Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent K_m values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent V_max values were 0.98, 0.19 and 0.44 pM·min⁻¹, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves.

L-theanine is used as an additive in food industry due to its unique health benefits to human being. Using γ-glutamyltranspeptidase (GGT) for L-theanine biosynthesis is one of the most effective methods. Fifty putative Bacillus strains were isolated from tea plant rhizosphere soil and could produce GGT but differed in GGT-producing activity. The capacity of forming L-theanine activity catalyzed by the GGTs from different strains was consistent with their activity of producing GGTs. Strain BSFT-42 was identified as Bacillus subtilis based on biochemical properties and 16S rDNA sequence alignment. The GGT produced by this strain had the highest L-theanine-forming activity. Under the optimized conditions, the GGT from the fermentation broth of strain BSFT-42 at 32 h could convert 87.45% of Gln to L-theanine and produced 17.49 mM of L-theanine. This study indicated the possibility to use GGT excreted by B. subtilis BSFT-42 for L-theanine production.