Development of Hypertolerant Strain of Yarrowia lipolytica Accumulating Succinic Acid Using High Levels of Acetate

Abstract

Acetate is emerging as a promising feedstock for biorefineries as it can serve as an alternate carbon source for microbial cell factories. In this study, we expressed acetyl-CoA synthase in Yarrowia lipolytica PSA02004PP, and the recombinant strain grew on acetate as the sole carbon source and accumulated succinic acid or succinate (SA). Unlike traditional feedstocks, acetate is a toxic substrate for microorganisms; therefore, the recombinant strain was further subjected to adaptive laboratory evolution to alleviate toxicity and improve tolerance against acetate. At high acetate concentrations, the adapted strain Y. lipolytica ACS 5.0 grew rapidly and accumulated lipids and SA. Bioreactor cultivation of ACS 5.0 with 22.5 g/L acetate in a batch mode resulted in a maximum cell OD 600 of 9.2, with lipid and SA accumulation being 0.84 and 5.1 g/L, respectively. However, its fed-batch cultivation yielded a cell OD 600 of 23.5, SA titer of 6.5 g/L, and lipid production of 1.5 g/L with an acetate uptake rate of 0.2 g/L h, about 2.86 times higher than the parent strain. Cofermentation of acetate and glucose significantly enhanced the SA titer and lipid accumulation to 12.2 and 1.8 g/L, respectively, with marginal increment in cell growth (OD 600 : 26.7). Furthermore, metabolic flux analysis has drawn insights into utilizing acetate for the production of metabolites that are downstream to acetyl-CoA. To the best of our knowledge, this is the first report on SA production from acetate by Y. lipolytica and demonstrates a path for direct valorization of sugar-rich biomass hydrolysates with elevated acetate levels to SA.

Insights into the Genomic and Phenotypic Landscape of the Oleaginous Yeast Yarrowia lipolytica

“…This has been the subject of numerous studies since 2015 when a floated cell strategy was employed to enhance lipogenesis [ 67 ]. ALE was generally used to enhance strain oleogenic capacities [ 67 , 68 ] and their production of carboxylic acids [ 69 ] or to improve tolerance to various toxic compounds [ 70 , 71 , 72 , 73 , 74 ] and high temperatures [ 75 ]. In our study, it appears that growth at 37 °C was a highly divergent trait.…”

Hydrolysis of lignocellulose to succinic acid: a review of treatment methods and succinic acid applications

“…Fungi (brown rot fungi, white rot fungi, and soft rot fungi) show excellent biodegradability in the process of lignin removal from wheat straw materials [29], making the biological pretreatment method environment-friendly. However, the long fermentation time and many by-products will increase the cost of fermentation, and more efficient biological strains need to be cultivated through strain improvement and fermentation medium optimization [30][31][32]. Additionally, different pretreatment methods show varying specificity for the change in the physical and chemical structure of crop straw [33].…”

Contributions of Adaptive Laboratory Evolution towards the Enhancement of the Biotechnological Potential of Non-Conventional Yeast Species

“…Lately, some studies have emerged taking advantage of the synergistic combination of ALE and metabolic engineering to use yeasts as cell factories to improve their features [ 83 ]. Narisetty et al [ 83 ] expressed a heterologous acetyl-CoA synthase in a Y. lipolytica strain and later subjected the constructed strain to ALE to improve its tolerance to increasing contents of acetate as the sole carbon source. The authors not only successfully evolved this yeast to resist high concentrations of acetate but also detected an enhancement of succinic acid production (5.1–6.5 g/L) with this strategy.…”