Microbial cell factories - efficient production of value added compounds

Succinate production with Corynebacterium glutamicum

Succinate is a C4-dicarboxylate with interesting prospects for a sustainable chemical industry when produced biotechnologically in large amounts from renewable carbon sources. It can serve as a precursor for the production of a great variety of important bulk chemicals, such as tetrahydrofurane (THF), 1,4-butanediol, γ-butyrolactone, or maleic anhydride, which are currently produced petrochemically (1-3). Therefore, succinate was identified as one of the top 12 building block chemicals from biomass by the U.S. Department of Energy. Within the framework of an ERA-IB project, we developed C. glutamicum strains by metabolic engineering for the synthesis of succinate both under aerobic and anaerobic conditions.

For succinate production from glucose under aerobic conditions, the succinate dehydrogenase genes sdhCAB were deleted first, leading to an accumulation of 4.7 g l-1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by-product. By deleting genes for all known acetate-producing pathways (pta-ackA, pqo, and cat) acetate production could be reduced by 83% and succinate production increased up to 7.8 g l-1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l-1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)-1 h-1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimisation of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain C. glutamicum BL1 (ΔpqoΔpta-ackAΔsdhCABΔcat) containing plasmid pAN6-pycP458Sppc led to an additional increase of the product yield to 0.45 mol succinate/mol glucose and a titre of 10.6 g l-1 (90 mM) succinate (4).

Besides glucose, also glycerol could be used as renewable carbon source for aerobic succinate production. For this purpose, strain BL1 was transformed with plasmid pVWEx1-glpFKD coding for glycerol utilisation genes of Escherichia coli (Fig. 1). This plasmid was constructed in the group of Volker Wendisch and enables growth of C. glutamicum with glycerol as sole carbon source. The resulting strains were tested in minimal medium for aerobic succinate production from glycerol, which is a by-product in biodiesel synthesis. Strain BL-1/pVWEx1-glpFKD formed 79 mM (9.3 g l-1) succinate from 375 mM glycerol, representing 42% of the maximal theoretical yield under aerobic conditions. A specific succinate production rate of 1.55 mmol g-1 (cdw) h-1 and a volumetric productivity of 3.59 mM h-1 were obtained, the latter value representing the highest one currently described in literature. The results demonstrate that metabolically engineered strains of C. glutamicum are well suited for aerobic succinate production from glycerol.

Scheme of the central metabolism of C. glutamicum tailored for aerobic succinate production from glycerol
Figure 1. Scheme of the central metabolism of C. glutamicum tailored for aerobic succinate production from glycerol. Enzymes whose genes were deleted are indicated by ‘X’. The reactions affected by these deletions and their products are displayed in grey. Enzymes whose genes were overexpressed are highlighted in grey boxes and the arrows for the corresponding reactions are thickened. Abbreviations: ACN, aconitase; AK, acetate kinase; CoAT, acetyl-CoA : CoA transferase; CS, citrate synthase; FUM, fumarase; GF, glycerol facilitator (from E. coli); GK, glycerol kinase (from E. coli); G-3-P DH, glycerol-3-phosphate dehydrogenase (from E. coli); ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MQO, malate : menaquinone oxidoreductase; MS, malate synthase; OAA, oxaloacetate; ODHC, 2-oxoglutarate dehydrogenase complex; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PEPCx, PEP carboxylase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PTA, phosphotransacetylase; PQO, pyruvate : menaquinone oxidoreductase; SCS, succinyl-CoA synthetase; SDH, succinate dehydrogenase.

C. glutamicum wild type is known to produce L-lactate, acetate, and succinate under oxygen-limiting conditions, whereby succinate is formed by the reduction of oxaloacetate to succinate. In our work, we addressed two shortfalls of anaerobic succinate production, which are the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 was constructed (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat, Δpqo, Δpta-ackA, ΔldhA). Chromosomal integration of the pyruvate carboxylase gene pycP458S into BOL-1 resulted in strain BOL-2, which catalysed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the co-substrate formate (Fig. 2), the fdh gene from Mycobacterium vaccae coding for an NAD+-coupled formate dehydrogenase (FDH) was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate (Figure 2), strain BOL-3/pAN6-gap accumulated 134 g L-1 succinate in 53 h with an average succinate production rate of 1.59 mmol g (cdw)-1 h-1. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose) (5).

