QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans^1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis^3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Background: Triterpenoids from birch (Betula platyphylla Suk.) exert antitumor and anti-HIV activities. Due to the complexity of plant secondary metabolic pathways, triterpene compounds in plants is not always determined by a single gene; they may be controlled by polygene quantitative traits. Secondary metabolism related to terpenoids involves tissue specificity and localisation of key biosynthetic enzymes. Terpene synthesis is influenced by light, hormones and other signals, as well as upstream transcription factor regulation. Results: Anchor Herein, we identified and characterised two birch MYB transcription factors (TFs) that regulate triterpenoid biosynthesis. BpMYB21 and BpMYB61 are R2R3 TFs that positively and negatively regulate responses to methyl-jasmonate (MeJA) and salicyclic acid (SA), respectively. Expression of BpMYB21 and BpMYB61 was elevated in leaves and stems more than roots during July/August in Harbin, China. BpMYB21 expression was increased by abscisic acid (ABA), MeJA, SA and gibberellins (GAs). BpMYB61 expression in leaves and BpMYB21 expression in stems was reduced by ABA, MeJA and SA, while GAs, ethylene, and injury increased BpMYB61 expression. BpMYB21 was localised in nuclei, while BpMYB61 was detected in cell membranes and nuclei. Promoters for both BpMYB21 (1302 bp) and BpMYB61 (850 bp) were active. BpMYB21 and BpMYB61 were ligated into pYES3, introduced into AnchorINVScl (yeast strain without exogenous genes), INVScl-pYES2-SSAnchorAnchor (transgenic yeast strain harbouring the SS gene from birch), and INVScl-pYES2-SE (transgenic yeast strain harbouring the SE gene from birch), and the squalene content was highest in AnchorINVScl-pYES-MYB21-SS (transgenic yeast strain harbouring SS and MYB21 genes) and INVScl-pYES3-MYB61 (transgenic yeast strain harbouring the MYB61 gene). In BpMYB21 transgenic birch key triterpenoid synthesis genes were up-regulated, and in BpMYB61 transgenic birch AnchorFPS (farnesyl pyrophosphate synthase) and SS (squalene synthase) were up-regulated, but HMGR (3-hydroxy-3-methylglutaryl coenzyme a reductase), BPWAnchor (lupeol synthase), SE (squalene epoxidase) and BPY (b-amyrin synthase) were down-regulated. Both BpMYB21 and BpMYB61 specifically activate SE and BPX (cycloartenol synthase synthesis) promoters. Conclusions: These findings support further functional characterisation of R2R3-MYB genes, and illuminate the regulatory role of BpMYB21 and BpMYB61 in the synthesis of birch triterpenoids.

Birch (Betula platyphylla Suk.) contains triterpenoids with anti-HIV and anti-tumor pharmacological activities. However, the natural abundance of these triterpenoids is low, and their chemical synthesis is costly. Transcription factors have the ability to regulate the metabolite pathways of triterpenoids via multi-gene control, thereby improving metabolite yield. Thus, transcription factors have the potential to facilitate the production of birch triterpenoids. Plant bHLH (basic helix-loop-helix) transcription factors play important roles in stress response and secondary metabolism. In this study, we cloned two genes, BpMYC4 and BpbHLH9, that encode bHLH transcription factors in Betula platyphylla Suk. The open reading frame (ORF) of BpMYC4 was 1452 bp and encoded 483 amino acids, while the ORF of BpbHLH9 was 1140 bp and encoded 379 amino acids. The proteins of BpMYC4 and BpbHLH9 were localized in the cell membrane and nucleus. The tissue-specific expression patterns revealed that BpMYC4 expression in leaves was similar to that in the stem and higher than in the roots. The expression of BpbHLH9 was higher in the leaves than in the root and stem. The expressions of BpMYC4 and BpbHLH9 increased after treatment with abscisic acid, methyl jasmonate, and gibberellin and decreased after treatment with ethephon. The promoters of BpMYC4 and BpbHLH9 were isolated using a genome walking approach, and 900-bp and 1064-bp promoter sequences were obtained for BpMYC4 and BpbHLH9, respectively. The ORF of BpbHLH9 was ligated into yeast expression plasmid pYES3 and introduced into INVScl and INVScl1-pYES2-SS yeast strains. The squalene and total triterpenoid contents in the different INVScl1 transformants decreased in the following order INVScl1-pYES-SS-bHLH9 > INVScl1-pYES3-bHLH9 > INVScl1-pYES2- BpSS > INVScl-pYES2. In BpbHLH9 transgenic birch, the relative expression of the genes that encodes for enzymes critical for triterpenoid synthesis showed a different level of up-regulation compair with wild birch(control), and the contents of betulinic acid, oleanolic acid and betulin in bHLH9–8 transgenic birch were increased by 11.35%, 88.34% and 23.02% compared to in wild birch, respectively. Our results showed that the modulation of BpbHLH9 by different hormones affected triterpenoid synthesis and triterpenoid contents. This is the first report of the cloning of BpbHLH9, and the findings are important for understanding the regulatory role of BpbHLH9 in the synthesis of birch triterpenoids.

Urban traffic flow has the characteristics of nonlinearity and time-variation, and how to accurately forecast short-term traffic flow has been an essential part in traffic field. Taking advantage of the Generalized Regression Neural Network (GRNN), the paper establishes the short-term forecasting model of traffic flow based on GRNN. The GRNN model selects the cross validation algorithm to train the network, takes the root mean square of forecasting error as the evaluation criterion of the network to determine the smoothing factor and uses the method of rolling forecasting to forecast the traffic flow. Compared with the forecasting models of RBF and BP neural network, GRNN has stronger approximation capability and higher forecasting accuracy.