Discovery of novel amino acid production traits by evolution of synthetic co-cultures

Rico Zuchowski, Simone Schito, Friederike Neuheuser, Philipp Menke, Daniel Berger, Niels Hollmann, Srushti Gujar, Lea Sundermeyer, Christina Mack, Astrid Wirtz, Oliver H. Weiergräber, Tino Polen, Michael Bott, Stephan Noack & Meike Baumgart

Background

Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities.

Results

Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain.

Conclusion

Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Metabolic engineering of Corynebacterium glutamicum for production of scyllo-inositol, a drug candidate against Alzheimer's disease

Paul Ramp, Alexander Lehnert, Susana Matamouros, Astrid Wirtz, Meike Baumgart, Michael Bott

Abstract

Scyllo-inositol has been identified as a potential drug for the treatment of Alzheimer's disease. Therefore, cost-efficient processes for the production of this compound are desirable. In this study, we analyzed and engineered Corynebacterium glutamicum with the aim to develop competitive scyllo-inositol producer strains. Initial studies revealed that C. glutamicum naturally produces scyllo-inositol when cultured with myo-inositol as carbon source. The conversion involves NAD+-dependent oxidation of myo-inositol to 2-keto-myo-inositol followed by NADPH-dependent reduction to scyllo-inositol. Use of myo-inositol for biomass formation was prevented by deletion of a cluster of 16 genes involved in myo-inositol catabolism (strain MB001(DE3)Δiol1). Deletion of a second cluster of four genes (oxiC-cg3390-oxiD-oxiE) related to inositol metabolism prevented conversion of 2-keto-myo-inositol to undesired products causing brown coloration (strain MB001(DE3)Δiol1Δiol2). The two chassis strains were used for plasmid-based overproduction of myo-inositol dehydrogenase (IolG) and scyllo-inositol dehydrogenase (IolW). In BHI medium containing glucose and myo-inositol, a complete conversion of the consumed myo-inositol into scyllo-inositol was achieved with the Δiol1Δiol2 strain. To enable scyllo-inositol production from cheap carbon sources, myo-inositol 1-phosphate synthase (Ino1) and myo-inositol 1-phosphatase (ImpA), which convert glucose 6-phosphate into myo-inositol, were overproduced in addition to IolG and IolW using plasmid pSI. Strain MB001(DE3)Δiol1Δiol2 (pSI) produced 1.8 g/L scyllo-inositol from 20 g/L glucose and even 4.4 g/L scyllo-inositol from 20 g/L sucrose within 72 h. Our results demonstrate that C. glutamicum is an attractive host for the biotechnological production of scyllo-inositol and potentially further myo-inositol-derived products.

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism

Viviënne Mol, Martyn Bennett, Benjamín J. Sánchez, Beata K. Lisowska, Markus J. Herrgård, Alex Toftgaard Nielsen, David J. Leak, Nikolaus Sonnenschein

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.

Novel insights into the transcriptional regulation of cell division in Corynebacterium glutamicum

Kim Julia Kraxner

In the first part of this doctoral thesis the transcriptional regulation of the odhI gene (cg1630) of Corynebacterium glutamicum was analyzed. OdhI in its unphoshorylated state functions as inhibitor of the 2-oxoglutarate dehydrogenase complex (ODHC) by binding to the OdhA subunit. Phosphorylation of OdhI by serine/threonine protein kinases abolishes this effect. Inhibition of ODHC activity by OdhI was shown to be crucial for overproduction and secretion of L-glutamate, which is used as a flavour enhancer. Since downstream of odhI two genes presumably encoding transcriptional regulators (cg1631 and cg1633) are located, it was speculated that these could be involved in transcriptional regulation of odhI. However, transcriptome analysis of deletion mutants lacking cg1631 or cg1633 and DNA affinity chromatography with the odhI promoter did not support this hypothesis. Furthermore, no other potential transcriptional regulators of odhI could be identified. Thus, there is currently no evidence for transcriptional regulation of odhI. The second part of this thesis addresses the regulation of cytokinesis in C. glutamicum. In contrast to e.g. Escherichia coli and Bacillus subtilis, knowledge about regulators of cytokinesis in Actinobacteria is very limited. In this study, the so far uncharacterized Cg1631 protein was discovered to be a transcriptional regulator of the ftsZ gene in C. glutamicum encoding the key player of bacterial cell division. Therefore, Cg1631 was named FtsR, standing for FtsZ regulator. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology, emphasizing an important function of FtsR in cell division or cell wall synthesis. The wild-type phenotype could be restored by plasmid-based complementation. Chromatin affinity purification with subsequent next generation sequencing (ChAP-Seq) identified a region in the ftsZ promoter as a major FtsR binding site, but revealed also additional potential target genes. With the ChAP-Seq results a putative DNA-binding motif could be identified for FtsR. Transcriptional activation of ftsZ expression by FtsR was underlined by DNA microarray experiments, electrophoretic mobility shift assays (EMSAs), and reporter gene studies. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ftsR mutant is not solely caused by reduced ftsZ expression but involves additional factors. In summary, FtsR was identified as the first transcriptional regulator of ftsZ in C. glutamicum. Furthermore, since FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, it can be assumed that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria. This makes FtsR a promising target for the development of new antimicrobial drugs against pathogenic relatives of C. glutamicum

The conserved actinobacterial transcriptional regulator FtsR controls expression of ftsZ and further target genes and influences growth and cell division in Corynebacterium glutamicum

Kim Julia Kraxner, Tino Polen, Meike Baumgart, and Michael Bott

Key mechanisms of cell division and its regulation are well understood in model bacteria such as Escherichia coli and Bacillus subtilis. In contrast, current knowledge on the regulation of cell division in Actinobacteria is rather limited. FtsZ is one of the key players in this process, but nothing is known about its transcriptional regulation in Corynebacterium glutamicum, a model organism of the Corynebacteriales.

In this study, we used DNA affinity chromatography to search for transcriptional regulators of ftsZ in C. glutamicum and identified the Cg1631 protein as candidate, which was named FtsR. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology. Plasmid-based expression of native ftsR or of homologs of the pathogenic relatives Corynebacterium diphtheriae and Mycobacterium tuberculosis in the ΔftsR mutant could at least partially reverse the mutant phenotype. Absence of ftsR caused decreased expression of ftsZ, in line with an activator function of FtsR. In vivo crosslinking followed by affinity purification of FtsR and next generation sequencing of the enriched DNA fragments confirmed the ftsZ promoter as in vivo binding site of FtsR and revealed additional potential target genes and a DNA-binding motif. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ΔftsR mutant is not solely caused by reduced ftsZ expression, but involves further targets.

In this study, we identified and characterized FtsR as the first transcriptional regulator of FtsZ described for C. glutamicum. Both the absence and the overproduction of FtsR had severe effects on growth and cell morphology, underlining the importance of this regulatory protein. FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, which suggests that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria.

The consequence of an additional NADH dehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504

D. Kostner, B. Luchterhand, A. Junker, S. Volland, R. Daniel, J. Büchs, W. Liebl & A. Ehrenreich

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum

Dirk Oehler, Anja Poehlein, Andreas Leimbach, Nicolai Müller, Rolf Daniel, Gerhard Gottschalk & Bernhard Schink

Background

Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life.

Results

The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome.

Conclusions

The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.