Graphic Anaerobic succinate production with C. glutamicum
Graphic Anaerobic succinate production with C. glutamicum
Figure 2. Anaerobic succinate production with C. glutamicum. The upper part shows the concept for obtaining 2 succinate from 1 glucose and 2 formate. The oxidation of formate provides not only carbon dioxide for PEP or pyruvate carboxylation but also reducing equivalents for reduction of oxaloacetate to succinate. The lower part shows an anaerobic fed-batch fermentation with C. glutamicum strain BOL-3/pAN6-gap with co-utilisation of glucose and formate.

Itaconate production with Corynebacterium glutamicum

Itaconic acid (2-methylidenebutanedioic acid) is an unsaturated dicarboxylic acid which has gained considerable interest in recent years as it was reported to be one of the top 12 building block chemicals that can be produced from biomass and replace fossil-based chemicals. Aspergillus terreus is the dominant production host for itaconate, which is synthesised via the decarboxylation of cis-aconitate by the enzyme cis-aconitate decarboxylase. Within the framework of an ERA-IB project we explored the capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolise it. Expression of the A. terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in the wild-type strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg-1 and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l-1), a molar yield of 0.4 mol mol-1, and a volumetric productivity of 2.1 mmol l-1 h-1. These results demonstrate the potential of C. glutamicum for itaconate production (Fig. 3) (6).

Graphic Development of an itaconate-producer strain of C. glutamicum by metabolic engineering
Graphic Development of an itaconate-producer strain of C. glutamicum by metabolic engineering
Figure 3. Development of an itaconate-producer strain of C. glutamicum by metabolic engineering. The upper part represents a simplified view of the pathway from glucose to itaconate. The lower part shows the four major steps of strain development, which were the overexpression of the cis-aconitate decarboxylase gene CAD1 of Aspergillus terreus, the improvement of Cad activity by fusion with the maltose-binding protein, the use of nitrogen limiting conditions, and the reduction of isocitrate dehydrogenase activity by exchange of the ATG start of the icd gene by TTG.

Production of L-leucine and its precursor ketoisocaproate with Corynebacterium glutamicum

The branched-chain amino acids (BCAAs) L-valine, L-isoleucine, and L-leucine are essential amino acids and used as components of pharmaceuticals, in animal feed industry, and as additives of infusion solutions and dietary products. Compared to L-glutamate or L-lysine, BCAAs are currently produced in much smaller quantities of 1000 - 3000 tons per year. However, the market is substantially growing, requiring efficient production processes. In an industry-financed project, we developed an efficient L-leucine production strain of C. glutamicum. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a Ki of 0.4 mM. We identified a feedback-resistant IMPS variant carrying two amino acid exchanges (R529H, G532D). The corresponding leuAfbr gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterised in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g L-1 (Fig. 4). The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol L-1 h-1. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application (7).

Graphic L-Leucine production with the metabolically engineered C. glutamicum strain MV-LeuF2
Figure 4. L-Leucine production with the metabolically engineered C. glutamicum strain MV-LeuF2. The left panel shows a representative fed-batch fermentation with the formation of L-leucine, L-valine, L-alanine, and L-lysine. The batch phase ended and the feed phase started after 24.5h. The cultivation was stopped after 56 h. The right pictures show that thick foam was observed at the end of the cultivation and after centrifugation of the culture broth, a whitish L-leucine sediment covered by the cells was observed.

2-Ketoisocaproate (KIC), the last intermediate in L-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in L-leucine production strains of C. glutamicum, KIC became the major product, however, the strains were auxotrophic for L-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried, besides the ilvE start codon exchange, three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM L-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l−1) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC L−1 h−1 (8). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol (9).

Development of a T7-RNA polymerase-based expression system for Corynebacterium glutamicum

Although C. glutamicum is a well-established model organism in white biotechnology, there are only a few systems for the adjustable (over)expression of homologous and heterologous genes available. Hence, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible expression system based on the well-known T7-RNA polymerase in the prophage-free strain C. glutamicum MB001 (Fig. 5A) (10). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter (Fig. 5B). The properties of the system were evaluated using eyfp as heterologous target gene (Fig. 6). Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. This system is now widespread and used for a broad range of projects.

