Qualitative Analysis of Phytochemicals by High-resolution Mass Spectrometry -Analysis for Prenylnaringenin-

Significance of the Topic

Microbial production of complex plant-derived compounds is a key strategy for sustainable manufacturing of high-value phytochemicals. Prenylnaringenin, a prenylated flavonoid with demonstrated anti-inflammatory, antioxidant, anti-influenza and potential anticancer and antidiabetic activities, represents an attractive target for low-carbon bioproduction. High-resolution mass spectrometry (HRMS) offers the speed and accuracy needed to evaluate engineered strains and accelerate discovery.

Objectives and Study Overview

This work aimed to:

• Construct a Saccharomyces cerevisiae strain capable of converting phenylalanine into prenylnaringenin by introducing plant and microbial genes.
• Demonstrate the use of a quadrupole time-of-flight HRMS (LCMS-9030) to distinguish prenylnaringenin isomers from impurities and to rapidly confirm compound identity.

Methodology and Instrumentation

Metabolic engineering and analytical workflow:

1. Pathway assembly: Six Arabidopsis thaliana genes (AtPAL1, AtC4H, AtCPR1, At4CL3, AtCHS3, AtCHI1) were expressed in yeast to produce naringenin from phenylalanine.
2. Prenyltransferase screening: Eleven candidate PT genes from plants and microbes were introduced to achieve naringenin prenylation.
3. Fermentation and extraction: Engineered yeast cultures were harvested and metabolites extracted for analysis.
4. Chromatography: Separation on a COSMOSIL 5C18-MSII column (2.0 mm×150 mm) with a water/acetonitrile gradient containing 0.1 % acetic acid.
5. High-resolution detection: LCMS-9030 Q-TOF in negative-ion ESI mode, MS scan (m/z 70–1000) and targeted MS/MS of m/z 339.124, collision energy 35 V ±17 V.

Main Results and Discussion

• Extracted ion chromatograms at ±0.25 Da revealed multiple peaks; narrowing the window to ±5 ppm isolated a single signal at 19.5 min corresponding to prenylnaringenin.
• Product ion spectra matched fragmentation patterns of known standards, narrowing candidates to 6- and 8-prenylnaringenin.
• Co-injection with authentic standards confirmed 8-prenylnaringenin production from the plant-derived PT SfN8DT-1.
• A PT from the fungus Neosartorya fischeri yielded a distinct peak consistent with 3'-prenylnaringenin, illustrating enzyme substrate promiscuity.

Benefits and Practical Applications

• High-resolution MS enables rapid deconvolution of isomeric products and discrimination of background impurities without extensive purification.
• Reduced reliance on multiple reference standards through accurate mass and fragmentation profiling.
• Accelerated strain screening supports iterative design in metabolic engineering workflows.

Future Trends and Applications

Emerging directions include:

• Expansion of prenyltransferase libraries through genome mining and directed evolution to access diverse prenylated flavonoids.
• Integration of HRMS with automated high-throughput fermentation platforms for real-time strain evaluation.
• Application of advanced data analytics and machine learning to MS/MS spectra for de novo identification of novel metabolites.

Conclusion

This study demonstrates that coupling metabolically engineered yeast with the high mass accuracy and resolution of the LCMS-9030 Q-TOF facilitates rapid identification and structural elucidation of prenylnaringenin isomers. Such a platform accelerates bioproduction research toward sustainable manufacturing of bioactive phytochemicals.

References

1. Cui L., Ndinteh D.T., Na M., Thuong P.T., Silike-Muruumu J., Njamen D., Mbafor J.T., Fomum Z.T., Ahn J.S., Oh W.K. Isoprenylated flavonoids from the stem bark of Erythrina abyssinica. Journal of Natural Products. 2007;70(7):1039–1042.
2. Wang S., Dunlap T.L., Howell C.E., Mbachu O.C., Rue E.A., Phansalkar R., Chen S.N., Pauli G.F., Dietz B.M., Bolton J.L. Hop (Humulus lupulus L.) extract and 6-prenylnaringenin induce P450 1A1 catalyzed estrogen 2-hydroxylation. Chemical Research in Toxicology. 2016;29(7):1142–1150.
3. Isogai S., Okahashi N., Asama R., Nakamura T., Hasunuma T., Matsuda F., Ishii J., Kondo A. Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases. Metabolic Engineering Communications. in press (2021).
4. Nikolic D., Li Y., Chadwick L.R., Grubjesic S., Schwab P., Metz P., van Breemen R.B. Metabolism of 8-prenylnaringenin, a potent phytoestrogen from hops (Humulus lupulus), by human liver microsomes. Drug Metabolism and Disposition. 2004;32(3):272–279.