Complete biosynthesis of QS-21 in engineered yeast.

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.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import.

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.

Melt Extrusion for a High Melting Point Compound with Improved Solubility and Sustained Release.

The objective of the current study was to develop an amorphous solid dispersion for a high melting point compound, griseofulvin (GRF), with an enhanced solubility and a controlled release pattern utilizing hot melt extrusion (HME) technology. Hypromellose acetate succinate (HPMCAS, Shin-Etsu AQOAT®, medium particle size) was explored as the polymeric carrier, while hypromellose (HPMC, Metolose® SR) was chosen as the release rate control agent. GRF presented an HPMCAS grade-dependent solubility: AS-HMP > AS-MMP > AS-LMP. At 10 wt.% loading, the release of GRF was prolonged to 6 h with the incorporation of 10% HPMC 90SH-100SR, while its solubility was enhanced up to sevenfold. Fourier transform infrared spectroscopy (FT-IR) identified the H-bonding between drug and polymers. Element analysis utilizing X-ray photoelectron spectroscopy (XPS) discovered that less GRF aggregated on the surface of binary powders compared with ternary powders containing HPMC, indicating the relatively poor wettability of the latter one. The morphology of extrudates was observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), illustrating a much smoother and uniform surface of binary extrudates. Immediate release tablets including 10% super-disintegrant L-HPC were able to achieve identical dissolution profile as the powders of extrudates.

Uncovering the protein translocon at the chloroplast inner envelope membrane.

Chloroplasts require protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to import thousands of cytoplasmically synthesized preproteins. However, the molecular identity of the TIC translocon remains controversial. Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. We purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. Thus, this complex constitutes a general TIC translocon.

Chloroplast envelope localization of EDS5, an essential factor for salicylic acid biosynthesis in Arabidopsis thaliana.

Chloroplasts are responsible for biosynthesis of salicylic acid (SA) an important signal molecule in plant immunity. EDS5 is a homolog of the MATE (multidrug and toxic compound extrusion) family of transporters, and is essential for SA biosynthesis. It has been speculated that EDS5 would be involved in the export of SA from chloroplasts. However, the subcellular localization of EDS5 remains largely uncharacterized. We demonstrate here that EDS5 is specifically localized to the chloroplast envelope membrane in Arabidopsis. In addition, we found that EDS5 is preferentially expressed in epidermal cells. These findings suggest that EDS5 is responsible for transport of SA from chloroplasts to the cytoplasm in epidermal cells.

Determination of the optimal cell-penetrating peptide sequence for intestinal insulin delivery based on molecular orbital analysis with self-organizing maps.

Our recent work has shown that the intestinal absorption of insulin can be improved significantly by coadministration of cell-penetrating peptides (CPPs), especially penetratin. However, a relatively high dose of penetratin is required to adequately stimulate the intestinal absorption of insulin. Therefore, in this study, we sought to determine the CPP that most effectively enhanced intestinal insulin absorption. An in situ loop absorption study using 26 penetratin analogues suggested that the chain length, hydrophobicity, and amphipathicity of the CPPs, as well as their basicity, contribute to their absorption-enhancing efficiency. Moreover, a molecular orbital method with self-organizing maps (SOMs) classification suggested that multiple factors, including the molecular weight, basicity, the lowest unoccupied molecular orbital energy, absolute hardness, and chemical potential of CPPs, are associated with their effects on intestinal insulin absorption. Furthermore, the new CPPs proposed by SOM clustering had a marked capacity to interact with insulin, and their ability to enhance insulin absorption was much stronger than that of the original penetratin. Therefore, the peptide sequence that optimally enhances intestinal insulin absorption could be defined by SOM with the molecular orbital method, and our present work emphasizes the utility of such methodologies in the development of effective drug delivery systems.

Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity.

Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction, and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localizations of phototropins are implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localization pattern to the chloroplast photorelocation movement. First, the fusion of a nuclear localization signal with phot2, which effectively reduced the amount of phot2 in the cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localized phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fractions of phot2, and of phot1 to a lesser extent, were localized on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which was previously shown to be defective in blue light-induced Golgi localization and avoidance response, affected the localization pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking from the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.

Menthosomes, novel ultradeformable vesicles for transdermal drug delivery: optimization and characterization.

