Joonsong Park

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.

Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum

Succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. However, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. In this study, we report the development of metabolically engineered Corynebacterium glutamicum for high production of succinate by release of end-product inhibition coupled with an increase of key metabolic flux. It was found that the rates of glucose consumption and succinate production were significantly reduced by extracellular succinate in an engineered strain, S003. To understand the mechanism underlying the inhibition by succinate, comparative transcriptome analysis was performed. Among the downregulated genes, overexpression of the NCgl0275 gene was found to suppress the inhibition of glucose consumption and succinate production, resulting in a 37.7% increase in succinate production up to 55.4g/L in fed-batch fermentation. Further improvement was achieved by increasing the metabolic flux from PEP to OAA. The final engineered strain was able to produce 152.2g/L succinate, the highest production reported to date, with a yield of 1.1g/g glucose under anaerobic condition. These results suggest that the release of end-product inhibition coupled with an increase in key metabolic flux is a promising strategy for enhancing production of succinate.

The role of Corynebacterium glutamicum spiA gene in whcA-mediated oxidative stress gene regulation

The Corynebacterium glutamicum WhcA protein, which inhibits the expression of oxidative stress response genes, is known to interact with the SpiA protein. In this study, we constructed and analyzed spiA mutant cells with the goal of better understanding the function of the spiA gene. A C. glutamicum strain overexpressing the spiA gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. Expression of the spiA and whcA genes was repressed by oxidant diamide, indicating coordinate regulation and dispensability of the genes in cells under oxidative stress. In the spiA-overexpressing cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Deletion of whcA in spiA-overexpressing cells (or vice versa) produced phenotypes similar to the wild-type strain. Collectively, these data demonstrate a negative regulatory role of the spiA gene in whcA-mediated oxidative stress response and provide additional clues on the mechanism by which the whcA gene is regulated.

SpiE interacts with Corynebacterium glutamicum WhcE and is involved in heat and oxidative stress responses

The gene whcE in Corynebacterium glutamicum positively responds to oxidative and heat stress. To search for proteins that interact with WhcE, we employed a two-hybrid system with WhcE as the bait. Sequencing analysis of the isolated clones revealed peptide sequences, one of which showed high sequence identity to a hydrophobe/amphiphile efflux-1 family transporter encoded by NCgl1497. The interaction of the NCgl1497-encoded protein with WhcE in vivo was verified using reporter gene expression by real-time quantitative PCR (RT-qPCR). The WhcE protein strongly interacted with the NCgl1497-encoded protein in the presence of oxidative and heat stress. Furthermore, purified WhcE and NCgl1497-encoded proteins interacted in vitro, especially in the presence of the oxidant diamide, and the protein–protein interaction was disrupted in the presence of the reductant dithiothreitol. In addition, the transcription of NCgl1497 was activated approximately twofold in diamide- or heat-treated cells. To elucidate the function of the NCgl497 gene, an NCgl1497-deleted mutant strain was constructed. The mutant showed decreased viability in the presence of diamide and heat stress. The mutant strain also exhibited reduced transcription of the thioredoxin reductase gene, which is known to be regulated by whcE. Based on the results, NCgl1497 was named spiE (stress protein interacting with WhcE). Collectively, our data suggest that spiE is involved in the whcE-mediated oxidative stress response pathway of C. glutamicum.

Corynebacterium glutamicum sdhA encoding succinate dehydrogenase subunit A plays a role in cysR-mediated sulfur metabolism

The Corynebacterium glutamicum CysR protein plays a critical regulatory role in sulfur metabolism. In this study, we isolated a protein interacting with CysR by employing a two-hybrid system. Subsequent analysis identified the gene as sdhA annotated to encode succinate dehydrogenase flavoprotein subunit A, a Krebs cycle enzyme. Deletion of the gene (ΔsdhA) severely affected cell growth and final cell yield, particularly in complex media. In addition, the ΔsdhA mutant strain was unable to use acetate as the sole carbon source, showing the identity of the gene. Transcription of the cysR gene and genes known to be regulated by cysR was affected in the ΔsdhA mutant strain, suggesting a positive role for sdhA on cysR. Furthermore, ΔsdhA cells showed increased sensitivity to oxidants, such as diamide, menadione, and hydrogen peroxide. In ΔsdhA cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Taken together, our findings show that the SdhA protein not only performs a role as a TCA enzyme but also communicates with sulfur metabolism, thereby regulating genes involved in redox homeostasis.