Insights into the structure-function relations of amorpha-4,11- diene synthase

Terpenoids are considered the biggest and most varied class of natural products. They are involved with a wide range of applications from food to cosmetics to medicine. All terpenoids originate from the same C5 isoprene building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), but have a diverse range of complex chemical structures with multiple chiral centers and/or chemical modi cations

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 C-30 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 C-30 carotenoid production

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

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

Hepatoprotective and Antioxidant Activities of Tribulus Terrestris

Tribulus terrestris L. has been used in folk medicine throughout history. The present study examined the acute toxicity of the total ethanolic extract of T. Terrestris followed by investigation of the hepatoprotective activity of the total ethanolic extract and different fractions of the aerial parts of the plant compared to silymarin against carbon tetrachloride- induced hepatic damage in rats. In addition, in vivo antioxidant activity was examined and linked to the previous in vitro DPPH free radical scavenging activity investigation. This study established the plant’s safety and the hepatoprotective effect of the total ethanolic extract of the aerial parts of the plant and its different fractions due to significant decrease in CCl4- induced rise in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin in rats. Treatment with the ethyl acetate fraction significantly reduced oxidative stress in CCl4- intoxicated rats, as evident by a decrease inmalondialdehyde (MDA) content associated with elevation of hepatic reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hence, this hepatoprotective effect could be due to the antioxidant activity of the plant which is mainly imparted by the two major di-p-coumaroylquinic acid derivatives isolated from the ethyl acetate fraction

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/I. 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

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

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. Abstract: 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. Graphical abstract: (Figure presented.)</p