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.

Increasing demands for bioactive compounds have motivated researchers to employ microorganisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized as safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up-to-date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as well as promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoids cell factory development.

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.

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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.

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Indonesia is highly diverse in culture, ethnicity, religion, and natural resources. The diversity is also obvious in its cuisine. Soto is an example of an iconic Indonesian dish found in many parts of the country. Despite sharing a similar name, the recipes vary according to their place of origin. Until now, how many variants of Soto are in Indonesia is not exactly known. This review aimed to discuss the characteristics of Soto based on ingredients. Soto is a soup composed of spices-flavored broth and various solid ingredients, such as vegetables and protein-and carbohydrate-rich foods. Every region in Indonesia has a different recipe of Soto; some use non-turmeric soup or coconut milk soup. The carbohydrate ingredients are mainly soun (cellophane noodle) and potato. The protein ingredients are mainly composed of chicken, egg, and beef. Vegetables consist of bean sprouts, tomatoes, and cabbage. Fried garlic, shallot, spring onion, and crackers are added as a garnish. The diversity of the ingredient makes the distinction among Soto: diversity of locality and diversity of ingredients. Approximately 46 types of Soto are reviewed in this article. Most of the Soto recipes use chicken, bean sprouts, soun, boiled egg, garlic, white pepper, red onion, ginger, and bay leaf as the main ingredients. Soto becomes an identity of its geographical origins and has distinction based on the main recipes.