Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

Abstract

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO₂-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

Keywords: acetate metabolism; metabolic engineering; orthogonal pathway; peroxisome; triterpenoids.

Fig. 1.

Engineering the cytosolic metabolic pathway for squalene production in Y. lipolytica. (A) Schematic view of the metabolic pathway for squalene biosynthesis in the cytosol. The biosynthetic pathway of squalene starting from acetyl-CoA was divided into three modules: up-pathway (shown in red), mid-pathway (shown in purple), and down-pathway (shown in blue). ERG10, acetyl-CoA acetyltransferase. ERG13, 3-hydroxy-3-methylglutary-CoA (HMG-CoA) synthase. tHMGR, truncated HMG-CoA reductase. ERG12, mevalonate kinase. ERG8, phosphomevalonate kinase. ERG19, mevalonate pyrophosphate decarboxylase. IDI, IPP:DMAPP isomerase. ERG20, farnesyl pyrophosphate synthetase. ERG1, squalene synthase. MvaE and MvaS, from Enterococcus faecalis. IPP, isopentenyl diphosphate. DMAPP, dimethylallyl diphosphate. FPP, farnesyl pyrophosphate. (B) Each module was overexpressed in Y. lipolytica, individually and in combination. Squalene titers were measured after 48 h of fermentation in YPD media. (C and D) Squalene titers (C) and intracellular content (D) were significantly enhanced when YPD media were supplemented with 0.2 M PBS. Samples in media without supplemented PBS were measured at 48-h cultivation, whereas samples in supplemented PBS media were measured at 72-h cultivation. (E) Fermentation time-course profiles indicated that PBS-added fermentation reached the highest squalene titers at 72-h cultivation, yet, at 48 h in the PBS-unconditioned fermentation. Statistical significance was tested using the two-sided Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001. All data are represented as mean ± SD of three biologically independent experiments.

Fig. 2.

Construction of the sequestered biosynthetic route for squalene in the peroxisome. (A) Schematic illustration of the orthogonal pathway for squalene production in the peroxisome. Conventional biosynthesis of squalene in Y. lipolytica relies on the endogenous cytosolic MVA pathway (shown in black), which tightly couples with sterol synthesis. Here, we build an orthogonal pathway for squalene synthesis in the peroxisome by introducing the complete squalene pathway starting from acetyl-CoA (shown in blue), in which the MVA pathway was assembled to harvest peroxisomal acetyl-CoA. (B) The entire squalene pathway was sequentially assembled in the peroxisome or overexpressed in the cytosol, respectively. Squalene titers were measured after 72 h of fermentation. (C) Time-course profiles of squalene content and glucose concentration in media of strain Sq06 overexpressing the cytosolic pathway and strain Sq10 harboring the peroxisomal pathway. Statistical significance was tested using the two-sided Student’s t test; ***P < 0.001; ns, not significant. All data are represented as mean ± SD of three biologically independent experiments.

Fig. 3.

Converting lipid by-product to peroxisomal squalene through lipid metabolism. (A) Schematic illustration of intracellular lipid metabolism, including its biosynthesis, hydrolysis, and degradation. Overexpressed genes in this study are shown in blue. PYC1, pyruvate carboxylase. YHM2, mitochondrial citrate carrier. MmACL, ATP:citrate lyase from Mus musculus. ACC1, acetyl-CoA carboxylase. GPD1, NAD⁺-dependent G3P dehydrogenase. DGA1, diacylglycerol acyltransferase. tlTGL, lipase from T. lanuginosus. POX1-6, acyl-CoA oxidases. MFE1, multifunctional β-oxidation protein. POT1, 3-ketoacyl-CoA thiolase. PEX10, peroxisome biogenesis factor. (B) Engineering lipid biosynthesis and hydrolysis by overexpressing rate-limiting enzymes increased squalene production in the peroxisome. (C) Engineering lipid degradation by strengthening the β-oxidation pathway increased squalene production in the peroxisome. (D) Fermentation profiles of squalene-producing strain Sq27 in a 3-L bioreactor. Statistical significance was tested using the two-sided Student’s t test; **P < 0.01 and ***P < 0.001; ns, not significant. All data are represented as mean ± SD of three biologically independent experiments.

Fig. 4.

Establishing an orthogonal acetyl-CoA shortcut in the peroxisome for squalene production. (A) Schematic illustration of the metabolic network based on acetate uptake. The blue arrows indicate the orthogonal squalene pathway starting from acetate in the peroxisome. The gray arrows indicate that the small fraction of glucose supplemented in substrate cofeeding system mainly support cell growth. SeACS, acetyl-CoA synthetase from Salmonella enterica. (B) Profiles of squalene production when stain Sq10 was grown in YPA media supplemented with PBS. (C and D) A decreased biomass was observed when strain Sq10 cultured in YPA media (C) relatively to that in YPD media, whereas squalene content in both YPA and YPD media is similar (D). (E and F) Introduction of SeACS (SeACS^L641P) in the peroxisome forming an orthogonal acetate utilization pathway in strain Sq28 increased significantly squalene production in terms of titers (E) and cellular content (F), compared to the parent strain Sq10. (G) Acetic acid in both salt and acid forms as carbon source was implemented in acetate fermentation in the bioreactor. In cofeeding glucose and acetate systems, glucose was continuously supplemented in small quantities to sustain a negligible concentration in the bioreactor and minimize catabolite repression. (H and I) Fermentation profiles of squalene-producing strain Sq28 on acetate-only culture (H) and on glucose-acetate mix cofeeding culture (I). Statistical significance was tested using the two-sided Student’s t test; ***P < 0.001; ns, not significant. All data are represented as mean ± SD of three biologically independent experiments.