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.