Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone

Abstract

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Keywords: 2-pyrone synthase; Rhodotorula toruloides; metabolic engineering; oilcane juice; triacetic acid lactone.

Figure 1

TAL production in Rhodotorula toruloides‐I12 using different substrates. (a) Commonly used sugars and (b) acetate spiking affects TAL production. TAl, triacetic acid lactone.

Figure 2

Metabolic pathway engineering for triacetic acid lactone biosynthesis in Rhodotorula toruloides. ACC1, acetyl‐CoA carboxylase; ACL1, ATP‐citrate lyase; ACS1, acetyl‐CoA synthetase; PDC1, pyruvate decarboxylase; ALD5, acetylaldehyde dehydrogenase; AMPD1, AMP deaminase; CIT2, peroxisomal citrate synthase; DGA1, diacylglycerol acyltransferase; GhPS, 2‐pyrone synthase from Gerbera hybrida; LRO1, lecithin cholesterol acyltransferase; ME1, malic enzyme; MLS1, cytosolic malate synthase; PDH, pyruvate dehydrogenase; PEX10, peroxisomal matrix protein; PYC1, pyruvate carboxylase; YLACL1, ATP‐citrate lyase from Yarrowia lipolytica. Some metabolites were not positioned following their intracellular compartmentalization.

Figure 3

TAL production from different metabolic engineering strategies. (a) Overexpression of selected gene targets. (b) Disruption of selected gene targets. (c) Multiple gene targets by combinatorial engineering. TAL, triacetic acid lactone.

Figure 4

Fed‐batch bioreactor fermentation of Rhodotorula toruloides. (a) The cell growth (OD₆₀₀), total consumed glucose and acetate under glucose‐based medium. (b) The TAL titer and corresponding yield to its theoretical yield under glucose‐based medium. (c) The cell growth (OD₆₀₀), total consumed glucose and acetate under oilcane juice‐based medium. (d) The TAL titer and corresponding yield to its theoretical yield under oilcane juice‐based medium. OD, optical density; TAL, triacetic acid lactone.