ABSTRACT
d-Lactic acid serves as a pivotal platform chemical in the production of poly d-lactic acid (PDLA) and other value-added products. This compound can be synthesized by certain bacteria, including Klebsiella pneumoniae. However, industrial-scale lactic acid production in Klebsiella pneumoniae faces challenges due to growth inhibition caused by lactic acid stress, which acts as a bottleneck in commercial microbial fermentation processes. To address this, we employed a combination of evolutionary and genetic engineering approaches to create an improved Klebsiella pneumoniae strain with enhanced lactic acid tolerance and production. In flask fermentation experiments, the engineered strain achieved an impressive accumulation of 19.56 g/L d-lactic acid, representing the highest production yield observed in Klebsiella pneumoniae to date. Consequently, this strain holds significant promise for applications in industrial bioprocessing. Notably, our genome sequencing and experimental analyses revealed a novel correlation between UTP-glucose-1-phosphate uridylyltransferase GalU and lactic acid resistance in Klebsiella pneumoniae. Further research is warranted to explore the potential of targeting GalU for enhancing d-lactic acid production.
ABSTRACT
BACKGROUND: Propionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess. RESULTS: Adaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR-Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids. CONCLUSION: Our study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.
ABSTRACT
Resistance to weak organic acids is important relative to both weak organic acid preservatives and the development of inhibitor tolerant yeast as industrial production organisms. The ABC transporter Pdr12 is important for resistance to sorbic and propionic acid, but its role in tolerance to other weak organic acids with industrial relevance is not well established. In this study, yeast strains with altered expression of PDR12 and/or CMK1, a protein kinase associated with post-transcriptional negative regulation of Pdr12, were exposed to seven weak organic acids: acetic, formic, glycolic, lactic, propionic, sorbic and levulinic acid. These are widely used as preservatives, present in lignocellulosic hydrolysates or attractive as chemical precursors. Overexpression of PDR12 increased tolerance to acids with longer chain length, such as sorbic, propionic and levulinic acid, whereas deletion of PDR12 increased tolerance to the shorter acetic and formic acid. The viability of all strains decreased dramatically in acetic or propionic acid, but the Δpdr12 strains recovered more rapidly than other strains in acetic acid. Furthermore, our results indicated that Cmk1 plays a role in weak organic acid tolerance, beyond its role in regulation of Pdr12, since deletion of both Cmk1 and Pdr12 resulted in different responses to exposure to acids than were explained by deletion of Pdr12 alone.