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D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

ABSTRACT: D-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify D-pantothenate. The wild type of C. glutamicum was found to accumulate 9 micrograms of this vitamin per liter. A strain was constructed (i) to abolish L-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to D-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and D-pantothenate (panBC) synthesis. With this strain a D-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10(5)-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the D-pantothenate-specific part of the pathway and that the availability of beta-alanine is essential for D-pantothenate formation.

SUBMITTER: Sahm H

PROVIDER: S-EPMC91285 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

Sahm H H Eggeling L L

Applied and environmental microbiology 19990501 5

D-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes a ...[more]

PMID: 10223988

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Project description:BackgroundCorynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.ResultsHere, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ε-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.ConclusionC. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.

Project description:The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and succinylation are limited. This is the first study investigating the acetylome and succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology.

Project description:BACKGROUND:Promoters are commonly used to regulate the expression of specific target genes or operons. Although a series of promoters have been developed in Corynebacterium glutamicum, more precise and unique expression patterns are needed that the current selection of promoters cannot produce. RNA-Seq technology is a powerful tool for helping us to screen out promoters with expected transcriptional strengths. RESULTS:The promoter PCP_2836 of an aldehyde dehydrogenase coding gene from Corynebacterium glutamicum CP was identified via RNA-seq and RT-PCR as a growth-regulated promoter. Comparing with the strong constitutive promoter Ptuf, the transcriptional strength of PCP_2836 showed a significant decrease that from about 75 to 8% in the stationary phase. By replacing the native promoters of the aceE and gltA genes with PCP_2836 in the C. glutamicum ATCC 13032-derived L-valine-producing strain AN02, the relative transcriptional levels of the aceE and gltA genes decreased from 1.2 and 1.1 to 0.35 and 0.3, and the activity of their translation products decreased to 43% and 35%, respectively. After 28 h flask fermentation, the final cell density of the obtained strains, GRaceE and GRgltA, exhibited a 7-10% decrease. However, L-valine production increased by 23.9% and 27.3%, and the yield of substrate to product increased 43.8% and 62.5%, respectively. In addition, in the stationary phase, the intracellular citrate levels in GRaceE and GRgltA decreased to 27.0% and 33.6% of AN02, and their intracellular oxaloacetate levels increased to 2.7 and 3.0 times that of AN02, respectively. CONCLUSIONS:The PCP_2836 promoter displayed a significant difference on its transcriptional strength in different cell growth phases. With using PCP_2836 to replace the native promoters of aceE and gltA genes, both the transcriptional levels of the aceE and gltA genes and the activity of their translation products demonstrated a significant decrease in the stationary phase. Thus, the availability of pyruvate was significantly increased for the synthesis of L-valine without any apparent irreversible negative impacts on cell growth. Use of this promoter can enhance the selectivity and control of gene expression and could serve as a useful research tool for metabolic engineering.

Dataset Information

D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

Publications

D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

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