Efficient metabolic evolution of engineered Yarrowia lipolytica for succinic acid production using a glucose-based medium in an in situ fibrous bioreactor under low-pH condition

Abstract

Background: Alkali used for pH control during fermentation and acidification for downstream recovery of succinic acid (SA) are the two largest cost contributors for bio-based SA production. To promote the commercialization process of fermentative SA, the development of industrially important microorganisms that can tolerate low pH has emerged as a crucial issue.

Results: In this study, an in situ fibrous bed bioreactor (isFBB) was employed for the metabolic evolution for selection of Y. lipolytica strain that can produce SA at low pH using glucose-based medium. An evolved strain named Y. lipolytica PSA3.0 that could produce SA with a titer of 19.3 g/L, productivity of 0.52 g/L/h, and yield of 0.29 g/g at pH 3.0 from YPD was achieved. The enzyme activity analysis demonstrated that the pathway from pyruvate to acetate was partially blocked in Y. lipolytica PSA3.0 after the evolution, which is beneficial to cell growth and SA production at low pH. When free-cell batch fermentations were performed using the parent and evolved strains separately, the evolved strain PSA3.0 produced 18.4 g/L SA with a yield of 0.23 g/g at pH 3.0. Although these values were lower than that obtained by the parent strain PSA02004 at its optimal pH 6.0, which were 25.2 g/L and 0.31 g/g, respectively, they were 4.8 and 4.6 times higher than that achieved by PSA02004 at pH 3.0. By fed-batch fermentation, the resultant SA titer of 76.8 g/L was obtained, which is the highest value that ever achieved from glucose-based medium at low pH, to date. When using mixed food waste (MFW) hydrolysate as substrate, 18.9 g/L SA was produced with an SA yield of 0.38 g/g, which demonstrates the feasibility of using low-cost glucose-based hydrolysate for SA production by Y. lipolytica in a low-pH environment.

Conclusions: This study presents an effective and efficient strategy for the evolution of Y. lipolytica for SA production under low-pH condition for the first time. The isFBB was demonstrated to improve the metabolic evolution efficiency of Y. lipolytica to the acidic condition. Moreover, the acetate accumulation was found to be the major reason for the inhibition of SA production at low pH by Y. lipolytica, which suggested the direction for further metabolic modification of the strain for improved SA production. Furthermore, the evolved strain Y. lipolytica PSA3.0 was demonstrated to utilize glucose-rich hydrolysate from MFW for fermentative SA production at low pH. Similarly, Y. lipolytica PSA3.0 is expected to utilize the glucose-rich hydrolysate generated from other carbohydrate-rich waste streams for SA production. This study paves the way for the commercialization of bio-based SA and contributes to the sustainable development of a green economy.

Keywords: In-situ fibrous bed bioreactor; Metabolic evolution; Succinic acid; Yarrowia lipolytica.

Fig. 1

Metabolic evolution of Y. lipolytica PSA02004 for SA production at low pH. a Cell growth and SA production at pH 6.0. b Cell growth and SA production at pH 5.0. c Cell growth and SA production at pH 4.0. d Cell growth and SA production at pH 3.0. e Cell growth and SA production at pH without control. W.C refers to without pH control. f Glucose consumption rate at different pH. g Relative specific growth rate at different pH, in which the specific growth rate of the first generation was set as 100%. Relative specific growth rate (%) = (specific growth rate in any generation)/(specific growth rate in the first generation) × 100. h, i SA productivity and SA yield at different pH (filled triangles represent glucose concentration; filled circles represent SA concentration; open squares represent DCW; shaded areas represent the corresponding pH; open triangles represent glucose consumption rate; filled square represents the relative specific growth rate; open circles represent SA productivity; open stars represent SA yield)

Fig. 2

Metabolic pathways for succinate production in Y. lipolytica

Fig. 3

Fed-batch fermentation for SA production by evolved strain PSA 3.0 at low pH. (dotted line represents pH value; filled triangles represent glucose concentration; filled circles represent SA concentration; open squares represent DCW)

Fig. 4

Cell growth and SA production of evolved PSA 3.0 under low-pH condition with mixed food waste hydrolysate as feedstock (dotted line represents pH value; filled triangles represent glucose concentration; filled circles represent SA concentration; open squares represent DCW)