Elevated β-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica

Abstract

β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

Pareto optimal metabolic engineering for the growth‐coupled overproduction of sustainable chemicals

“…Liu et al (2021) suggested a modern strategy to optimize the β‐carotene production by constructing codon‐adapted genes and minimizing the intermediate accumulation, which plays an important role in metabolic balance. The metabolic balance means no accumulation of intermediates at the connecting node when combining upstream and downstream pathways.…”

Engineering of Yarrowia lipolytica for terpenoid production

“…Indeed, the cultivation of an engineered Y. lipolytica strain recently resulted in 39.5 g/L and 494 mg/g DCW β-carotene. These production metrics exceed what has been reported in scientific and patent literature on β-carotene production by recombinant and native microbes ( Costa Perez et al, 2017 ; M. Liu et al, 2021 ).

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“…The overexpression of GGPPSp resulted in a 4-fold increase in β-carotene titer ( Kildegaard et al, 2017 ). In another study, the expression of the archaeal Archaeoglobus fulgidus GGPPS increased carotenoid yield 2.6-fold, while the combined overexpression of ERG20p and native GGPPSp only increased carotenoid yield by 1.9-fold (M. Liu et al, 2021 ). Likewise, expression of Synechococcus sp.…”

“… Liu et al, 2019 ) α-farnesene Glucose Glycerol Po1f ↑FS-L-ERG20 ↑tScHMG1 ↑IDI ↑HMG ↑ERG19 2.57 g/L (bioreactor) (S. C. Liu et al, 2020 ) α-farnesene Oleic acid Po1f ↑VHb ↑MdFS-L-ERG20 ↑ERG12 ↑IDI ↑ERG8,19 10.2 g/L (bioreactor) (Y. Liu et al, 2021 ) β-farnesene Glucose ATCC 20460 ↑HMG ↑ERG12 ↑ACL1 ↑SeACS L641P ↑IDI ↑ERG20 ↑AaBFS 955 mg/L (glass tube) Arnesen et al (2020) β-farnesene Glucose Po1f ΔDGA1 ΔDGA2 ↑tHMG ↑BFS ↑ERG8,10,12, 13,19,20 IDI Δgut2 Δpox3,4,5,6 22.8 g/L (bioreactor) (T. Shi et al, 2021 ) α-humulene Glucose Po1f ↑POT1 ↑AcACHS2 PTS ↑RpHMG PTS ↑ANT1 ↑(ERG12,8,20,10,13,19) PTS ↑IDI PTS 3.2 g/L (bioreactor) Guo et al (2021) α-santalene Glucose ATCC 201249 ↑ClSTS ↑ERG8 ↑tHMG 27.92 mg/L (bioreactor) Jia et al (2019) Nootkatone Glucose ATCC 201249 ↑CnVS ↑CnCYP706M1-tAtATR1 ↑tScHMG ↑ERG20 Nootkatone: 978.2 μg/L Guo et al (2018) Valencene Valencene: 22.8 mg/L (shake flask) Valencene …”

“…Lastly, expression of Vitreoscilla hemoglobin (Vhbp) was demonstrated to improve α-farnesene production by 12.7%, likely by enhancing oxygen delivery to the cells (Y. Liu et al, 2021 ).…”