Introduction
β-carotene
(C40H56), one of many carotenoids, is
the precursor of vitamin A. β-carotene has become an essential
ingredient in food additives, cosmetics, and pharmaceuticals.β-carotene
has the function of enhancing immune functions, anti-oxidation, and
anti-cancer activities (Hejazi, Holwerda, & Wijffels, 2004)
(Bogacz-Radomska & Harasym, 2018). The global β-carotene market size is
anticipated to reach USD 583 million by the end of 2024 (Nester,
2019).
Currently, the primary sources of β-carotene include chemical synthesis,
plant extracts, and
microbial
fermentation.
Among these, the
microbial
fermentation is considered to be the best way to meet market demands, as
it is environment-friendly and economical.
The most commonly used microorganisms to produce
β-carotene
are Blakeslea trispora (He et al., 2017), engineeredYarrowia lipolytica (Larroude et al., 2018),Saccharomyces
cerevisiae (R. Wang et al., 2017),
andEscherichia coli (Wu et al., 2017). β-carotene is a lipid-soluble
compound and stored in the lipid bodies of engineered oleaginous yeastY. lipolytica .
Compared
with the non-oleaginous Saccharomyces cerevisiae orEscherichia coli,Y.
lipolytica is more suitable for the production of
β-carotene.Y. lipolytica is generally recognized as a safe strain and a
potential industrial host for the production of bio-ingredients
(Beopoulos, Desfougéres, Sabirova, & Nicaud, 2010). An engineeredY.
lipolytica strain to maximize β-carotene production and 1.47 mg/L of
β-carotene
was obtained after six days of cultivation in a flask (Jang, Yu, Jang,
Jegal, & Lee, 2018). Another engineered Y. lipolytica strain was
constructed by incorporating two genes, bi-functional phytoene
synthase/lycopene cyclase (crtYB) and phytoene desaturase (crtI) from
the red yeast Xanthophyllomyces dendrorhous . After 72h
cultivation, 31.1 mg/L β-carotene was obtained (Kildegaard et al.,
2017). Although β-carotene-producing engineered Y. lipolytica has
made significant progress, the fermentation process has not been
systematically optimized.
The production of β-carotene in Y. lipolytica requires aerobic
culture. When the glucose concentration in the culture medium is
high, the Crabtree effect often
occurs in aerobic conditions during most yeast fermentation, leading to
the production of alcohol and acetate through substrate-level
phosphorylation. Excessive alcohols and acids compete with β-carotene
synthesis for the substrate of acetyl-CoA.
The
Crabtree effect eventually leads to a reduction in the yield of the
target product (Li et al., 2019a). Fed-batch cultures provide a carbon
source at a low level by feeding essential nutrients incrementally.
Therefore,
this culturing technique is used to overcome the Crabtree effect (Wen,
Zhang, & Tan, 2006).
Fed-batch
strategies include constant dissolved oxygen value feeding
(DO-stat)
(Hu, Zheng, & Shen, 2010; B. Kim, Binkley, Kim, & Lee, 2018; Y. Wang
et al.,
2016),
constant specific growth rate feeding
(μ-stat)
(Maghsoudi et al., 2012),
constant
PH feeding
(pH-stat)
(B. S. Kim, Lee, Lee, Chang, & Chang, 2004; Li et al., 2019b), and
constant carbon source concentration feeding (Khatri & Hoffmann, 2006).
Besides the Crabtree effect, oxygen in short supply is another obstacle
in aerobic culture.
With cells growing, oxygen consumption exceeds the maximum oxygen
transfer capacity, which becomes a limiting factor for cell growth (Van
Hoek, 1998). Thus, a solution for microbes to receive an adequate amount
of oxygen is by decreasing the specific growth rate and amount of oxygen
consumption. Usually, DO-stat can be used to reduce the high rate of
oxygen consumption because it can control dissolved oxygen at a constant
value using fed substrate at a specific rate. Once the carbon source is
exhausted in the logarithmic growth phase, the O2 value
rapidly increases due to cell death from hypoxia. If the carbon source
was fed timely, the O2 value decreases as the cells
re-utilize carbon source and restore growth. Subsequently, a constant DO
level can be maintained by continuous feeding and keep a balance between
oxygen consumption and supply. The
DO-stat
strategy typically works well in a defined media where nutrient
depletion results in cell death and a rapid elevating DO (Picotto et
al., 2017). Many authors have used the DO-stat feeding strategy to
achieve high yield. The DO-stat fed-batch fermentation strategy was used
to produce tyrosine phenol
lyase
by recombinant Escherichia coli , the final biomass was 35.6 g/L,
and the volumetric activity reached 12292 U/L after 30 h cultivation
(Kawaguchi et al., 2019a). The DO-stat feeding strategy was a promising
strategy together with the use of ammonium hydroxide for pH control to
improve P(3HB) volumetric productivity (Cruz, Gouveia, Dionísio,
Freitas, & Reis, 2019). This particular method has been widely used in
the aerobic culture to produce highly valuable chemicals and biofuels.
Our laboratory has previously constructed an engineered β-carotene
producing strain of Y. lipolytica . The purpose of this study was
to develop a DO-stat culture strategy to improve biomass and β-carotene
yield. The changes of ATP and NADP+/NADPH during
culture process was also explored. A simple kinetic model relating the
cell growth to the limiting substrate (glucose) and major product
(β-carotene)
was constructed. This study established an effective method to increase
the yield of β-carotene and provides a new vehicle for the
industrialized β-carotene
production.