Insights into taxadiene synthase catalysis and promiscuity facilitated by mutability landscape and molecular dynamics.

MAIN CONCLUSION: Protein modeling, carbocation docking, and molecular dynamics along with structure-based mutability landscapes provided insight into taxadiene synthase catalysis (first step of the anticancer Taxol biosynthesis), protein structure-function correlations, and promiscuity. Plant terpenes belong to one of the largest and most diverse classes of natural products. This diversity is driven by the terpene synthase enzyme family which comprises numerous different synthases, several of which are promiscuous. Taxadiene synthase (TXS) is a class I diterpene synthase that catalyzes the first step in the biosynthesis pathway of the diterpene Taxol, an anticancer natural product produced by the Taxus plant. Exploring the molecular basis of TXS catalysis and its promiscuous potential garnered interest as a necessary means for understanding enzyme evolution and engineering possibilities to improve Taxol biosynthesis. A catalytically active closed conformation TXS model was designed using the artificial intelligence system, AlphaFold, accompanied by docking and molecular dynamics simulations. In addition, a mutability landscape of TXS including 14 residues was created to probe for structure-function relations. The mutability landscape revealed no mutants with improved catalytic activity compared to wild-type TXS. However, mutations of residues V584, Q609, V610, and Y688 showed high degree of promiscuity producing cembranoid-type and/or verticillene-type major products instead of taxanes. Mechanistic insights into V610F, V584M, Q609A, and Y688C mutants compared to the wild type revealed the trigger(s) for product profile change. Several mutants spanning residues V584, Q609, Y688, Y762, Q770, and F834 increased production of taxa-4(20),11(12)-diene which is a more favorable substrate for Taxol production compared to taxa-4(5),11(12)-diene. Finally, molecular dynamics simulations of the TXS reaction cascade revealed residues involved in ionization, carbocation stabilization, and cyclization ushering deeper understanding of the enzyme catalysis mechanism.

Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.

Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory.

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.

Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering.

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis. Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene.

A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis.

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C30 carotenoid production.

Metabolic Engineering of Bacillus subtilis Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production.

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the B. subtilis genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (dxs, ispD, ispF, ispH, ispC, ispE, ispG) together with ispA (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of Pantoea ananatis) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of B. subtilis. The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in B. subtilis. We determined that the expression of GGPPS through the crtE gene is essential for the formation of sufficient precursor, GGPP, in B. subtilis as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.

Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering.

Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved kcat by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.

Insights into the Three-Dimensional Structure of Amorpha-4,11-diene Synthase and Probing of Plasticity Residues.

Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure-function relationships of all residues in the active site to obtain further insight into the ADS mechanism.

Metabolic engineering of Bacillus subtilis for terpenoid production.

Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.

Enhanced C30 carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes.

Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functionally and structurally diverse group of natural products from which numerous medicines and relevant fine chemicals are derived. The well-characterized and broadly used production organism Bacillus subtilis forms an ideal background for creating and studying novel synthetic routes. In comparison to other bacteria, B. subtilis emits the volatile compound isoprene, the smallest representative of isoprenoids, in high concentrations and thus represents an interesting starting point for an isoprenoid cell factory. In this study, the effect of systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway on isoprenoid production in B. subtilis was investigated. B. subtilis strains harboring a plasmid containing C30 carotenoid synthetic genes, crtM and crtN, were combined with pHCMC04G plasmids carrying various synthetic operons of the MEP pathway genes. The levels of produced carotenoids, diaponeurosporene and diapolycopene, were used as indication of the role of the various enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids can be increased significantly by overexpressing the MEP pathway enzymes. More broadly, the strains developed in this study can be used as a starting point for various isoprenoid cell factories.

Chemical constituents from Tribulus terrestris and screening of their antioxidant activity.

Two oligosaccharides (1,2) and a stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of Tribulus terrestris along with five known compounds (4-8). The structures of the compounds were established as O-ß-D-fructofuranosyl-(2→6)-α-D-glucopyranosyl-(1→6)-ß-D-fructofuranosyl-(2→6)-ß-D-fructofuranosyl-(2→1)-α-D-glucopyranosyl-(6→2)-ß-D-fructofuranoside (1), O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→2)-ß-D-fructofuranoside (2), 4,5-di-p-cis-coumaroylquinic acid (3) by different spectroscopic methods including 1D NMR ((1)H, (13)C and DEPT) and 2D NMR (COSY, TOCSY, HMQC and HMBC) experiments as well as ESI-MS analysis. This is the first report for the complete NMR spectral data of the known 4,5-di-p-trans-coumaroylquinic acid (4). The antioxidant activity represented as DPPH free radical scavenging activity was investigated revealing that the di-p-coumaroylquinic acid derivatives possess potent antioxidant activity so considered the major constituents contributing to the antioxidant effect of the plant.