Isolation and Expression of Lactate Dehydrogenase Genes from Rhizopus oryzae

ABSTRACT Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD+-dependent l-lactate dehydrogenases (LDH) (EC 1.1.1.27 ), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts fromldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize thatldhB encodes a second NAD+-dependent LDH that is capable of converting l-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. BothldhA and ldhB restored fermentative growth toEscherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.

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Immobilization of Rhizopus oryzae in a modified polyvinyl alcohol gel for L(+)-lactic acid production

Annals of Microbiology ◽

10.1007/s13213-012-0549-x ◽

2012 ◽

Vol 63 (3) ◽

pp. 957-964 ◽

Cited By ~ 8

Author(s):

Peng Wang ◽

Zhen Chen ◽

Juan Li ◽

Li Wang ◽

Guohong Gong ◽

...

Keyword(s):

Lactic Acid ◽

Polyvinyl Alcohol ◽

Acid Production ◽

Rhizopus Oryzae ◽

Lactic Acid Production

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Optimization of Process Parameters and Culture Medium for L-(+)-Lactic Acid Production by Rhizopus oryzae

JOURNAL OF CHEMICAL ENGINEERING OF JAPAN ◽

10.1252/jcej.08we105 ◽

2009 ◽

Vol 42 (8) ◽

pp. 589-595 ◽

Cited By ~ 2

Author(s):

Sule Bulut ◽

Murat Elibol ◽

Dursun Ozer

Keyword(s):

Lactic Acid ◽

Process Parameters ◽

Acid Production ◽

Culture Medium ◽

Rhizopus Oryzae ◽

Lactic Acid Production ◽

Optimization Of Process Parameters

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First Report of Cucurbit aphid-borne yellows virus in Spain

Plant Disease ◽

10.1094/pdis.2004.88.8.907a ◽

2004 ◽

Vol 88 (8) ◽

pp. 907-907 ◽

Cited By ~ 22

Author(s):

M. Juarez ◽

V. Truniger ◽

M. A. Aranda

Keyword(s):

Nucleotide Sequence ◽

Nucleotide Sequence Identity ◽

Rt Pcr ◽

Sequence Comparisons ◽

E Coli ◽

Sequence Identity ◽

Tissue Print ◽

Sigma Chemical ◽

Beet Pseudo Yellows Virus ◽

Cucumis Melo L

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5′-GAATACGGTCGCGGCTAGAAATC-3′ (CE9) and 5′-CTATTTCGGGTTCTGGACCTGGC-3′ (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) H. Guilley et al. Virology 202:1012, 1994. (3) H. Lecoq et al. Plant Pathol. 41:749, 1992.

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Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

Applied and Environmental Microbiology ◽

10.1128/aem.00683-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5492-5499 ◽

Cited By ~ 82

Author(s):

Minoska Valli ◽

Michael Sauer ◽

Paola Branduardi ◽

Nicole Borth ◽

Danilo Porro ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Lactic Acid ◽

Intracellular Ph ◽

Cell Sorting ◽

Acid Production ◽

Lactic Acid Production ◽

Low Ph ◽

The Mean ◽

High Level ◽

Lactate Dehydrogenases

ABSTRACT Yeast strains expressing heterologous l-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.

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