Combined metabolic and enzymatic engineering for de novo biosynthesis of δ-tocotrienol in Yarrowia lipolytica

Abstract

δ-Tocotrienol, an isomer of vitamin E with anti-inflammatory, neuroprotective and anti-coronary arteriosclerosis properties, is widely used in health care, medicine and other fields. Microbial synthesis of δ-tocotrienol offers significant advantages over plant extraction and chemical synthesis methods, including increased efficiency, cost-effectiveness and environmental sustainability. However, limited precursor availability and low catalytic efficiency of key enzymes remain major bottlenecks in the biosynthesis of δ-tocotrienol. In this study, we assembled the complete δ-tocotrienol biosynthetic pathway in Yarrowia lipolytica and enhanced the precursor supply, resulting in a titre of 102.8 mg/L. The catalytic efficiency of the rate-limiting steps in the pathway was then enhanced through various strategies, including fusion expression of key enzymes homogentisate phytyltransferaseand and tocopherol cyclase, semi-rational design of SyHPT and multi-copy integration of pathway genes. The final a δ-tocotrienol titre in a 5 L bioreactor was 466.8 mg/L following fed-batchfermentation. This study represents the first successful de novo biosynthesis of δ-tocotrienol in Y. lipolytica, providing valuable insights into the synthesis of vitamin E-related compounds.

Keywords: Fed-batch fermentation; Metabolic engineering; Yarrowia lipolytica; enzyme engineering; δ-tocotrienol.

Graphical abstract

Fig. 1

Biosynthetic pathway of δ-tocotrienol in Y. lipolytica. The δ-tocotrienol biosynthetic pathway is divided into three modules: the shikimate pathway (pink box), MVA pathway (beige box) and δ-tocotrienol biosynthesis pathway (blue box). Enzymes and intermediates involved include: ARO3, 3-deoxy-7-phosphoheptulonate synthase; ARO4, 3-deoxy-7-phosphoheptulonate syn-thase; ARO1, pentafunctional AROM polypeptide; ARO7, chorismite mutase; ARO8, aromatic aminotransferase I; ARO9, aromatic aminotransferase II; ERG10, acetyl-CoA C-acetyltransferase; ERG13, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase; tHMG1, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, diphosphomevalonate decarboxylase; IDI1, isopentenyl diphosphate delta-isomerase; ERG20, farnesyl pyrophosphate synthetase; GGPS, geranylgeranyl diphosphate synthase; HPD, hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyl transferase; VTE1, tocopherol cyclase; G6P, glucose-6-phosphate; 6PGL, 6-phosphoglucono-δ-lactone; Ru5P, ribulose 5-phosphate; R5P, ribose-5-phosphate; Xu5P, d-xylulose 5-phosphate; G3P, 3-phosphoglycerate; S7P, sedoheptulose-7-phosphate; F6P, fructose-6-phosphate; F-1,6BP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-arabino-heptulonate-7-phosphate; 4-HPP, 4-hydroxyphenylpyruvate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl pyrophosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl pyrophosphate; HGA, homogentisic acid; MGGBQ, 2-methyl-6-geranylgeranyl benzoquinol.

Fig. 2

Expression of δ-tocotrienol biosynthesis pathway enzymes from various sources. (A) Gene integration at distinct loci: HGA synthesis genes at the D17 locus; MGGBQ and δ-tocotrienol synthesis genes at the E4 locus. (B) Effects of expressing δ-tocotrienol biosynthesis pathway genes from different sources. "+" denotes gene integration into the chromosome; "—" denotes no integration. (C) HPLC chromatogram of the δ-tocotrienol standard. (D) HPLC chromatogram of the fermentation broth. Values are averages of three independent experiments. Error bars indicate standard deviation.

Fig. 3

Effect of enhancing the shikimic acid pathway on the synthesis of δ-tocotrienol. (A) Schematic diagram of the HGA biosynthesis pathway in Y. lipolytica. PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-arabino-heptulonate-7-phosphate; 4-HPP, 4-hydroxyphenylpyruvate; PPY, phenylpyruvate; HGA, homogentisic acid. (B) Combinatorial expression of the genes ARO1, ARO4, ARO7, ARO4^K221L and ARO7^G139S in Y. lipolytica. Values are averages of three independent experiments. Error bars indicate standard deviation.

Fig. 4

Effect of enhancing the MVA pathway on the synthesis of δ-tocotrienol. (A) Schematic diagram of GGPP synthesis via the endogenous MVA pathway in Y. lipolytica. HMG-CoA, 3-hydroxy-3-Menthyl-Glutaryl-CoA; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl pyrophosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl pyrophosphate. (B) Overexpression of key genes in the MVA pathway (RG12, YlGGPS, tHMG1 and IDI1). "+" indicates that the gene has been integrated into the strain's chromosome, while "—" indicates that the gene has not been integrated. (C) Comparison of the effects of GGPP synthases from Sulfolobus acidocaldarius and Xanthophyllomyces dendrorhous on the synthesis of δ-tocotrienol. "+" indicates that the gene has been integrated into the strain's chromosome, while "—" indicates that the gene has not been integrated. Values are averages of three independent experiments. Error bars indicate standard deviation.

Fig. 5

Effect of linker type in SyHPT-AtVTE1 fusions on δ-tocotrienol biosynthesis. Fusion protein linkers TPTP, (TPTP)₂ (TPTPTPTP), E3AK (EAAAK), (E3AK)₂ (EAAAKEAAAK), G4S (GGGGS), (G4S)₂ (GGGGSGGGGS), GSG and (GSG)₂ (GSGGSG) were tested. Values are averages of three independent experiments. Error bars indicate standard deviation.

Fig. 6

Structure of the SyHPT protein and preliminary mutation results. (A) Predicted structure of the SyHPT protein and amino acid residues in its active site pocket. Substrates are represented by blue sticks and amino acid residues by grey sticks. (B) Relative activity of alanine scanning mutants of SyHPT. Values are averages of three independent experiments. Error bars indicate standard deviation. (C) Mutation of lysine 77 to acidic and aromatic amino acids. Values are average of three independent experiments. Error bars indicate standard deviation. (D) Integration of SyHPT^K77Y and AtVTE1 mutants into the multi-copy sites of Yarrowia lipolytica, and complementation with LEU2 and URA3 markers. Values are averages of three independent experiments. Error bars indicate standard deviation. (E) Root mean squared deviation of atomic positions for WT (blue) and SyHPT^K77Y (red) proteins in 100 ns molecular dynamics (MD) simulations. (F) Number of hydrogen atoms in WT (blue) and SyHPT^K77Y (red) proteins.

Fig. 7

Fermentation of δ-tocotrienol by strain VE-23 in a 5 L bioreactor. Time courses are shown for glucose concentration, cell density, δ-tocotrienol and HGA production in fed-batch fermentation in a 5 L bioreactor at pH 5.0 with 20 % dissolved oxygen.