Improved squalene production through increasing lipid contents in Saccharomyces cerevisiae

Abstract

Squalene, a valuable acyclic triterpene, can be used as a chemical commodity for pharmacology, flavor, and biofuel industries. Microbial production of squalene has been of great interest due to its limited availability, and increasing prices extracted from animal and plant tissues. Here we report genetic perturbations that synergistically improve squalene production in Saccharomyces cerevisiae. As reported previously, overexpression of a truncated HMG-CoA reductase 1 (tHMG1) led to the accumulation 20-fold higher squalene than a parental strain. In order to further increase squalene accumulation in the tHMG1 overexpressing yeast, we introduced genetic perturbations-known to increase lipid contents in yeast-to enhance squalene accumulation as lipid body is a potential storage of squalene. Specifically, DGA1 coding for diacylglycerol acyltranferase was overexpressed to enhance lipid biosynthesis, and POX1 and PXA2 coding for acyl-CoA oxidase and a subunit of peroxisomal ABC transporter were deleted to reduce lipid β-oxidation. Simultaneous overexpression of tHMG1 and DGA1 coding for rate-limiting enzymes in the mevalonate and lipid biosynthesis pathways led to over 250-fold higher squalene accumulation than a control strain. However, deletion of POX1 and PXA2 in the tHMG1 overexpressing yeast did not improve squalene accumulation additionally. Fed-batch fermentation of the tHMG1 and DGA1 co-overexpressing yeast strain resulted in the production of squalene at a titer of 445.6 mg/L in a nitrogen-limited minimal medium. This report demonstrates that increasing storage capacity for hydrophobic compounds can enhance squalene production, suggesting that increasing lipid content is an effective strategy to overproduce a hydrophobic molecule in yeast.

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

“…(A) Squalene biosynthesis via MVA pathway in yeast, fungi, and algae. The engineering strategies for enhanced squalene production are as follows: overexpression of HMGR (Polakowski et al, 1998; Tokuhiro et al, 2009; Mantzouridou and Tsimidou, 2010; Dai et al, 2012, 2014; Zhuang and Chappell, 2015; Rasool et al, 2016a,b; Kwak et al, 2017; Paramasivan and Mutturi, 2017; Han et al, 2018; Huang et al, 2018; Wei et al, 2018) and SQS (Dai et al, 2014; Zhuang and Chappell, 2015; Rasool et al, 2016a,b), downregulation of SQE (Garaiová et al, 2014; Hull et al, 2014; Zhuang and Chappell, 2015; Rasool et al, 2016a,b; Han et al, 2018) in yeast; downregulation of SQE in algae (Kajikawa et al, 2015). (B) Squalene biosynthesis via MEP pathway in bacteria.…”

“…Rapid and remarkable advances in genetic engineering, metabolic engineering and synthetic biology approaches over the last few decades have facilitated the insertion of heterologous genes and editing of the genome of an organism, enabling successful attempts to produce innumerable molecules of industrial importance (Martin et al, 2009; Stephanopoulos, 2012; Singh, 2014; Jullesson et al, 2015). Lately, a number of microorganisms have been engineered by inserting a squalene biosynthetic pathway or by the modification of existing biosynthetic pathway for over-production of squalene (Ghimire et al, 2009; Katabami et al, 2015;Han et al, 2018; Wei et al, 2018).…”

Yarrowia lipolytica Strains Engineered for the Production of Terpenoids

“…Overexpression of the native diacylglycerol O-acyltransferase (DGA1) together in the squalene platform strain resulted in 320 ± 16.2 mg/L squalene, less than when only expressing SQS. Overexpression of the native DGA1 in combination with a truncated version of HMG in S. cerevisiae increased squalene titers, probably due to an increased lipid content providing storage for squalene accumulation (Wei et al, 2018). However, overexpression of DGA1 in Y. lipolytica did not benefit squalene production in the context of this study, likely due to the diversion of acetyl-CoA to lipid biogenesis (Figure 1).…”

Self-Redirection of Metabolic Flux toward Squalene and Ethanol Pathways by Engineered Yeast

“…The excessive cytoplasmic accumulation of olefin oils such as squalene and terpene inflict toxic effects and metabolic stress on the yeast cell [43,44]. However, lipid droplets in yeast can only store small quantities of olefin oil due to the tight regulation of their biogenesis [45,46].…”

“…DGA1 encodes diacylglycerol O-acyltransferase, catalyzes the critical step involved in the production of triacylglycerol (TAG) and stimulation of biogenesis of lipid droplets in yeast [49]. The previous study has shown that overexpression of DGA1 enhanced the accumulation of squalene via stimulating the biogenesis of lipid droplets in yeast [44].…”