Menthosomes, novel deformable carriers for the enhancement of transdermal delivery are introduced in this study. Meloxicam (MX)-loaded menthosomes were formulated, and their physicochemical characteristics and skin permeability were evaluated. A two-factor spherical and second-order composite experimental design was used to prepare the formulation of the menthosomes. Ten formulations of menthosomes composed of a phospholipid as the lipid bilayer carrier, cholesterol (Chol) as a stabilizer and cetylpyridinium chloride (CPC) and L-menthol as penetration enhancers were prepared. The amounts of Chol and CPC were selected as causal factors. Physicochemical characteristics (particle size, size distribution, zeta potential, elasticity and drug content) and an in vitro skin-permeation study of meloxicam-loaded menthosomes were evaluated. The concentrations of MX that permeated the skin at 2-12 h and the flux were selected as response variables. The optimal formulation was estimated using a nonlinear response-surface method incorporating thin-plate spline interpolation. The experimental values were very close to the values predicted by the computer programs in this study. A Bayesian network analysis was applied to gain a mechanistic understanding of the relationships between causal factors and response variables.

Relationship between diffusivity of water molecules inside hydrating tablets and their drug release behavior elucidated by magnetic resonance imaging.

We reported previously that sustained release matrix tablets showed zero-order drug release without being affected by pH change. To understand drug release mechanisms more fully, we monitored the swelling and erosion of hydrating tablets using magnetic resonance imaging (MRI). Three different types of tablets comprised of polyion complex-forming materials and a hydroxypropyl methylcellulose (HPMC) were used. Proton density- and diffusion-weighted images of the hydrating tablets were acquired at intervals. Furthermore, apparent self-diffusion coefficient maps were generated from diffusion-weighted imaging to evaluate the state of hydrating tablets. Our findings indicated that water penetration into polyion complex tablets was faster than that into HPMC matrix tablets. In polyion complex tablets, water molecules were dispersed homogeneously and their diffusivity was relatively high, whereas in HPMC matrix tablets, water molecule movement was tightly restricted within the gel. An optimal tablet formulation determined in a previous study had water molecule penetration and diffusivity properties that appeared intermediate to those of polyion complex and HPMC matrix tablets; water molecules were capable of penetrating throughout the tablets and relatively high diffusivity was similar to that in the polyion complex tablet, whereas like the HPMC matrix tablet, it was well swollen. This study succeeded in characterizing the tablet hydration process. MRI provides profound insight into the state of water molecules in hydrating tablets; thus, it is a useful tool for understanding drug release mechanisms at a molecular level.

Reliability evaluation of nonlinear design space in pharmaceutical product development.

Formulation design space of indomethacin tablets was investigated using a nonlinear response surface method incorporating multivariate spline interpolation (RSM-S). In this study, a resampling method with replacement was applied to evaluate the reliability of border on the design space estimated by RSM-S. The quantities of lactose, cornstarch, and microcrystalline cellulose were chosen as the formulation factors. Response surfaces were estimated using RSM-S, and the nonlinear design space was defined under the restriction of more than 3 kgf hardness and more than 70% dissolution 30 min before and after an accelerated test. The accuracy of the resampling method was elucidated and high correlation coefficients were produced. However, the distribution of the border on the design space generated by the resampling method was far from normal, and the confidence interval of the border was estimated using a nonparametric percentile technique. Consequently, the reliability of the design space was decreased by approaching the edge of the experimental design. RSM-S and this resampling method might be useful for estimating the reliability of nonlinear design space.

One- and two-dimensional blue native-PAGE and immunodetection of low-abundance chloroplast membrane protein complexes.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful method for separating protein complexes from biological membranes under native conditions. BN-PAGE provides much higher resolution than gel filtration or sucrose density gradient centrifugation, and it can be used to estimate the molecular mass of protein complexes. First, membrane protein complexes need to be solubilized with a mild nonionic detergent such as digitonin or dodecyl maltoside. Coomassie brilliant blue G-250, a negatively charged dye that binds to the surface of the solubilized complexes, is then added so these can be resolved according to their size by non-denaturing (native) electrophoresis. BN-PAGE can be combined with a second dimension SDS-PAGE step (two-dimensional (2D)-BN/SDS-PAGE), so that the subunits making up these complexes are also separated according to their size. Here, we present our 2D-BN/SDS-PAGE method, and subsequent immunoblotting method, for the detection of relatively low-abundance proteins from plant chloroplasts.

Modeling of latent structure of indomethacin solid dispersion tablet using Bayesian networks.

