A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation.

NADP(+)-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol.

Whole organism biocatalysis.

The use of biocatalysts for the industrial synthesis of chemicals has been attracting much attention as an environment-friendly synthetic method. Microbial cells play a leading role in 'chemo-enzymatic synthesis' because of their great diversity. Several microbes with unique catalytic abilities have been found through intensive screening and put to practical use. Besides, advanced molecular biological techniques are powerful tools for developing more satisfactory biocatalysts.

Wax ester production by bacteria.

The enzymological and genetic aspects of microbial metabolism of hydrocarbons have been extensively revealed. Such molecular information is useful for understanding the bioremediation of oil spill environments and production of hydrocarbon-specific fine chemicals.

Two acyl-CoA dehydrogenases of Acinetobacter sp. strain M-1 that uses very long-chain n-alkanes.

Two genes encoding acyl-CoA dehydrogenases, acdA and acdB, arranged in tandem, were found in the chromosomal DNA of Acinetobacter sp. strain M-1. AcdA was purified from the parental strain and AcdB was purified from an Escherichia coli strain expressing the cloned gene. The substrate specificities of the two enzymes suggest that AcdA is a medium-chain acyl-CoA dehydrogenase and that AcdB is a long-chain acyl-CoA dehydrogenase. Characterization of n-alkane metabolism in Acinetobacter sp. strain M-1 has revealed parallel pathways as well as enzymes with overlapping specificities in a single pathway. The two acyl-CoA dehydrogenases described here provide another example of the physiological complexity underlying n-alkane utilization.

Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols.

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.

Ethanol catabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.

Two-component systems of Corynebacterium glutamicum: deletion analysis and involvement of the PhoS-PhoR system in the phosphate starvation response.

Corynebacterium glutamicum contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (Pi) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under Pi limitation, and therefore the genes lacking in this strain were named phoS (encoding the sensor kinase) and phoR (encoding the response regulator). DNA microarray analyses with the C. glutamicum wild type and the DeltaphoRS mutant supported a role for the PhoRS system in the adaptation to Pi starvation. In contrast to the wild type, the DeltaphoRS mutant did not induce the known Pi starvation-inducible (psi) genes within 1 hour after a shift from Pi excess to Pi limitation, except for the pstSCAB operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the pst operon. Primer extension analysis of selected psi genes (pstS, ugpA, phoR, ushA, and nucH) confirmed the microarray data and provided evidence for positive autoregulation of the phoRS genes.

The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses.

The phosphate (P(i)) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays. Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from P(i)-sufficient to P(i)-limiting conditions led to identification of five groups comprising 92 genes. Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium. One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to P(i) limitation. Among these genes, the RNA levels of the pstSCAB (ABC-type P(i) uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5'-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes. Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation. Several genes also involved in P uptake and metabolism were not affected by P(i) starvation; these included the genes encoding a PitA-like P(i) uptake system and a putative Na(+)-dependent P(i) transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate. In summary, a global, time-resolved picture of the response of C. glutamicum to P(i) starvation was obtained.

Wax ester production from n-alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase.

Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.