Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling

Abstract

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

FIG. 1.

Pantothenate biosynthetic pathway in C. glutamicum ATCC 13032 and its integration into the synthesis of branched-chain amino acids. The genes and enzymes involved in the biosynthetic steps are indicated. Abbreviations: TDH, threonine dehydratase; AHAS, acetohydroxy acid synthase; AHAIR, acetohydroxy acid isomeroreductase; DHAD, dihydroxy acid dehydratase; BCAT, branched-chain amino acid transaminase; KPHMT, ketopantoate hydroxymethyltransferase; PS, pantothenate synthetase; PK, pantothenate kinase; ADC, aspartate-α-decarboxylase; IPMS, isopropylmalate synthase.

FIG. 2.

Evolution of different substrates and products throughout cultivation of C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1ilvBNCD)(pMM55). Biomass accumulation and glucose consumption are shown in panel A. Most of the results are expressed in mM; the only exception is biomass, which is expressed in grams per liter.

FIG. 3.

Metabolic flux distribution at the pyruvate node throughout cultivation of C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1ilvBNCD)(pMM55). (A) Absolute flux values expressed as mmol/(g [dry weight] × h). (B) Relative values expressed as molar percentages of the glucose consumption rate. Fluxes from glycolysis and the phosphotransferase system arriving at pyruvate ⧫, from pyruvate to acetolactate via acetohydroxy acid synthase ○, from pyruvate to acetyl-CoA via pyruvate dehydrogenase ▪, and from pyruvate to biomass ▴ were determined. The flux from pyruvate to alanine is not shown since the value was less than 2% throughout cultivation. The flux to biomass includes the direct flux from pyruvate and the anaplerotic flux leading to oxaloacetate (OAA).

FIG. 4.

Metabolic flux distribution at the ketoisovalerate node throughout cultivation of C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1ilvBNCD)(pMM55). (A) V1, V2, V3, V4, V5, V6, V7, and V8 are absolute values expressed as mmol/(g [dry weight] × h). (B) V9, V10, V11, V12, and V13 are absolute values expressed as mmol/(g [dry weight] × h). (C) V2, V3, V4, V5, V7, V8, V10, V12, and V13 are relative values expressed as molar percentages of the glucose consumption rate. The following fluxes were determined: from pyruvate to dl-2,3-dihydroxy-isovalerate (V1); dl-2,3-dihydroxy-isovalerate excretion (V2); from dl-2,3-dihydroxy-isovalerate to ketoisovalerate (V3); valine excretion (V4); ketoisovalerate excretion (V5); from ketoisovalerate to ketoisocaproate (V6); leucine excretion (V7); ketoisocaproate excretion (V8); from ketoisovalerate to ketopantoate (V9); ketopantoate excretion (V10); from ketopantoate to pantoate (V11); pantoate excretion (V12); and pantothenate excretion (V13). The following enzyme reactions corresponded to the fluxes: V1, acetohydroxy acid synthase and acetohydroxy acid isomeroreductase; V2, dl-2,3-dihydroxy-isovalerate export; V3, dihydroxy acid dehydratase; V4, branched-chain amino acid transaminase and valine export; V5, ketoisovalerate export; V6, isopropylmalate synthase, isopropylmalate isomerase, and 3-isopropylmalate dehydrogenase; V7, branched-chain amino acid transaminase and leucine export; V8, ketoisocaproate export; V9, ketopantoate hydroxymethyltransferase; V10, ketopantoate export; V11, acetohydroxy acid isomeroreductase; V12, pantoate export; and V13, pantothenate synthetase and pantothenate export.

FIG. 5.

Agglomerative hierarchical clustering of the DNA microarray hybridization data. RNA samples from both the pantothenate producer (P) C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1ilvBNCD)(pMM55) and the control strain (C) C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1)(pMM36) were cohybridized with a “reference pool.” Samples were taken from batch cultivations at six times (7 h, 10 h, 14 h, 18 h, 22 h, and 30 h). The identified gene clusters, clusters A to C, are indicated; a specific region of cluster C is marked C1. An excerpt of the most relevant genes belonging to clusters A and C is shown on the right. All members of cluster B are specified by a relative increase in gene expression in the pantothenate producer throughout the fermentation.

FIG. 6.

Relative expression of plasmid-encoded genes in C. glutamicum ATCC 13032ΔilvA P-ilvEM3(pJC1ilvBNCD)(pMM55). Gene expression was measured with the LightCycler instrument using total RNA from the pantothenate production strain and the reference strain with empty cloning vectors. Relative gene expression was normalized using the values determined for the reference strain. The values are means of four measurements, and the error bars indicate standard deviations.