BACKGROUND: When designing pharmaceutical products, the relationships between causal factors and pharmaceutical responses are intricate. A Bayesian network (BN) was used to clarify the latent structure underlying the causal factors and pharmaceutical responses of a tablet containing solid dispersion (SD) of indomethacin (IMC). METHOD: IMC, a poorly water-soluble drug, was tested with polyvinylpyrrolidone as the carrier polymer. Tablets containing a SD or a physical mixture of IMC, different quantities of magnesium stearate, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose, and subjected to different compression force were selected as the causal factors. The pharmaceutical responses were the dissolution properties and tensile strength before and after the accelerated test and a similarity factor, which was used as an index of the storage stability. RESULT: BN models were constructed based on three measurement criteria for the appropriateness of the graph structure. Of these, the BN model based on Akaike's information criterion was similar to the results for the analysis of variance. To quantitatively estimate the causal relationships underlying the latent structure in this system, conditional probability distributions were inferred from the BN model. The responses were accurately predicted using the BN model, as reflected in the high correlation coefficients in a leave-one-out cross-validation procedure. CONCLUSION: The BN technique provides a better understanding of the latent structure underlying causal factors and responses.

Latent structure analysis in pharmaceutical formulations using Kohonen's self-organizing map and a Bayesian network.

A latent structure analysis of pharmaceutical formulations was performed using Kohonen's self-organizing map (SOM) and a Bayesian network. A hydrophilic matrix tablet containing diltiazem hydrochloride (DTZ), a highly water-soluble model drug, was used as a model formulation. Nonlinear relationship correlations among formulation factors (oppositely charged dextran derivatives and hydroxypropyl methylcellulose), latent variables (turbidity and viscosity of the polymer mixtures and binding affinity of DTZ to polymers), and release properties [50% dissolution times (t50s) and similarity factor] were clearly visualized by self organizing feature maps. The quantities of dextran derivatives forming polyion complexes were strongly related to the binding affinity of DTZ to polymers and t50s. The latent variables were classified into five characteristic clusters with similar properties by SOM clustering. The probabilistic graphical model of the latent structure was successfully constructed using a Bayesian network. The causal relationships among the factors were quantitatively estimated by inferring conditional probability distributions. Moreover, these causal relationships estimated by the Bayesian network coincided well with estimations by SOM clustering, and the probabilistic graphical model was reflected in the characteristics of SOM clusters. These techniques provide a better understanding of the latent structure between formulation factors and responses in DTZ hydrophilic matrix tablet formulations.

In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV.

Protein translocation across the inner envelope of plastids is mediated by the TIC (translocon at the inner envelope membrane of chloroplasts) protein translocation machinery. Tic20 has been shown to function as a central component of TIC machinery. The Arabidopsis genome encodes four Tic20 homologous proteins, AtTic20-I, AtTic20-II, AtTIC20-IV and AtTic20-V, among which only AtTic20-I has been extensively characterized and demonstrated to be essential for protein import into chloroplasts. AtTic20-I is more closely related to AtTic20-IV than to AtTic20-II or AtTic20-V, whereas AtTic20-II and AtTic20-V show higher similarities to each other than to AtTic20-I or AtTic20-IV. Here, we show that AtTic20-IV is expressed mainly in roots whereas AtTic20-I is more abundant in shoots than in roots. Although AtTic20-IV is dispensable for viability in the wild-type background, interestingly, expression of AtTic20-IV is markedly elevated in both shoots and roots in the tic20-I knockout mutant that exhibits severe albino and seedling-lethal phenotypes. The albino tic20-I seedlings do not accumulate any of the photosynthetic proteins analyzed, but the plastids can still import non-photosynthetic housekeeping proteins. This residual import ability of the tic20-I mutant can be attributed to partial compensation by the elevated expression of AtTic20-IV, since a double knockout mutant of AtTic20-I and AtTic20-IV exhibits more severe embryonic lethality. Further overexpression of AtTic20-IV in the tic20-I mutant can only marginally rescue the accumulation of photosynthetic proteins in the albino seedlings. These data demonstrate an absolute requirement of at least one of the two closely related Tic20 proteins in protein translocation across the inner envelope of plastids and also suggest their distinct substrate preferences.

Self-organizing map analysis using multivariate data from theophylline powders predicted by a thin-plate spline interpolation.

