3.1 Debottlenecking mevalonate pathway for squalene production
In yeast, squalene was primarily synthesized from the mevalonate (MVA)
pathway (Fig. 1). Staring with acetyl-CoA condensation, yeast uses a
number of critical enzymes to synthesize squalene, including
acetoacetyl-CoA thiolase (Erg 10, YALI0B08536g), HMG-CoA reductase
(YALI0E04807g), mevalonate kinase (Erg 12, YALI0B16038g),
phosphomevalonate kinase (Erg 8, YALI0E06193g), mevalonate pyrophosphate
decarboxylase (MVD1, YALI0F05632g), farnesyl pyrophosphate synthase
(Erg20, YALI0E05753g), geranyl pyrophosphate synthase (YALI0D17050g) and
squalene synthase (SQS1, YALI0A10076g). Genome annotation indicates thatY. lipolytica contains the complete mevalonate pathway (Fig. 1).
In MVA pathway, HMG-CoA reductase was reported as the rate-limiting
metabolic step in squalene accumulation (RODWELL, NORDSTROM, &
MITSCHELEN, 1976). In addition, there was almost no squalene accumulated
by native Y. lipolytica due to the quick consumption of squalene
by downstream ergosterol synthase. After we overexpressed the endogenous
squalene synthase gene (SQS ), squalene production was increased
to 17.25 mg/L at 120 h with chemically-defined complete synthetic media
(CSM-leu) in test tube. With this as a starting strain, we investigated
the effect of three HMG-CoA reductases (encoded by HMG) on squalene
production. The three HMGs were derived from Saccharomyces
cerevisiae , Silicibacter pomeroyi and Y. lipolytica .
Truncated form of HMG-CoA reductase
devoid of N-terminal membrane targeting signal has been proven to be
effective in improving isoprenoid production in Saccharomyces
cerevisiae (encoded by SctHMG ) (Polakowski, Stahl, & Lang,
1998; Thompson, Kwak, & Jin, 2014). When co-expressed with endogenous
SQS, the strain with the truncated HMG1 (SctHMG) led to squalene
production at 83.76 mg/L (Fig. 2A), indicating that overexpression of
HMG-CoA reductase was beneficial for squalene production. To test
whether other sources of HMG-CoA reductase could display better
functions, we co-expressed SpHMG from Silicibacter
pomeroyi and endogenous ylHMG with SQS, respectively. A low
yield of squalene (9.24 mg/L) was produced in the strain expressingSpHMG . This result was consistent with previous findings that HMG
from Silicibacter pomeroyi was highly specific for NADH (Meadows
et al., 2016) and this bacterial-derived enzymes could not be directly
translated to yeast system. When endogenous ylHMG was
co-expressed with SQS (strain HLYaliS01 ), the engineered strain
yielded 121.31 mg/L squalene at 120 h in test tube, demonstrating the
potential of using Y. lipolytica as a platform to synthesize
various terpenes. We also tested the truncated form of ylHMGsequence (YALI0E04807p), of which the first 495 nucleotides that encode
the 165 amino acid N-terminal domain responsible for membrane
localization (ER targeting) were removed. The remaining C-terminal
residues containing the catalytic domain and an NADPH-binding region
(Gao et al., 2017) were overexpressed. We then overexpressed the
truncated ylHMG (t495ylHMG ) to compare how the variation
of ylHMG may improve squalene synthesis. Contrary to our
hypothesis, removal of the N-terminal 495bp of ylHMG exhibits
adverse effect on both squalene production and cell growth (Fig. 2A),
indicating that the N-terminal membrane-binding domain plays a critical
role in squalene synthesis.
In addition to the overexpression of the endogenous SQS and ylHMGgenes, we also tested whether the expression of other genes in the MVA
pathway would improve squalene production, including ylErg8 encoding
phosphomevalonate kinase, ylErg10 encoding acetoacetyl-CoA thiolase,
Erg12 encoding mevalonate kinase, and ylErg20 encoding farnesyl
pyrophosphate synthetase; ylGPS encoding geranyl pyrophosphate synthase.
And ylErg8, ylErg10, Erg12 and ylErg20 from S. cerevisiae were
also overexpressed to compare how the variation of these genes may
enhance squalene synthesis. As shown in Fig. 2B, co-overexpression of
ylErg8, ylErg10, Erg12 could not further improve squalene synthesis,
regardless of the source of the gene. Among all of these combinations
(Fig. 2B), the highest squalene production was obtained for the strain
in which ylGPS and SQS-ylHMG1 were overexpressed, with titer of 107.08
mg/L and a specific production of 36.24 mg/g DCW, which is still lower
than the strain only expressing SQS-ylHMG1. These results indicate that
sequential overexpression of the genes involved in the MVA pathway could
not further improve the carbon flux toward squalene, possibly due to the
stringent regulation of MVA pathway at multiple nodes, including
ergosterol-mediated feedback inhibition or SREBP-related transcriptional
repression.