Kubomura, A. | bioRxiv Science

bioRxiv2026

An aromatic substrate prenyltransferase involved in the chemical diversification of flavonoids in Glycyrrhiza glabra

Prenylated isoflavonoids are widely distributed specialized metabolites within the Fabaceae and contribute to various characteristic biological activities for both plants and humans. Several aromatic prenyltransferases (PTs) have been identified in Glycyrrhiza species, which are the most widely consumed crude drugs in traditional Chinese medicine. However, these enzymes do not sufficiently explain the structural diversity of prenylated flavonoids produced in the Glycyrrhiza genus. To identify additional novel PTs, we used elicited cultured Glycyrrhiza glabra roots as source material, in which elicitor treatment of cultured roots increased the accumulation of multiple prenylated flavonoids. To identify the responsible enzyme, PT candidates were screened using G. uralensis transcriptomes, currently the sole publicly available transcriptomic resource within the genus, and a homolog designated GgBSPT1 (BSPT; a broad-substrate prenyltransferase) was subsequently isolated from elicited cultured G. glabra roots. GgBSPT1 differed from previously identified Glycyrrhiza PTs in both amino acid sequence and enzymatic properties. GgBSPT1 catalyzed 3'-prenylation of isoliquiritigenin and 6-prenylation of five flavonoids, i.e., this PT displayed broad substrate acceptance across 20 distinct flavonoid structures. Overall, elicited cultured G. glabra roots enabled the identification of a previously unrecognized PT that is functionally distinct from earlier reported Glycyrrhiza PTs. This study provides a new insight into the metabolic plasticity of Glycyrrhiza species and expands the enzymatic toolkit for future metabolic engineering of prenylated phytochemicals by the unusually broad substrate specificity of GgBSPT1. Main conclusionUsing cultured Glycyrrhiza glabra roots, we identified a new prenyltransferase involved in the formation of a variety of flavonoids, thereby revealing novel prenylated isoflavonoid pathways in licorice.

molecular biology↗

bioRxiv2025

Genome-scale dissection of phase-variable gene function in Campylobacter jejuni using a stabilized phasotype library

Phase variation (PV) enables bacterial pathogens to rapidly alter their surface structures through reversible mutations in simple sequence repeats, promoting immune evasion and environmental adaptation. In Campylobacter jejuni, the stochastic nature of PV has hindered the systematic functional analysis of phase-variable genes (PVGs). Here, we introduce PV-GenShift, a genome-scale screening platform built on a genetically stabilized library of phase-locked C. jejuni variants. By fixing the ON/OFF states of 15 PVGs, PV-GenShift enables reproducible, high-resolution analysis of phasotypes, defined as unique ON/OFF combinations across multiple PVGs, under defined selective pressures. Using models of human serum exposure, murine colonization, and chicken gut passage, we identified distinct phasotypes associated with serum resistance and with enrichment during mouse colonization, particularly involving capsular polysaccharide modifications such as O-methyl phosphoramidation and methylation. In contrast, chicken gut passage resulted in heterogeneous ON/OFF shifts without a dominant phasotype. These findings highlight the combinatorial impact of PVG expression states on bacterial adaptation and establish PV-GenShift as a broadly applicable framework for dissecting PV-driven phenotypic diversity. This approach provides a scalable strategy for exploring genotype-phenotype relationships and offers insights relevant to vaccine design and targeted therapeutics. Significance StatementPhase variation generates phenotypic diversity that enables pathogens to evade immunity and adapt to changing environments; however, its random nature has long obscured functional analysis. This study introduces PV-GenShift, a genome-scale platform that stabilizes phase-variable gene expression in Campylobacter jejuni, allowing the systematic identification of gene combinations that influence survival under selective pressures. Using PV-GenShift, we identified phasotypes associated with serum resistance and enrichment during mouse colonization, while chicken passage produced diverse but non-specific shifts. These results demonstrate how combinatorial ON/OFF states of multiple genes shape bacterial adaptation and provide a generalizable strategy for studying phase variation across pathogens, with implications for vaccine design and targeted therapeutics.

microbiology↗