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QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. 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 synthesis3,4. 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.QS-21—an FDA-approved vaccine adjuvant—and several structural analogues of QS-21 can be synthesized in engineered yeast strains, and this process is much less laborious compared with the conventional mode of extraction from the Chilean soapbark tree. [ABSTRACT FROM AUTHOR]

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-d-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches. Methyl jasmonate triggers saponin production in Saponaria vaccaria. Using transcriptome data and heterologous expression, the authors identify P450s and glycosyltransferases that modify triterpenoids. They also discover the pathway for UDP-D-fucose biosynthesis. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of natural products. RiPP precursor peptides can undergo extensive enzymatic tailoring to yield structurally and functionally diverse products, and their biosynthetic logic makes them attractive bioengineering targets. Recent work suggests that unrelated RiPP-modifying enzymes contain structurally similar precursor peptide-binding domains. Using profile hidden Markov model comparisons, we discovered related and previously unrecognized peptide-binding domains in proteins spanning the majority of known prokaryotic RiPP classes, and we named this conserved domain the RiPP precursor peptide recognition element (RRE). Through binding studies we verified RRE's roles for three distinct RiPP classes: linear azole-containing peptides, thiopeptides and lasso peptides. Because numerous RiPP biosynthetic enzymes act on peptide substrates, our findings have powerful predictive value as to which protein(s) drive substrate binding, thereby laying a foundation for further characterization of RiPP biosynthetic pathways and the rational engineering of new peptide-binding activities. [ABSTRACT FROM AUTHOR]

Three CF and three CF subunits of the chloroplast H-ATP synthase are encoded on the chloroplast genome. The chloroplast atp genes are organized as two operons in plants but not in the green alga, Chlamydomonas reinhardtii. The atpBE or β operon shows a relatively simple organisation and transcription pattern, while the atpIHFA or α operon is transcribed into a large variety of mRNAs. The atp genes are related to those of cyanobacteria and, more distantly, to those of non-photosynthetic bacteria such as E. coli, suggesting a common origin of most FF ATP synthase subunits. Both the chloroplast and cyanobacterial ATP synthases have four F subunits, not three as in the E. coli complex. The proton pore of the CF is proposed to be formed by the interaction of subunits III and IV. [ABSTRACT FROM AUTHOR]

Transgenic tobacco ( Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content. [ABSTRACT FROM AUTHOR]

Transgenic tobacco ( Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k, of 3.5+0.2 mol CO·(mol sites)·s at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO·(mol sites)· in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO and O, the rate of CO assimilation was measured at 25° C at different intercellular partial pressures of CO and O. These measurements were combined with carbon-isotope analysis (C/C) of CO in the air passing over the leaf to estimate the conductance for transfer of CO from the substomatal cavities to the sites of carboxylation (0.3 mol·m·s·bar) and thus the partial pressure of CO at the sites of carboxylation. The calculated Michaelis-Menten constants for CO and O were 259 ±57 μbar (8.6±1.9μM) and 179 mbar (226 μM), respectively, and the effective Michaelis-Menten constant for CO in 200 mbar O was 549 μbar (18.3 μM). From measurements of the photocompensation point (Γ = 38.6 ubar) we estimated Rubisco's relative specificity for CO, as opposed to O to be 97.5 in vivo. These values were dependent on the size of the estimated CO-transfer conductance. [ABSTRACT FROM AUTHOR]