Graphic The T7-RNA polymerase-based expression system for Corynebacterium glutamicum
Figure 5. The T7-RNA polymerase-based expression system for Corynebacterium glutamicum. A. genomic region of C. glutamicum MB001(DE3) carrying the DE3 insertion. A 4.5 kb DNA fragment was amplified from chromosomal DNA of E. coli BL21(DE3) and inserted into the intergenic region of cg1121-cg1122 of MB001(DE3). The fragment contains lacI, lacZα and T7 gene 1, the latter two under the transcriptional control of the lacUV5 promoter and its three LacI operator sites O1-O3. B. Map of the expression plasmid pMKEx2, which is based on pJC1 and an expression cassette from pET52b. The region between the T7 promoter and the T7 terminator is shown in detail. Taken from (10)

Graphic T7 RNAP-dependent expression of eyfp in C. glutamicum MB001(DE3)/pMKEx2-eyfp
Figure 6. T7 RNAP-dependent expression of eyfp in C. glutamicum MB001(DE3)/pMKEx2-eyfp. The strain was cultivated for 24 h aerobically in 2xTY medium using a BioLector at 1200 rpm and 30 °C. Gene expression was induced 2 hours after starting the cultivation by addition of 0 - 250 µM IPTG. The upper part shows fluorescence microscopy images of the cultures with 0 µM, 10 µM, 25 µM, or 250 µM IPTG. Images were taken with an exposure time of 40 ms. The red bar represents a length of 5 µm. The lower part of the figure shows a FACS analysis of the same cultures. Dot plots of eYFP fluorescence versus forward scatter are shown. Adapted from (10).

Construction of C. glutamicum chassis strains

In 2011 we started a joint project with several partners (Volker Wendisch, Jörn Kalinowski, Wolfgang Wiechert, Stephan Noack, Reinhard Krämer, Evonik) with the aim to construct genome-reduced C. glutamicum chassis strains. For this project we chose the so called “top-down” approach, which means that we started with the wild type genome and deleted DNA regions that are not relevant. In contrast to other minimal genome projects aiming at maximal genome reduction and therefore requiring rich complex media for cultivation, we aimed at the construction of chassis strains with a reduced complexity, but maintaining the same growth rate in glucose minimal medium as the parent wild type. These strains should therefore be suitable for biotechnological applications, in which media components are an important cost factor. The term “relevant” therefore defines genes that are not required for growth in glucose minimal medium with a growth rate of about 0.4 h-1 in shake flasks. The first milestone was reached with the construction of the prophage-free strain MB001 (11), which is already a well-established strain for various projects. For further genome reduction we evaluated every native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, 41 gene clusters were determined ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful, but 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level (12). In contrast, 26 deleted clusters were found to include exclusively “irrelevant” genes for wild type-like growth in glucose minimal medium. The combination of several of these clusters led to the construction of chassis strain C1* with a 13.4% reduced genome without any negative impact on the biological fitness under defined conditions in comparison to the wild type (Fig. 7) (13).

Graphic Progress in the C. glutamicum genome reduction project
Figure 7: Progress in the C. glutamicum genome reduction project. From the wild type ATCC 13032 via the prophage-free strain MB001 to C1* with a 13.4% smaller genome. Taken from (13).

Robustness of Corynebacterium glutamicum during 1,5-diaminopentane production

Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important characteristic of industrial production organisms. In a close collaboration with the group of Marco Oldiges in our institute we studied the robustness of a 1,5-diaminopentane-producing strain C. glutamicum in a two compartment scale-down device to create short-term environmental changes, in particular oxygen limitation, simulating industrial scale cultivation conditions (Fig. 8). Using multi omics-based methods we showed, amongst other things, that L-lactate serves as a reversible and flexible external buffer for carbon and redox equivalents, thus allowing C. glutamicum to deal with oxygen inhomogeneity in large scale processes (14).

