Enhancing the recombinant protein productivity of Yarrowia lipolytica using insitu fibrous bed bioreactor.

In this study, the ability of Yarrowia lipolytica to produce the recombinant lipase CalB from Candida antarctica, used as a model protein has been compared across different bioreactor processes using glycerol, a byproduct from the biodiesel industry as the main carbon source. Batch, pulsed fed-batch (PFB), and continuous fed-batch (CFB) strategies were first compared using classical stirred tank (STR) bioreactors in terms of biomass production, carbon source uptake, and lipase production. Additionally, an in situ fibrous bed bioreactor (isFBB) was developed using sugarcane bagasse as a cell immobilization support. The maximum lipase titer achieved using the isFBB culture mode was 38%, 33%, and 49% higher than those obtained using the batch, PFB, and CFB cultures, respectively. The lipase productivity in isFBB mode (142U/mL/h) was 1.4-fold higher than that obtained using batch free cell cultures. These results highlight that isFBB is an efficient technology for the production of recombinant enzymes.

Green and sustainable succinic acid production from crude glycerol by engineered Yarrowia lipolytica via agricultural residue based in situ fibrous bed bioreactor.

In situ fibrous bed bioreactor (isFBB) for efficient succinic acid (SA) production by Yarrowia lipolytica was firstly developed in our former study. In this study, agricultural residues including wheat straw, corn stalk and sugarcane bagasse were investigated for the improvement of isFBB, and sugarcane bagasse was demonstrated to be the best immobilization material. With crude glycerol as the sole carbon source, optimization for isFBB batch fermentation was carried out. Under the optimal conditions of 20g sugarcane bagasse as immobilization material, 120gL-1 crude glycerol as carbon source and 4Lmin-1 of aeration rate, the resultant SA concentration was 53.6gL-1 with an average productivity of 1.45gL-1h-1 and a SA yield of 0.45gg-1. By feeding crude glycerol, SA titer up to 209.7gL-1 was obtained from fed batch fermentation, which was the highest value that ever reported.

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.

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.