The quality by design concept in pharmaceutical formulation development requires establishment of a science-based rationale and a design space. We integrated thin-plate spline (TPS) interpolation and Kohonen's self-organizing map (SOM) to visualize the latent structure underlying causal factors and pharmaceutical responses. As a model pharmaceutical product, theophylline powders were prepared based on the standard formulation. The angle of repose, compressibility, cohesion, and dispersibility were measured as the response variables. These responses were predicted quantitatively on the basis of a nonlinear TPS. A large amount of data on these powders was generated and classified into several clusters using an SOM. The experimental values of the responses were predicted with high accuracy, and the data generated for the powders could be classified into several distinctive clusters. The SOM feature map allowed us to analyze the global and local correlations between causal factors and powder characteristics. For instance, the quantities of microcrystalline cellulose (MCC) and magnesium stearate (Mg-St) were classified distinctly into each cluster, indicating that the quantities of MCC and Mg-St were crucial for determining the powder characteristics. This technique provides a better understanding of the relationships between causal factors and pharmaceutical responses in theophylline powder formulations.

A statistical approach to the development of a transdermal delivery system for ondansetron.

Transdermal delivery of drugs has gained attention as an alternative to intravenous and oral methods of delivery. However, the skin permeation of drugs is generally poor. To overcome this problem, many permeation enhancers have been developed. In this study, ondansetron hydrogels were prepared, and their skin permeation and pharmacological effects were evaluated in mice. To prepare the hydrogels, a Box-Behnken design was introduced. Fifteen formulations of ondansetron hydrogels composed of hydroxyethylcellulose and hydroxypropylcellulose as gel bases, l-menthol as a penetration enhancer and isopropanol (IPA), N-methyl-2-pyrrolidone (NMP) and water as a solvent were prepared. The quantities of IPA (X(1)), l-menthol (X(2)) and NMP (X(3)) were selected as causal factors. We performed an in vitro skin permeation study and an in vivo skin irritation study with the test hydrogels. The flux and the total irritation score were selected as response variables. The optimal formulation, one that has an appropriate penetration and an acceptable skin irritation score, was estimated using a nonlinear response surface method incorporating thin-plate spline interpolation. The optimal formulation also delivered the desired pharmacological activity. These results indicated the feasibility of delivering ondansetron transdermally.

Role of individual test samples in optimal solutions in pharmaceuticals predicted using a nonlinear response surface method.

Establishing a method to evaluate the reliability of an optimal solution is an exciting challenge for the nonlinear response surface method. We reported previously that the bootstrap (BS) resampling technique and Kohonen's self-organizing map are promising tools for meeting this challenge. To understand the usefulness of these techniques further, we employed a formulation optimization study of photocrosslinked polyacrylic acid (PAA) hydrogel as a case study. In a series of experiments, a large number of optimal solutions were generated with BS resampling and they were classified into three distinct clusters with SOM clustering. Using analysis of Bayesian estimation, we clarified the mode of generating clusters; e.g., cluster 2 was distinguished by the difference in features between the BS optimal solutions and the original optimal solution, whereas cluster 3 was distinguished by the substantial change in the shape of the response surfaces. We concluded that cluster 1 represents the global optimal solution, and then estimated 95% confidence intervals of the optimal solutions using the BS optimal solutions. These findings prove that our method is a valid approach for evaluating nonlinear optimal solutions. This method has applications for establishing a science-based rationale for, and a design space in, pharmaceutical formulation development.

Multivariate statistical approach to optimizing sustained-release tablet formulations containing diltiazem hydrochloride as a model highly water-soluble drug.

A multivariate statistical technique was applied to designing a tablet for the sustained release over 24h of diltiazem hydrochloride, a model highly water-soluble drug. Tablets of a hydrophilic matrix composed of dextran derivatives and hypromellose were prepared. The formulations were optimized using a nonlinear-response surface method incorporating thin-plate spline interpolation (RSM-S). A bootstrap (BS) resampling method was used to estimate the confidence intervals of the optimal formulations. The response surfaces estimated by RSM-S visualized the effects of the formulation factors, and the optimal release profile for diltiazem was predicted quantitatively as a function of the quantities of the formulation factors, using RSM-S. The simultaneous optimal solutions and their confidence intervals were estimated using RSM-S and BS resampling. The results clearly indicate nonlinear relationships between the formulation factors and the response variables. The observed responses of the optimal preparation coincided well with the predicted responses. The optimal hydrophilic matrix tablet allowed almost zero-order release of diltiazem hydrochloride for 24h. In conclusion, an oral sustained-release tablet formulation, active over a long period, was successfully optimized using RSM-S, and the reliability of the optimal solution was evaluated using BS resampling.