Dual cytoplasmic‐peroxisomal engineering for high‐yield production of sesquiterpene α‐humulene in Yarrowia lipolytica

Abstract

The sesquiterpene α-humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti-inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α-humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α-humulene production. First, a chassis with high α-humulene output in the cytoplasm was constructed by integrating α-humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl-CoA pathways.Subsequently, the strategy of dual cytoplasmic-peroxisomal engineering was adopted in Y. lipolytica; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α-humulene, a 100-fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu 2+ -repressible promoters was firstly established, which significantly improved the α-humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α-humulene in a 5-L bioreactor, 6.8-fold higher than the highest α-humulene titer reported before this study.Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica.

Enhancing Trans-Nerolidol Productivity in Yarrowia lipolytica by Improving Precursor Supply and Optimizing Nerolidol Synthase Activity

“…To circumvent nonproductive consumption of intermediates and reduce cell toxicity caused by excessive metabolites, compartmentalizing is usually a good solution for heterologous biosynthesis. , The peroxisome is considered to have a high content of the acetyl-CoA pool from the β-oxidation of fatty acids, rendering it more propitious for MVA pathway compartmentalization . Here, the enhanced peroxisome targeting sequence (ePTS1) was employed to target enzymes of the trans -nerolidol biosynthetic pathway into the peroxisome.…”

“…47,48 The peroxisome is considered to have a high content of the acetyl-CoA pool from the β-oxidation of fatty acids, rendering it more propitious for MVA pathway compartmentalization. 6 Here, the enhanced peroxisome targeting sequence (ePTS1) 49 was employed to target enzymes of the trans-nerolidol biosynthetic pathway into the peroxisome. To validate the efficiency of ePTS1 in Y. lipolytica, the N-terminus of the peroxisomal matrix protein PEX22 (111) from S. cerevisiae, which is anchored to the peroxisomal membrane, was assembled to the red fluorescent protein (mRuby) for visualizing the peroxisomal location in cells, 50 and the C-terminal ePTS1 was fused with the green fluorescent protein (hrGFP).…”

“…For example, harnessing the organelle-derived acetyl-CoA was proven to be able to enhance acetyl-CoA availability and successfully increased the titers of α-humulene and amorpha-4,11-diene to 21.7 g/L and 427 mg/L in Yarrowia lipolytica and S. cerevisiae with the combination of cytoplasmic and peroxisomal or mitochondrial engineering. , Besides, integration of strengthening the MVA pathway and repressing the competitive sterol biosynthesis strongly supported the FPP flux toward target sesquiterpene production in S. cerevisiae or other yeasts, such as trans -nerolidol, − amorphadiene, , bisabolene, farnesene, , α-santalene, , caryophyllene, and longifolene .…”

“…Due to these outstanding traits, Y. lipolytica has been engineered for efficiently producing terpenoid compounds and fatty acid-derived chemicals. ,− ,,,− However, the cytosolic acetyl-CoA in Y. lipolytica is mainly directed toward lipid biosynthesis instead of the MVA pathway .…”

High-yield α-humulene production in Yarrowia lipolytica from waste cooking oil based on transcriptome analysis and metabolic engineering

“…For example, Zhang et al overexpressed the entire α-humulene synthesis pathway and the rate-limiting enzymes in C. tropicalis , which achieved 4115.42 mg/L α-humulene in fed-batch fermentation [ 5 ]. Furthermore, Zhang et al and Guo et al employed organelle engineering in S. cerevisiae and Y. lipolytica respectively, which realized a high yield of α-humulene [ 6 , 8 , 9 ]. However, the reduction of production expenses is also an extremely critical factor in the sustainable and industrial production of α-humulene.…”