Pathway mining-based integration of critical enzyme parts for de novo biosynthesis of steviolglycosides sweetener in Escherichia coli

The development of microbial synthetic biology has been revolutionizing the industrial production of important plant terpenoids since the dawn of the century^1,2. By reconstructing the downstream biosynthetic pathways and systematically engineering the supply of common terpenoid precursors, the engineered microorganism chassis (e.g., Escherichia coli and Saccharomyces cerevisiae) can produce desired terpenoids in a more controllable manner. This strategy is also adapted to enrich the knowledge of cell metabolism via either discovery of novel or proof of postulated pathways³.

The 13α-hydroxylation of kaurenoic acid to generate steviol is the first divergent step specific to the SGs pathway. Although the critical kaurenoic acid 13α-hydroxylase (KAH) was reported to be partially purified and its N-terminal sequence was described, efforts in cloning its encoding cDNA have been unsuccessful^4,5. On the other hand, sequences of several potential KAHs from S. rebaudiana have been deposited in Genebank database, including KAH_ACD93722, KAH_AEH65420, KAH_AEH65421, KAH_AEH65422, KAH_AEH65423, and KAH_AEH65424, without any functional characterization⁵. To comprehensively survey the genes associated with SG biosynthesis, RNA-seq was performed for two samples of Stevia leaves, sr-1 (30-day-old leaves with a low SG content) and sr-2 (90-day-old leaves with a high SG content). All these potential KAHs mentioned above were detected in our Stevia RNA-seq data. Phylogenetic analysis showed that KAH_ACD93722, KAH_AEH65422, and KAH_AEH65424 belong to the CYP716 family, KAH_AEH65420 belongs to the CYP701 family, and KAH_AEH65421 and KAH_AEH65423 belong to the CYP82 family (Figure 1B). When the resting cells of recombinant E. coli BL21(DE3) co-expressing various KAH and its NADPH-dependent cytochrome P450 reductase (CPR) were used to transform the substrate kaurenoic acid, only KAH_ACD93722 exhibited low activity, producing 0.067 mg l⁻¹ steviol (Figure 1B and 1C, Supplementary information, Figure S1). Recently, a CYP714 family enzyme CYP714A2, which contributes to the production of diverse GA compounds through various oxidations of C and D rings of the ent-kaurene scaffold in Arabidopsis thaliana, was reported to bear a KAH activity when expressed in yeast⁷. We thus screened for sequences similar to CYP714A2 in our RNA-seq database, and identified a novel KAH belonging to the CYP714 family designated as KAHn2, showing 40% identity with that of CYP714A2 (Figure 1B). KAHn2 was nearly 15-fold more efficient than KAH_ACD93722 and the steviol yield reached 1.01 mg l⁻¹ (Figure 1C). Interestingly, contrary to other SG pathway genes (Supplementary information, Figure S2), the transcription of KAHn2 in mature leaves was downregulated rather than upregulated in our RNA-seq data.

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