Application of PCR-HRM method for microsatellite polymorphism genotyping in the LDHA gene of pigeons (Columba livia)

High-resolution melting (HRM) is a post-PCR method that allows to discriminate genotypes based on fluorescence changes during the melting phase. HRM is used to detect mutations or polymorphisms (e.g. microsatellites, SNPs, indels). Here, the (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene in pigeons was analysed using the HRM method. Individuals (123 homing pigeons) were genotyped using conventional PCR. Birds were classified into groups based on genotype type and the results were tested by qPCR-HRM and verified using sequencing. Based on the evaluated protocol, five genotypes were identified that vary in the number of TTTAT repeat units (3/3, 4/4, 3/4, 4/5, and 5/5). Sequencing have confirmed the results obtained with qPCR-HRM and verified that HRM is a suitable method for identification of three-allele microsatellite polymorphisms. It can be concluded that the high-resolution melting (HRM) method can be effectively used for rapid (one-step) discrimination of the (TTTAT)3-5 microsatellite polymorphism in the pigeon’s LDHA gene.

The ldhA Gene Encoding Fermentative L-Lactate Dehydrogenase in Corynebacterium glutamicum Is Positively Regulated by the Global Regulator GlxR

Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes L-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding L-lactate dehydrogenase is solely responsible for L-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and L-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, L-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.

FSMP-13. HYPOXIC REGULATION OF LACTATE DEHYDROGENASE GENES (LDHA/B) IN T98 GLIOBLASTOMA MULTIFORME CELLS

Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a poor prognosis. GBM cells exhibit extensive metabolic alterations that enhance survival and proliferation in the mixed normoxic-hypoxic tumor microenvironment. Lactate dehydrogenase (LDH) enzymes are critical mediators of the normoxic to hypoxic transition in cells. Two LDH genes (A/B) encode monomers that combine to form five isoenzymes (LDH1-5) with different properties for pyruvate to lactate interconversion. Hypoxic induction of LDHA in all cells appears to occur via HIF-1 mediated transcription. However, little is known about hypoxic regulation of LDHB in cancer. We report on hypoxic regulation of LDHA/B in T98G, a rare cell line that has both normal and neoplastic features. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Relative expression of LDH isoforms 1-5 was assessed using native gel electrophoresis. Expression of the LDHA and LDHB genes was measured using qRT-PCR. LDHA-dominant isoforms (4/5) were detected in T98G cells subjected to normoxia and hypoxia via gel electrophoresis, however, LDHB-dominant isoforms (1/2) were not. The LDHA/B-equimolar isoform (3) was decreased in T98G cells subjected to hypoxia. LDHA gene expression was over two-fold greater than LDHB in normoxia (p = .00256 by one-tailed Mann-Whitney U test), and over nine-fold greater in hypoxia (p = .00256). LDHA:LDHB expression in hypoxia compared to normoxia was significantly different (p = .00256). LDHA expression increased three-fold in hypoxia (p = .00256), while LDHB expression decreased 0.3-fold in hypoxia (p = .03288). We document LDHB dysregulation in T98G cells as the gene is minimally responsive to oxygen. Therapeutic strategies aimed at promoting LDHB expression may complement inhibition of LDHA and reduce GBM survival in hypoxia.

Strengthening the TCA cycle to alleviate metabolic stress due to blocking by-products synthesis pathway in Klebsiella pneumoniae

ABSTRACT 1,3-Propanediol (1,3-PDO) is an important synthetic monomer for the production of polytrimethylene terephthalate (PTT). Here, we engineered Klebsiella pneumoniae by a multi-strategy to improve 1,3-PDO production and reduce by-products synthesis. First, the 2,3-butanediol (2,3-BDO) synthesis pathway was blocked by deleting the budB gene, resulting in a 74% decrease of 2,3-BDO titer. The synthesis of lactate was decreased by 79% via deleting the ldhA gene, leading to a 10% increase of 1,3-PDO titer. Further, reducing ethanol synthesis by deleting the aldA gene led to a 64% decrease of ethanol titer, and the 1,3-PDO titer and yield on glycerol increased by 12 and 10%, respectively. Strengthening the TCA cycle by overexpressing the mdh gene improved 1,3-PDO synthesis effectively. Under 5-L fed-batch fermentation conditions, compared to wild type strain, the production of 2,3-BDO, lactate and ethanol in the mutant strain decreased by 73, 65 and 50%, respectively. Finally, the production of 1,3-PDO was 73.5 g/L with a molar yield of 0.67 mol/mol glycerol, improved 16% and 20%, respectively. This work provides a combined strategy for improving 1,3-PDO production by strengthening the TCA cycle to relieve metabolic stress by deleting genes of by-products synthesis, which was also beneficial for the extraction and separation of downstream products.