Graphic Comparison of mRNA and proteins levels of a cadaverine-producing C. glutamicum strain during cultivation in two-compartment scale-down device (SD-I and SD-II) vs. cultivation in a reference process (REF) with sufficient oxygen supply.
Graphic Comparison of mRNA and proteins levels of a cadaverine-producing C. glutamicum strain during cultivation in two-compartment scale-down device (SD-I and SD-II) vs. cultivation in a reference process (REF) with sufficient oxygen supply. Figure 8. Comparison of mRNA and proteins levels of a cadaverine-producing C. glutamicum strain during cultivation in two-compartment scale-down device (SD-I and SD-II) vs. cultivation in a reference process (REF) with sufficient oxygen supply. Red and green arrows represent increased and decreased mRNA or protein levels in the scale-down device compared to the reference process, respectively. Targets where both the mRNA level and the protein level of a particular gene was increased or decreased are shown in the intersection. Taken from (14).

References

1. Litsanov, B., Brocker, M., Oldiges, M., and Bott, M. (2014) Succinic acid. in Bioprocessing of renewable resources to commodity bioproducts (Kondo, A., and Bisaria, V. eds.), John Wiley & Sons, Hoboken, New Jersey, USA. pp 437-474

2. Eikmanns, B. J., and Bott, M. (2015) Engineering Corynebacterium glutamicum for the production of organic acids and alcohols. in Corynebacterium glutamicum: From systems biology to biotechnological applications (Burkovski, A. ed.), Caister Academic Press, Norfolk, UK. pp 111-137

3. Wendisch, V. F., Bott, M., and Eikmanns, B. J. (2006) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Curr. Opin. Microbiol. 9, 268-274

4. Litsanov, B., Kabus, A., Brocker, M., and Bott, M. (2012) Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum. Microb. Biotechnol. 5, 116-128

5. Litsanov, B., Brocker, M., and Bott, M. (2012) Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate. Appl. Environ. Microbiol. 78, 3325-3337

6. Otten, A., Brocker, M., and Bott, M. (2015) Metabolic engineering of Corynebacterium glutamicum for the production of itaconate. Metab. Eng. 30, 156-165

7. Vogt, M., Haas, S., Klaffl, S., Polen, T., Eggeling, L., van Ooyen, J., and Bott, M. (2014) Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metab. Eng. 22, 40-52

8. Vogt, M., Haas, S., Polen, T., van Ooyen, J., and Bott, M. (2015) Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes. Microb. Biotechnol. 8, 351-360

9. Vogt, M., Brüsseler, C., Ooyen, J. V., Bott, M., and Marienhagen, J. (2016) Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum. Metab. Eng. 38, 436-445

10. Kortmann, M., Kuhl, V., Klaffl, S., and Bott, M. (2015) A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level. Microb. Biotechnol. 8, 253-265

11. Baumgart, M., Unthan, S., Rückert, C., Sivalingam, J., Grünberger, A., Kalinowski, J., Bott, M., Noack, S., and Frunzke, J. (2013) Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology. Appl. Environ. Microbiol. 79, 6006-6015

12. Unthan, S., Baumgart, M., Radek, A., Herbst, M., Siebert, D., Brühl, N., Bartsch, A., Bott, M., Wiechert, W., Marin, K., Hans, S., Krämer, R., Seibold, G., Frunzke, J., Kalinowski, J., Rückert, C., Wendisch, V. F., and Noack, S. (2015) Chassis organism from Corynebacterium glutamicum – a top-down approach to identify and delete irrelevant gene clusters. Biotechnol. J. 10, 290-301

13. Baumgart, M., Unthan, S., Kloß, R., Radek, A., Polen, T., Tenhaef, N., Müller, M. F., Küberl, A., Siebert, D., Brühl, N., Marin, K., Hans, S., Krämer, R., Bott, M., Kalinowski, J., Wiechert, W., Seibold, G., Frunzke, J., Rückert, C., Wendisch, V. F., and Noack, S. (2017) Corynebacterium glutamicum chassis C1*: building and testing a novel platform host for synthetic biology and industrial biotechnology. ACS Synth. Biol., In press

14. Limberg, M. H., Schulte, J., Aryani, T., Mahr, R., Baumgart, M., Bott, M., Wiechert, W., and Oldiges, M. (2017) Metabolic profile of 1,5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor inhomogeneities. Biotechnol. Bioeng. 114, 560-575

Last Modified: 30.08.2023