Construction of Canthaxanthin-Producing Yeast by Combining Spatiotemporal Regulation and Pleiotropic Drug Resistance Engineering

Abstract

The ketocarotenoid canthaxanthin has important applications in the feed industry. Its biosynthesis using microbial cell factories is an attractive alternative to the current chemical synthesis route. Canthaxanthin-producing Saccharomyces cerevisiae was constructed by introducing the β-carotene ketolase variant OBKTM29 into a β-carotene producer. Subcellular re-localization of OBKTM29 was explored, together with copy number adjustment both in the cytoplasm and on the periplasmic membrane, to accelerate the conversion of β-carotene to canthaxanthin. Moreover, pleiotropic drug resistance (PDR) regulators Pdr1 and Pdr3 were overexpressed to improve the stress tolerance of the yeast strain, leading to obviously enhanced canthaxanthin production. The synthetic pathway was then regulated by a temperature-responsive GAL system to separate product synthesis from cell growth. Finally, 1.44 g/L canthaxanthin was harvested in fed-batch fermentation. This work demonstrated the power of spatial and temporal regulation and the efficiency of PDR engineering in heterologous biosynthesis.

Efficient Secretory Production of δ-Tocotrienol by Combining Pathway Modularization and Transportation Engineering

“…cerevisiae significantly boosted β-carotene production and secretion, while overexpression of the transcriptional activator PDR3 in the PDR family led to enhanced production of β-carotene, astaxanthin, and canthaxanthin in S. cerevisiae . Our previous study found that overexpressing PDR11 and YOL075C significantly increased the production and secretion of tocotrienols .…”

“…16 Overexpression of PDR10, SNQ2, and YOL075C in S. cerevisiae significantly boosted β-carotene production and secretion, 17 while overexpression of the transcriptional activator PDR3 in the PDR family led to enhanced production of β-carotene, astaxanthin, and canthaxanthin in S. cerevisiae. 18 Our previous study found that overexpressing PDR11 and YOL075C significantly increased the production and secretion of tocotrienols. 19 However, the efficiency of the transporters depends on the specific structures of the cargos, so the best transporter(s) for δ-tocotrienol may be different from those for the mixture of tocotrienols.…”

“…Similar observations have been reported for canthaxanthin production where the overexpression of PDR1 and PDR3 significantly promoted the synthesis of canthaxanthin. 18 However, the overexpression of PDR1 resulted in 23.7% decrease in biomass, which could be due to metabolic stress caused by overexpression of PDR1controlled membrane proteins, such as the PDR transporters (Figure S4). Among the PDR transporters, overexpression of PDR10 led to highest increase in extracellular δ-tocotrienol production (56.9%), but the total δ-tocotrienol production decreased by 9.3% and the biomass decreased by 14.61% (Figure 9A).…”

Compartmentalization engineering of yeasts to overcome precursor limitations and cytotoxicity in terpenoid production

“…The plasma membrane provides storage space for the accumulation of heterologous hydrophobic target compounds (Wu et al, 2017), such as carotenoids (Liu et al, 2016). Targeting the key enzymes to the plasma membrane can effectively alleviate the toxicity of hydrophobic compounds and increase production (Bian et al, 2021;Chen M. et al, 2022). Ye et al (2018) fused a β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) and targeted the fusion protein to the cell membrane, which resulted in a 215.4% increase in astaxanthin production.…”

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…In addition, due to the unique molecular structure of terpenoids, their chemical synthesis routes often involve tedious synthesis steps, leading to low final yields and environmental concerns . Attempts have been made to synthesize terpenoids by introducing the plant-origin pathway genes into model microbial chassis, such as Escherichia coli , and Saccharomyces cerevisiae. − But prokaryotes such as E. coli often have difficulty in expressing P450 enzymes properly, which play important roles in the synthesis of many terpenoids.…”