Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L‐lactic acid

Abstract

Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

“…For the latter assays, specific commercial D-and L-lactate dehydrogenases were used. As expected, more than 90% of the total lactate measured for the wild-type strain corresponded to D-lactate at all time points (S. cerevisiae does normally not produce significant amounts of L-lactate (45)). The same was true for the dld1⌬ and dld2⌬ strains.…”

Long-term adaptive evolution of Leuconostoc mesenteroides for enhancement of lactic acid tolerance and production

“…Furthermore, upregulated phosphoglycerate mutase (LEUM_0251) probably contributes to smooth ATP production from the carbohydrate metabolism during carbon (glucose) utilization. Generally, LAB under acid stress are known to upregulate the pathway of glucose metabolism (glycolysis) to produce ATP efficiently, as substrate-level phosphorylation rather than oxidative phosphorylation, and this metabolic ploy can fulfill the requirements for ATP hydrolysis of F 0 F 1 ATPase [26, 38]. This notion is in line with a lower amount of ATP in the mutants here, especially in LMS70, than in the wild type.…”

“…The ammonia levels in all three mutants increased significantly as compared to the wild type. Redox power plays a pivotal role in lactic acid production because pyruvate is converted to lactic acid with consumption of NADH [37, 38]. Notably, the mutants showed an increased NADH/NAD + ratio in the stationary phase and larger total amounts of NADH and NAD + in both log and stationary phases.…”

Biobased organic acids production by metabolically engineered microorganisms

“…Further rewiring cellular metabolic fluxes to the production of lactic acid by reducing competing pathways that lead to ethanol and glycerol formation ( Figure 1) increased lactic acid production up to 35 g/L. Moreover, manipulating the availability of intracellular redox by deleting the external NADH dehydrogenase genes has proven to be important to achieve high L-lactic acid production, resulting in a strain capable of producing 117 g/L of L-lactic acid in a fed-batch mode with pH controlled at 3.5 [14]. Cargill has developed a yeast-based process (undisclosed yeast species) for lactic acid production that was commercially implemented in 2008 [11].…”

“…Introduction of L-lactate dehydrogenase gene from Pelodiscus sinensis (LDH, Figure 1) and fine tuning its expression enabled S. cerevisiae to accumulate 27.6 g/L of L-lactic acid [14]. Further rewiring cellular metabolic fluxes to the production of lactic acid by reducing competing pathways that lead to ethanol and glycerol formation ( Figure 1) increased lactic acid production up to 35 g/L.…”