3.2 Augmenting NAPDH and acetyl-CoA precursor pathways to
improve squalene production
NADPH as the primary biological reducing equivalent protects cell from
oxidative stress and extend carbon-carbon backbones, which was also
reported as the major rate-limiting precursor in fatty acids synthesis
in oleaginous species (Qiao et al., 2017; Wasylenko, Ahn, &
Stephanopoulos, 2015). HMG-CoA reductase (HMGR) is the first
rate-limiting enzyme in the mevalonate pathway and plays critical role
in regulating squalene biosynthesis (Ma et al., 2019).
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is reductively
hydrolyzed to mevalonate by releasing coenzyme A with NADPH as reducing
equivalent (Cao, Wei, Lin, & Hua, 2017). Based on previous work, source
of cytosolic NADPH in the Baker’s yeast may originate from various
alternative routes depending on the carbon source and genetic background
of the yeast strain (Huan Liu, Marsafari, Deng, & Xu, 2019; Minard,
Jennings, Loftus, Xuan, & McAlister-Henn, 1998; Minard &
McAlister-Henn, 2005). With glucose as carbon sources, cytosolic NADPH
primarily relies on the pentose phosphate pathway. Other cytosolic NADPH
pathways include NADP-specific isocitrate dehydrogenase (IDP2), malic
enzyme (ylMAE), mannitol dehydrogenase (ylMnDH1, ylMnDH2),
6-phosphogluconate dehydrogenase (ylGND2) and succinate semialdehyde
dehydrogenase (ylUGA2) (Huan Liu, Monireh Marsafari, Li Deng, et al.,
2019) (Fig. 1). In this work, we tested a collection of auxiliary
cytosolic NADPH pathways and investigated how these pathways may enhance
squalene production and cellular fitness on the basis of co-expressionSQS-ylHMG (Fig. 3A). Among these chosen NADPHs, mannitol
dehydrogenase (ylMnDH2, encoded by YALI0D18964g) presented the best
results to improve squalene production. Mannitol, a more reduced sugar
alcohol compared to glucose, played an essential role in modulating
cytosolic NADPHs through the mannitol cycle. This could partially
explain why mannitol was the major byproduct during lipid accumulation
phase in Y. lipolytica (P. Xu, Qiao, & Stephanopoulos,
2017). When ylMnDH2 was overexpressed with SQS and ylHMG
(strain HLYaliS02 , Supplymentary Table S2), the engineered strain
produced 11% more squalene with volumetric production titer increased
to 135.22 mg/L, despite relatively decreased yield of 32.33 mg/g DCW
(Fig. 3A). This is possibly ascribed to the increased cell fitness and
lipid content after enhancing the supplement of NADPH.
Apart from NADPH, acetyl-CoA, is an essential metabolic intermediate
connecting glycolysis, Krebs cycle, and glyoxylate shunt pathways.
Acetyl-CoA is also the intermediate metabolite participated in lipid
synthesis, peroxisomal lipid oxidation and amino acid degradation
pathways. It links both anabolism and catabolism, is the starting
molecule in MVA pathway. Cytosolic acetyl-CoA was found as a critical
precursor to boost secondary metabolite production (Huan Liu, Marsafari,
Wang, Deng, & Xu, 2019). For example, engineering alternative cytosolic
acetyl-CoA pathways were proven to be efficient strategies to improve
fatty acids and isoprenoid production in both Bakers’ yeast and Y.
lipolytica (Hu Liu, Fan, Wang, Li, & Zhou, 2019; Meadows et al., 2016;
van Rossum, Kozak, Pronk, & van Maris, 2016). Therefore, we next
investigated whether endogenous and various heterologous acetyl-CoA
pathways could improve squalene production. First, we investigated the
pyruvate decarboxylase (PDC), acetylaldehyde dehydrogenase (ALD) and
acetyl-CoA synthase (ACS) bypass (Fig. 1) and compared the efficiency of
this route from Y. lipolytica , S. cerevisiae andE.coli (Fig. 3B). By overexpression of pyruvate decarboxylase
(ScPDC) from S. cerevisiae and acetylaldehyde dehydrogenase
(EcPuuc) from E.coli , we obtained only 106.54 mg/L of squalene
(Fig. 3B). We observed that the cell growth fitness was negatively
impacted due to the expression of heterologous genes, possibly due to
the accumulation of the toxic aldehyde intermediate. We next attempted
the endogenous ATP citrate lyase, which is the primary acetyl-CoA route
to Y. lipolytica metabolism. ATP citrate lyase (ACL) was mainly
used for supply of the cytosolic acetyl-CoA, which was proven to have
two isoforms encoded by two separate genes in Y. lipolytica (ACL1
and ACL2) (Nowrousian, Kück, Loser, & Weltring, 2000). Endogenous
ylACL1 (YALI0E34793g) and ylACL2 (YALI0D24431g) genes were subsequently
tested. A 19.5% increase in squalene synthesis was obtained in the
resulting strains HLYaliS03 with ylACL2 overexpressed along with
SQS and ylHMG, leading to the titer of squalene 144.96 mg/L (Fig. 3B).
The increase was probably a result of the pushing strategies for
acetyl-CoA enrichment by expressing ACL2 so that adequate cytosolic
acetyl-CoA could be pushed into the MVA pathway for the synthesis of
squalene. Surprisingly, the specific yield reduced to 25.27 mg/g DCW
which was caused by the enhancement of cell growth due to the increased
lipid content. This increased lipid content may also serve as the
storage space to sequestrate squalene in our engineered cell.