Application of metabolic controls for the maximization of lipid production in semicontinuous fermentation

Abstract

Acetic acid can be generated through syngas fermentation, lignocellulosic biomass degradation, and organic waste anaerobic digestion. Microbial conversion of acetate into triacylglycerols for biofuel production has many advantages, including low-cost or even negative-cost feedstock and environmental benefits. The main issue stems from the dilute nature of acetate produced in such systems, which is costly to be processed on an industrial scale. To tackle this problem, we established an efficient bioprocess for converting dilute acetate into lipids, using the oleaginous yeast Yarrowia lipolytica in a semicontinuous system. The implemented design used low-strength acetic acid in both salt and acid forms as carbon substrate and a cross-filtration module for cell recycling. Feed controls for acetic acid and nitrogen based on metabolic models and online measurement of the respiratory quotient were used. The optimized process was able to sustain high-density cell culture using acetic acid of only 3% and achieved a lipid titer, yield, and productivity of 115 g/L, 0.16 g/g, and 0.8 g·L −1 ·h −1 , respectively. No carbon substrate was detected in the effluent stream, indicating complete utilization of acetate. These results represent a more than twofold increase in lipid production metrics compared with the current best-performing results using concentrated acetic acid as carbon feed.lipid production | acetate | tangential filtration | dynamic modeling | exhaust gas analysis

The role of temperature, pH and nutrition in process development of the unique oleaginous yeast Metschnikowia pulcherrima

“…). However, at a four‐fold increase of YE, the maximum stirring speed was increased by 33% compared to the reference fermentation due to the additional oxygen required for a stable DO (50%), indicating the need of advanced oxygen transfer and/or higher power consumption when increasing the productivities …”

“…Cutaneotrichosporon oleaginosus , for example, can be cultured under nutrient‐rich conditions on acetate achieving lipid contents up to 56.7% ( w / w ) . Genetically modified Y. lipolytica has been cultured at low carbon concentrations in semi‐continuous operation rather than under nitrogen starvation achieving a lipid productivity of 0.80 g L −1 h −1 – also on acetate . Such strategies are not required with M. pulcherrima , which – as shown on multiple substrates – still accumulates considerable amounts of lipids on nutrient‐rich feedstock (Fig.…”

“…19 Genetically modified Y. lipolytica has been cultured at low carbon concentrations in semi-continuous operation rather than under nitrogen starvation achieving a lipid productivity of 0.80 g L −1 h −1also on acetate. 46 Such strategies are not required with M. pulcherrima, whichas shown on multiple substrates 13,21still accumulates considerable amounts of lipids on nutrient-rich feedstock (Fig. 1, Figs S3 and S4, Table 1).…”

“…However, at a four-fold increase of YE, the maximum stirring speed was increased by 33% compared to the reference fermentation due to the additional oxygen required for a stable DO (50%), indicating the need of advanced oxygen transfer and/or higher power consumption when increasing the productivities. 45,46 The minor dependency of M. pulcherrima biomass yields on micronutrients ( Fig. 1) can become an advantage in low-cost lipid production, for example when using wastewater as feedstock.…”

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing inYarrowia lipolytica

“…Compared to S. cerevisiae, Y. lipolytica lacks Crabtree effects, without generation of ethanol under high glucose conditions. The low pH tolerance (Cui, Gao et al 2017), strictly aerobic nature (Abghari andChen 2014, Ledesma-Amaro, Lazar et al 2016) and versatile substrate-degradation profile (Ledesma-Amaro, Lazar et al 2016, Li and Alper 2016, Rodriguez, Hussain et al 2016) enable its robust growth from a wide range of renewable feedstocks, including pentose (Ledesma-Amaro, Lazar et al 2016, Li and Alper 2016, Rodriguez, Hussain et al 2016, crude glycerol , Dimitris, Zoe et al 2019, glacial acetic acids (Xu, Liu et al 2017) and volatile fatty acids (VFA) (Spagnuolo, Shabbir Hussain et al 2018) et al Unlike bacteria, the spatially-organized subcellular compartment and hydrophobic lipid bodies in oleaginous yeast provide the ideal environment for the regioselectivity and stereoselectivity of many plant-specific P450 enzymes (Lv, Koffas et al 2019). Due to the strong endogenous acetyl-CoA and malonyl-CoA flux, Y. lipolytica has been harnessed as an industrial workhorse for efficient synthesis of complex plant secondary metabolites including polyketides (Markham, Palmer et al 2018, flavonoids (Lv, Koffas et al 2019), carotenoids (Gao, Tong et al 2017) and terpenoids (Jin, Zhang et al 2019).…”