Peroxisomal KAT2 (3-ketoacyl-CoA thiolase 2) gene has a key role in gingerol biosynthesis in ginger (Zingiber officinale Rosc.)

Abstract

Ginger is an important spice crop with medicinal values and gingerols are the most abundant pungent polyphenols present in ginger, responsible for most of its pharmacological properties. The present study focuses on the molecular mechanism of gingerol biosynthesis in ginger using transcriptome analysis. Suppression Subtractive Hybridization (SSH) was done in leaf and rhizome tissues using high gingerol-producing ginger somaclone B3 as the tester and parent cultivar Maran as the driver and generated high-quality leaf and rhizome Expressed Sequence Tags (ESTs). The Blast2GO annotations of the ESTs revealed the involvement of leaf ESTs in secondary metabolite production, identifying the peroxisomal KAT2 gene (Leaf EST 9) for the high gingerol production in ginger. Rhizome ESTs mostly coded for DNA metabolic processes and differential genes for high gingerol production were not observed in rhizomes. In the qRT-PCR analysis, somaclone B3 had shown high chalcone synthase (CHS: rate-limiting gene in gingerol biosynthetic pathway) activity (0.54 fold) in the leaves of rhizome sprouts. The presence of a high gingerol gene in leaf ESTs and high expression of CHS in leaves presumed that the site of synthesis of gingerols in ginger is the leaves. A modified pathway for gingerol/polyketide backbone formation has been constructed explaining the involvement of KAT gene isoforms KAT2 and KAT5 in gingerol/flavonoid biosynthesis, specifically the KAT2 gene which is otherwise thought to be involved mainly in β-oxidation. The results of the present investigations have the potential of utilizing KAT/thiolase superfamily enzymes for protein/metabolic pathway engineering in ginger for large-scale production of gingerols.

Supplementary information: The online version contains supplementary material available at 10.1007/s13562-022-00825-x.

Keywords: 3-ketoacyl-CoA thiolase/KAT; Expressed Sequence Tags/ESTs; Ginger; Gingerol; Suppression subtractive hybridization; cDNA library.

Fig. 1

a Agarose-formaldehyde gel electrophoresis showing total RNA isolated from ginger leaves of somaclone B3 and control cultivar Maran using modified TRIzol method. Lane1: Transcript RNA marker (0.2–10 kb), Lane 2: Total RNA from somaclone B3, and Lane 3: Total RNA from control cultivar Maran b Second PCR product after experimental subtraction of leaf tester sample. Lane 1 and Lane 6: ϕX174 DNA/Hae III digest marker, Lane 3 and Lane 4: Subtracted and unsubtracted leaf tester cDNA. c Second PCR product after experimental subtraction of rhizome tester sample. Lane 1 and Lane 5: ϕX174 DNA/Hae III digest marker, Lane 2 and Lane 3: Subtracted and unsubtracted rhizome tester cDNA

Fig. 2

Leaf ESTs coding for a biological processes and b molecular functions

Fig. 3

Gene Ontology (GO) counts for a biological processes and b molecular functions in leaf ESTs

Fig. 4

Enzyme code distribution for leaf ESTs of somaclone B3

Fig. 5

Gene Ontology counts for a biological processes and b molecular functions in rhizome ESTs

Fig. 6

Rhizome ESTs coding for a biological processes and b molecular functions

Fig. 7

Expression fold change for CHS gene with Maran as calibrator. Somaclone B3 shows the highest CHS (rate limiting gene in gingerol biosynthesis) gene expression at the sprouting stage of rhizome

Fig. 8

Flow chart showing possible pathway for gingerol biosynthesis and the role of KAT2 and KAT5 enzymes in generation of extender unit, acetyl-CoA for gingerol production. The enzymes are highlighted in yellow. ACS, acetyl-CoA synthetase; ACAD, acyl-CoA dehydrogenase; ECH, enoyl-CoA hydratase; HCD, hydroxyacyl-CoA dehydrogenase; PAL, Phe ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase (as modified from de Souza et al. 2020). The predicted route for KAT2 generated peroxisomal acetyl-CoA and its regeneration as cytosolic acetyl-CoA is highlighted in bold red arrows. Cytosolic acetyl-CoA is carboxylated by ACC to malonyl-CoA which is the extender unit for flavonoids/gingerol