3.4 Shake flask cultivation of engineered strain with pH and
carbon/nitrogen ratio optimization
Despite that glucose is the preferred carbon source for cell growth, a
similar level of squalene production was detected in our engineered
strains (HLYaliS01 and HLYaliS02 , HLYaliS03 ).Y. lipolytica is a natural lipid producer, accumulating up to
30%~60% cell weight as lipid, which leads to a strong
competition for the precursor acetyl-CoA (P. Xu et al., 2017).
Meanwhile, cultivation pH and media C/N ratio were two critical factors
that affect cellular morphology and growth in Y. lipolytica(Szabo, 1999).
In our previous work, we observed a quick declining of cultivation pH
from 6 to 3.5 in polyketide synthesis, due to the accumulation of citric
acid when glucose was utilized. The pH variations negatively affect
strain physiology, alter cell membrane permeability and limit nutrients
transportation due to the loss of proton driving force. A significant
improvement of polyketide titer was observed by combining PBS buffer
with 1 mg/L cerulenin supplementations. Cerulenin is known to
irreversibly form a covalent adduct with the active site (cysteine
residue) of β-ketoacyl-ACP synthase, inhibiting the elongation of the
fatty acid backbone (Huan Liu, Monireh Marsafari, Fang Wang, et al.,
2019). Thus, a similar strategy was applied to promote squalene
production by strain HLYaliS02 (shown in Fig. 4 A and
Supplementary Fig. S3). When the engineered strain was cultivated in the
minimal YNB media with 0.2 M phosphoric buffer solution (PBS, pH 6.0),
squalene production was increased to 354.44 mg/L at 168 h (Supplementary
Fig. S3), which was an increase of 88.4%, compared with the results
from pH uncontrolled (Supplementary Fig. S1 C) experiment. The
improvement is ascribed to the better growth fitness under pH control
and the biomass of strain HLYaliS02 reached 13.98 g/L DCW with
the squalene specific yield at 25.35 mg/g DCW (Supplementary Table S3).
The major byproduct mannitol accumulated up to 2.2 g/L at 48 hour and
citric acid reached 7.81 g/L at 96 h; both mannitol and citrate were
subsequently reincorporated into cell metabolism (Supplementary Fig.
S3). But only ~50% of glucose was utilized in this
process which was consistent with our previous work, indicating the
supplementation of PO43- buffer may
negatively impact the glucose uptake rate. To further enhance squalene
synthesis, 1 mg/L cerulenin was supplemented to the minimal YNB-PBS
media at 48 h and the squalene production increased to 384.13 mg/L at
188 h, an 8.4% increase compared with the result without cerulenin
(Fig. 4 A). A similar fermentation profile of glucose consumption,
mannitol, and citric acid accumulation was found: half of glucose was
utilized while 14.9 g/L DCW was obtained with the squalene specific
yield at 25.78 mg/g DCW (Supplementary Table S3). Byproduct mannitol
reached 1.9 g/L at 48 h, but citric acid increased to 9.7 g/L, which was
higher than that in the YNB-PBS media without cerulenin supplemented,
possibly due to the fact that inhibition of the endogenous fatty acid
synthase may prevent citrate from being converted to acetyl-CoA and
oxaloacetate by ATP-citrate lyase (encoded by ACL).
We next investigated the effect of C/N ratio on squalene production in
YNB-PBS media supplemented with cerulenin (Fig. 4 B). Various C/N ratios
including 10:1, 20:1, 40:1, 60:1 and 80:1 was studied (Supplementary
Fig. S4). When the C/N ratio was set at 60:1, a similar fermentation
profile of glucose consumption, mannitol, dry cell weight, citric acid
was obtained, compared to the metabolic profile for C/N 80:1. Squalene
titer at C/N 60:1 reached 396 mg/L at 120 h with increased productivity.
The highest squalene titer was achieved in the media with C/N ratio
40:1, reaching 502.75 mg/L at 120 h with the yield to 32.6 mg/g DCW
(Fig. 4 B, supplementary Table S3), which was 30.8% higher than the
squalene production form C/N ratio 80:1 media. We speculated that
acetyl-CoA flux was enlarged and flowed to MVA pathway, since we
observed less citric acid accumulation (1.9 g/L) at the end of
fermentation. However, when the C/N ratio was further reduced to 20:1 or
10:1, adverse effect was obtained with decreasing squalene production
(Supplementary Fig. S4). We speculated that the superfluous nitrogen
provision may lead more carbons flowing to cell growth. These results
illustrated that C/N ratio plays an important role in the redistribution
of carbon flux and strongly influenced the accumulation of squalene.
Further downregulation of acetyl-CoA carboxylase (ACC) may be required
to improve squalene production. ACCase, as the malonyl-CoA source
pathway and the acetyl-CoA sink pathway during lipogenesis, was
primarily controlled through the phosphorylation of serine residues by
Snf1-mediated AMP kinase. Inhibition of fatty acid synthase pathway and
nitrogen starvation was proven to be effective strategies to activate
Snf1 kinase and slows down ACC1 activity (Seip, Jackson, He, Zhu, &
Hong, 2013; Zhang, Galdieri, & Vancura, 2013). It was consistent with
our findings that medium C/N ratio was beneficial for squalene
synthesis. By applying these engineering strategies, we have obtained an
oleaginous yeast strain with a similar squalene level to the strainS. cerevisiae (Han, Seo, Song, Lee, & Choi, 2018; Huang et al.,
2018). This work highlights the potential of engineering Y.
lipolytica as a promising microbial platform for efficient synthesis of
squalene and terpene-related compounds.