Fermentative production of Vitamin E tocotrienols in Saccharomyces cerevisiae under cold-shock-triggered temperature control

Abstract

The diverse physiological functions of tocotrienols have listed them as valuable supplementations to α-tocopherol-dominated Vitamin E products. To make tocotrienols more readily available, tocotrienols-producing S. cerevisiae has been constructed by combining the heterologous genes from photosynthetic organisms with the endogenous shikimate pathway and mevalonate pathway. After identification and elimination of metabolic bottlenecks and enhancement of precursors supply, the engineered yeast can produce tocotrienols at yield of up to 7.6 mg/g dry cell weight (DCW). In particular, proper truncation of the N-terminal transit peptide from the plant-sourced enzymes is crucial. To further solve the conflict between cell growth and tocotrienols accumulation so as to enable high-density fermentation, a cold-shock-triggered temperature control system is designed for efficient control of two-stage fermentation, leading to production of 320 mg/L tocotrienols. The success in high-density fermentation of tocotrienols by engineered yeast sheds light on the potential of fermentative production of vitamin E tocochromanols.

Microbial Cell Factories for Green Production of Vitamins

“…Further, the output of vitamin products in different dimensions will be increased by transforming the complex and multi-enzyme pathways required for the production of vitamins, establishing microbial flora with controllable functions and stability, and application of some advanced engineering technology, such as the cold-shock-triggered temperature control system, dynamic control of gene expression systems, different types of biosensors, cell-free systems and computer-aided design, etc. (Koo et al, 2020;Marucci et al, 2020;Sachsenhauser et al, 2020;Shen et al, 2020;Glasscock et al, 2021). Additionally, the modular and orthogonal strategies are increasingly supporting the construction of vitamin cell factories (Liu et al, 2015).…”

“…To balance cell growth and product synthesis, Shen et al (2020) recently combined heterologous genes from photosynthetic organisms with the endogenous shikimate and mevalonate pathways (MEP) to construct a strain of S. cerevisiae that produces tocotrienols (Figure 3A). By incorporating a newly designed cold-shock-triggered temperature control system, the phased control of cell biomass and tocotrienol accumulation by the engineered strains was successfully realized.…”

“…By incorporating a newly designed cold-shock-triggered temperature control system, the phased control of cell biomass and tocotrienol accumulation by the engineered strains was successfully realized. The final total tocotrienol titer reached 320 mg/L in a 5 L fermenter, which laid the foundation for the production of natural vitamin E in a fully fermentative process (Figure 3B; Shen et al, 2020).…”

Recent trends in biocatalysis

“…299 The engineering of S. cerevisiae to produce artemisinic acid (precursor for the anti-malarial drug artemisinin) by the Keasling group and Amyris is a landmark in metabolic engineering/ synthetic biology. 300 More recent progress includes engineering S. cerevisiae for the production of farnesene (bulk chemical) 301 and tocotrienols (vitamin E), 302 as well as engineering E. coli for the production of taxadiene (precursor for Taxol) 303 and viridiflorol (fine chemical). 304 However, achieving a productive synthesis of terpenoids (e.g., 410 g L À1 ) in microbes is often very challenging and requires extensive engineering and optimisation efforts due to the toxicity of intermediates/products and the complex regulation of native pathways.…”

Advances in production and structural derivatization of the promising molecule ursolic acid

“…[ 100 ] In conducting tocotrienol synthesis, a cold shock triggered temperature control system based on cold temperature (24°C) activated Gal4M9 system combined with PGAL system was designed to control the two‐stage fermentation effectively, further resolving the contradiction between cell growth and tocotrienol accumulation to achieve high‐density fermentation. [ 101 ] In response to the problem of low heat resistance of cell factories during fermentation, Jia et al. developed an intelligent microbial thermoregulation engine (IMHeRE) through heat tolerant systems by different heat shock proteins (HSPs).…”