation tandem mass spectra. These results
reveal the potential wide application of this technology for the real-time
monitoring of aerosolized active pharmaceutical ingredients that can
be administered through the inhalation route.
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