From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production

Here, we describe the development of a genetically defined strain of l-lysine hyperproducing Corynebacterium glutamicum by systems metabolic engineering of the wild type. Implementation of only 12 defined genome-based changes in genes encoding central metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L(-1) lysine and a productivity of 4.0 g L(-1) h(-1) in fed-batch culture. The specific glucose uptake rate of the wild type could be completely maintained during the engineering process, providing a highly viable producer. For these key criteria, the genetically defined strain created in this study lies at the maximum limit of classically derived producers developed over the last fifty years. This is the first report of a rationally derived lysine production strain that may be competitive with industrial applications. The design-based strategy for metabolic engineering reported here could serve as general concept for the rational development of microorganisms as efficient cellular factories for bio-production.     

Evolutionary engineering of Corynebacterium glutamicum

A unique feature of biotechnology is that we can harness the power of evolution to improve process performance. Rational engineering of microbial strains has led to the establishment of a variety of successful bioprocesses, but it is hampered by the overwhelming complexity of biological systems. Evolutionary engineering represents a straightforward approach for fitness‐linked phenotypes (e.g., growth or stress tolerance) and is successfully applied to select for strains with improved properties for particular industrial applications. In recent years, synthetic evolution strategies have enabled selection for increased small molecule production by linking metabolic productivity to growth as a selectable trait. This review summarizes the evolutionary engineering strategies performed with the industrial platform organism Corynebacterium glutamicum. An increasing number of recent studies highlight the potential of adaptive laboratory evolution (ALE) to improve growth or stress resistance, implement the utilization of alternative carbon sources, or improve small molecule production. Advances in next‐generation sequencing and automation technologies will foster the application of ALE strategies to streamline microbial strains for bioproduction and enhance our understanding of biological systems.     

Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.     

Phosphotransferase-dependent glucose transport in Corynebacterium glutamicum

G.M. MALIN AND G.I. BOURD. 1991. The transport system for glucose and its non-metabolizable analogue methyl-α-D-glucoside (MG) has been described in Corynebacterium glutamicum. The initial product of the transport reaction was shown to be a phosphate ester of MG (MGP). Free MG appeared inside the cells as a result of MGP dephosphorylation. The bacteria transported MG with an apparent K_m of 0.08 ± 0.017 mmol/l and V_max of 21 ± 2.3 nmol/(min × mg dry wt). Toluenized cells and crude cell extracts catalysed phosphoenolpyruvate (PEP)-dependent phosphorylation of MG and glucose. Both the membrane and the cytoplasmic fractions of bacterial extracts were required for phosphotransferase reaction. Most of the spontaneous mutants resistant to 2-deoxyglucose (DG), xylitol and 5-thioglucose were defective both in transport and in PEP-dependent phosphorylation of MG. Some strains were defective only in glucose utilization and some were also unable to grow on a number of other sugars. The phosphotransferase activity in extracts from mutant cells was restored by the addition of either membrane or cytoplasmic fraction from wild type bacteria. It was concluded that Corynebacterium glutamicum accumulated glucose and MG by means of a PEP-dependent phosphotransferase system (PTS).     

Comparisons of potentials for L-lysine production among different Corynebacterium glutamicum strains

Corynebacterium glutamicum is an industrially important organism that is most widely used for the production of various amino acids. A defined L-lysine-producing mutant was generated by introduction of the lysC mutation (T311I) into each of six representative C. glutamicum strains. The resulting six isogenic mutants were compared for L-lysine production under traditional 30 degrees C conditions and industrially more advantageous 40 degrees C conditions. It was found that there were significant differences in yield and productivity, especially at 40 degrees C. These results indicate the diversity among C. glutamicum strains in fermentative characters, as well as the importance of selecting a strain with industrially best performance.     

Large-scale engineering of the Corynebacterium glutamicum genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.     

Optimisation of a culture medium containing fish silage for L-lysine production by Corynebacterium glutamicum

In a first step the effects of 10 components of a culture medium designed for L-lysine production were evaluated with a 2(10-6) factorial design. Among them, glucose, fish silage, and ammonium sulphate showed a significant effect. In a second step, an orthogonal-central composite experimental design and response surface methodology was performed with five from the 10 initial compounds. The determination coefficient (R2) of the fitted second-order model was 0.990. L-lysine production with the optimised medium increased 2.6 times as compared with the original medium.     

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs

Journal of Industrial Microbiology & Biotechnology - Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other...     

Isolation and prominent characteristics of an L-lysine hyperproducing strain of Corynebacterium glutamicum

Summary A strain of Corynebacterium glutamicum was isolated that accumulated up to 44 g/l of L-lysine-HCl from 100 g/l of glucose × H₂O in a simple mineral salts medium. This strain was obtained from the wildtype by two mutagenesis steps. In the first step the aminoethyl-cysteine-resistant strain MH20 was obtained and in the second step the Leu ^— derivative MH20-22B. Enzymatic analysis of the hyperproducer MH20-22B revealed that this strain has feedback-resistant aspartate kinase and is devoid of isopropylmalate dehydratase. In addition, this strain has an extraordinarily high secretion rate of lysine (0.57 mmol/g dry weight and h), whereas strain MH20 has a low secretion rate (0.19 mmol/g per hour), and both strains have comparable cytosolic lysine concentrations. This suggests that the secretory step is influenced n the hyperproducer. Applying gene-directed mutagenesis, the aspartate kinase gene of the isolated strain (coding for feedback-resistant enzyme) was replaced by the gene coding of feedback-sensitive wild-type enzyme. The resulting strain still secretes lysine, although in low amounts (2 g/l). This is proof of the superior role of kinase regulation in metabolite flow and is indicative of unknown mutations, one of which is probably in the secretory system. Correspondence to: L. Eggeling     

Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation

Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.     

Metabolic analysis of glutamate production by Corynebacterium glutamicum

The dynamic behavior of the metabolism of Corynebacterium glutamicum during L-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. Glutamate production was induced by an increase of the temperature and a final concentration of 80 g/l was attained. During the production phase, various other compounds, notably lactate, trehalose, and DHA were secreted to the medium. Intracellular metabolites analysis showed important variations of glycolytic intermediates and NADH, NAD coenzymes levels throughout the production phase. Two phenomena occur during the production phase which potentially provoke a decrease in the glutamate yield: Both the intracellular concentrations of glycolytic intermediates and the NADH/NAD ratio increase significantly during the period in which the overall metabolic rates decline. This correlates with the decrease in glutamate yield due in part to the production of lactate and also to the period of the fermentation in which growth no longer occurred.     

Metabolic engineering of Corynebacterium glutamicum for L-serine production

Although L-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via L-serine formation, previous attempts to obtain a strain producing L-serine from glucose have not been successful. We functionally identified the genes serC and serB from Corynebacterium glutamicum, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic serA gene, serA(delta197), encoding an L-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of L-serine, as did the overexpression of these genes in a strain with the L-serine dehydratase gene sdaA deleted. However, reduced expression of the serine hydroxymethyltransferase gene glyA, in combination with the overexpression of serA(delta197), serC, and serB, resulted in a transient accumulation of up to 16 mM L-serine in the culture medium. When sdaA was also deleted, the resulting strain, C. glutamicum delta sdaA::pK18mobglyA'(pEC-T18mob2serA(delta197)CB), accumulated up to 86 mM L-serine with a maximal specific productivity of 1.2 mmol h(-1) g (dry weight)(-1). This illustrates a high rate of L-serine formation and also utilization in the C. glutamicum wild type. Therefore, metabolic engineering of L-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism.     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering

The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates.     

Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.     

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.