D-Lactic Acid Production from Sugarcane Bagasse by Genetically Engineered Saccharomyces cerevisiae

Abstract

Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, Saccharomyces cerevisiae harboring the D-lactate dehydrogenase (DLDH) gene from Leuconostoc mesenteroides was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of gpd1, gpd2, and adh1 to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔgpd and CEN.PK2_DLDHΔgpdΔadh1, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔgpd was crossed with the weak acid-tolerant S. cerevisiae BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of S. cerevisiae for the production of industrially potent products.

L‐lactate production in engineered Saccharomyces cerevisiae using a multistage multiobjective automated design framework

“…In fact, studies such as that of Van maris et al argue that to increase LA production, it is necessary to suppress pyruvate decarboxylase activity (van Maris et al, 2004). A third one, GPD1, which encodes glycerol-3-phosphate dehydrogenase implicated in the balance of NADH oxidation and intracellular accumulation of glycerol (Albertyn et al, 1994); as in the case of the PYC genes, we also found evidence in the literature for the GPD1 gene indicating that knockout of the latter is functional for LA optimization, because the knockout of the GPD1 gene leads to minimize the production of ethanol and glycerol (Sornlek et al, 2022).…”

Global trends and future prospects of lactic acid production from lignocellulosic biomass

“…The engineered strain, when subjected to SSF using alkaline-pretreated sugarcane bagasse, achieved an impressive d -LA yield of 0.33 g per g-glucan. 30 This outcome underscores its potential for the production of industrially valuable products. The genetic-engineering approaches have been exploited in a big way for the improvement of LA yield and optical purity by various microbial producers.…”

“…28 Furthermore, author keywords like “metabolic engineering”, “ Saccharomyces cerevisiae ”, and “ Escherichia coli ” hold the 10th, 11th, and 24th rankings, respectively. Of notable significance, extensive research has been undertaken on the production of LA from LCB through metabolically engineered S. cerevisiae 29,30 and E. coli . 31–33 Additionally, LA often emerges as a byproduct in systems where S. cerevisiae produces ethanol from LCB.…”

Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris

“…The quantification of D-lactic acid and other metabolites was performed as previously described, with some modifications [2,30]. The engineered strains were prepared by preculturing 5 mL aliquots in a yeast minimal medium (MGYH; 0.1 M phosphate buffer pH 6.0, yeast nitrogen base without amino acids (13.6 g/L), glycerol (20 g/L), D-biotin (0.4 mg/L), thiamine-hydrochloride (133.3 mg/L), and L-histidine (20 mg/L)).…”

“…High-performance liquid chromatography (HPLC) was used to measure the amount of D-lactic acid and other fermentation metabolites, including ethanol, glycerol, pyruvate, acetate and glucose, as previously described [2,30]. One mL of yeast culture was passed through a 0.2-micron nylon syringe filter (Filtrex, Bangkok, Thailand).…”