Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil

Abstract

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., Escherichia coli) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in E. coli, and the best engineered E. coli NA-IS 3D strain could produce α-santalene at a titer of 412 mg•L −1 . Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indoleassociated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of tnaA could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg•L −1 in E. coli tnaA − NA-IS 3D . Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

“…9 Recently, SAS was rationally engineered to produce different santalene isomers (e.g., α-santalene and βsantalene). 10 In addition to S. cerevisiae, several other chassis organisms have been engineered for the biosynthesis of αsantalene, such as Escherichia coli, 11 Yarrowia lipolytica, 12 and Nicotiana tabacum. 13 While S. cerevisiae is the preferred cell factory for the biosynthesis of natural products, 14 the methylotrophic yeast Komagataella phaffii (previously known as Pichia pastoris) demonstrates the advantages in expressing heterologous genes to high levels.…”

“…In addition to S. cerevisiae, several other chassis organisms have been engineered for the biosynthesis of α-santalene, such as Escherichia coli, Yarrowia lipolytica, and Nicotiana tabacum …”

Biotechnologies in Perfume Manufacturing: Metabolic Engineering of Terpenoid Biosynthesis

“…Recently, Wang et al ( 2021) [44] and Zhang et al ( 2022) [45] focused on the production of the α-santalol precursor α-santalene, using E. coli as host. The E. coli gene encoding FPP synthase (IspA) and a plant (Clausena lansium) α-santalene synthase gene (sts) were combined into a single operon, and associated with a heterologous MVA module both under an IPTG-inducible promoter [44].…”

“…Recently, Wang et al ( 2021) [44] and Zhang et al ( 2022) [45] focused on the production of the α-santalol precursor α-santalene, using E. coli as host. The E. coli gene encoding FPP synthase (IspA) and a plant (Clausena lansium) α-santalene synthase gene (sts) were combined into a single operon, and associated with a heterologous MVA module both under an IPTG-inducible promoter [44]. On this synthetic system, different combinatorial set of ribosome binding sites were explored to balance expression of coded proteins and to improve the isoprenyl diphosphate production and the synthesis of α-santalene in E. coli, reaching a titer of 412 mg/L in a flask culture [44].…”

Reassessing acetyl‐CoA supply and NADPH availability for mevalonate biosynthesis from glycerol in Escherichia coli

“…It is essential to build stable and efficient cell factories, which can sustain affordable bioprocess. The mevalonate pathway has been adopted to synthesize isoprenoids in the engineered microbial hosts (Martin et al, 2003; Meng et al, 2020; Peralta‐Yahya et al, 2011; Y. Wang et al, 2021). This pathway initiates mevalonate synthesis with a two‐step condensation of acetyl‐CoA by acetoacetyl‐CoA thiolase and 3‐hydroxy‐3‐methylglutaryl CoA (HMG‐CoA) synthase to HMG‐CoA, and HMG‐CoA reductase catalyzes a reductive diacylation of HMG‐CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) to generate mevalonate (Figure 1), which can undergo further phosphorylation and decarboxylation to form isopentenyl diphosphate and dimethylallyl diphosphate, the ubiquitous precursors for diverse isoprenoids (Liao et al, 2016; Miziorko, 2011).…”