Robotic workflows for automated long-term adaptive laboratory evolution: improving ethanol utilization by Corynebacterium glutamicum.

BACKGROUND: Adaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation. RESULTS: Here, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW-1 h-1 and a growth rate of 0.15 ± 0.01 h-1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements. CONCLUSIONS: The established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

Cellular localization of the hybrid pyruvate/2-oxoglutarate dehydrogenase complex in the actinobacterium Corynebacterium glutamicum.

For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

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

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.

Characteristics of the GlnH and GlnX Signal Transduction Proteins Controlling PknG-Mediated Phosphorylation of OdhI and 2-Oxoglutarate Dehydrogenase Activity in Corynebacterium glutamicum.

In Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment. IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process.

Communities of Niche-optimized Strains (CoNoS) - Design and creation of stable, genome-reduced co-cultures.

Current bioprocesses for production of value-added compounds are mainly based on pure cultures that are composed of rationally engineered strains of model organisms with versatile metabolic capacities. However, in the comparably well-defined environment of a bioreactor, metabolic flexibility provided by various highly abundant biosynthetic enzymes is much less required and results in suboptimal use of carbon and energy sources for compound production. In nature, non-model organisms have frequently evolved in communities where genome-reduced, auxotrophic strains cross-feed each other, suggesting that there must be a significant advantage compared to growth without cooperation. To prove this, we started to create and study synthetic communities of niche-optimized strains (CoNoS) that consists of two strains of the same species Corynebacterium glutamicum that are mutually dependent on one amino acid. We used both the wild-type and the genome-reduced C1* chassis for introducing selected amino acid auxotrophies, each based on complete deletion of all required biosynthetic genes. The best candidate strains were used to establish several stably growing CoNoS that were further characterized and optimized by metabolic modelling, microfluidic experiments and rational metabolic engineering to improve amino acid production and exchange. Finally, the engineered CoNoS consisting of an l-leucine and l-arginine auxotroph showed a specific growth rate equivalent to 83% of the wild type in monoculture, making it the fastest co-culture of two auxotrophic C. glutamicum strains to date. Overall, our results are a first promising step towards establishing improved biobased production of value-added compounds using the CoNoS approach.

A Tetratricopeptide Repeat Scaffold Couples Signal Detection to OdhI Phosphorylation in Metabolic Control by the Protein Kinase PknG.

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of posttranslational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here, we present an investigation using in vivo experiments, as well as biochemical and structural methods, of the molecular basis of the regulation of PknG from Corynebacterium glutamicum (CgPknG), in the light of previous knowledge available for the kinase from Mycobacterium tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPRs) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain to a ß-hairpin within the flexible N-terminal region that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCE Bacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. Actinobacteria, one of the largest bacterial phyla of major importance for biotechnology, medicine, and agriculture, developed a unique control process that revolves around a key protein, the protein kinase PknG. Here, we use genetic, biochemical, and structural approaches to study PknG in a system that regulates glutamate production in Corynebacterium glutamicum, a species used for the industrial production of amino acids. The reported findings are conserved in related Actinobacteria, with broader significance for microorganisms that cause disease, as well as environmental species used industrially to produce amino acids and antibiotics every year.

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase.

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l-1 5-KF were formed from 150 g l-1 fructose within 23 h, corresponding to a space-time yield of 5.6 g l-1 h-1 . Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.

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

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.

Regulation of γ-Aminobutyrate (GABA) Utilization in Corynebacterium glutamicum by the PucR-Type Transcriptional Regulator GabR and by Alternative Nitrogen and Carbon Sources.

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid mainly formed by decarboxylation of L-glutamate and is widespread in nature from microorganisms to plants and animals. In this study, we analyzed the regulation of GABA utilization by the Gram-positive soil bacterium Corynebacterium glutamicum, which serves as model organism of the phylum Actinobacteria. We show that GABA usage is subject to both specific and global regulatory mechanisms. Transcriptomics revealed that the gabTDP genes encoding GABA transaminase, succinate semialdehyde dehydrogenase, and GABA permease, respectively, were highly induced in GABA-grown cells compared to glucose-grown cells. Expression of the gabTDP genes was dependent on GABA and the PucR-type transcriptional regulator GabR, which is encoded divergently to gabT. A ΔgabR mutant failed to grow with GABA, but not with glucose. Growth of the mutant on GABA was restored by plasmid-based expression of gabR or of gabTDP, indicating that no further genes are specifically required for GABA utilization. Purified GabR (calculated mass 55.75 kDa) formed an octamer with an apparent mass of 420 kDa and bound to two inverted repeats in the gabR-gabT intergenic region. Glucose, gluconate, and myo-inositol caused reduced expression of gabTDP, presumably via the cAMP-dependent global regulator GlxR, for which a binding site is present downstream of the gabT transcriptional start site. C. glutamicum was able to grow with GABA as sole carbon and nitrogen source. Ammonium and, to a lesser extent, urea inhibited growth on GABA, whereas L-glutamine stimulated it. Possible mechanisms for these effects are discussed.

Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome-reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High-throughput phenotyping using robotics-integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass-specific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801-cg2828 and rrnC-cg3298 could not have been inferred a priori. Strikingly, bioreactor fed-batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a 'high-performance' strain from batch screening into a 'low-performance' strain in fed-batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions.

HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression.

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.

Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose.

BACKGROUND: 5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. RESULTS: For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. CONCLUSION: We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.

Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum.

In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ΔcyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ΔcyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1F O -ATP synthase and numerous transport processes. The impaired membrane potential of the ΔcyaB mutant could be due to a decreased expression of the cytochrome bc 1-aa 3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ΔcyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ΔcyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ΔcyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.

NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface.

Alcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 106 clones created by random mutagenesis we identified the variant LbADHK71E. Kinetic analysis of the purified enzyme revealed that LbADHK71E had a ~ 16% lowered KM value and a 17% higher Vmax for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Pyruvate carboxylase from Corynebacterium glutamicum : purification and characterization.

Pyruvate carboxylase of Corynebacterium glutamicum serves as anaplerotic enzyme when cells are growing on carbohydrates and plays an important role in the industrial production of metabolites derived from the tricarboxylic acid cycle, such as L-glutamate or L-lysine. Previous studies suggested that the enzyme from C. glutamicum is very labile, as activity could only be measured in permeabilized cells, but not in cell-free extracts. In this study, we established conditions allowing activity measurements in cell-free extracts of C. glutamicum and purification of the enzyme by avidin affinity chromatography and gel filtration. Using a coupled enzymatic assay with malate dehydrogenase, Vmax values between 20 and 25 µmol min-1 mg-1 were measured for purified pyruvate carboxylase corresponding to turnover numbers of 160 - 200 s-1 for the tetrameric enzyme. The concentration dependency for pyruvate and ATP followed Michaelis-Menten kinetics with Km values of 3.76 ± 0.72 mM and 0.61 ± 0.13 mM, respectively. For bicarbonate, concentrations ≥5 mM were required to obtain activity and half-maximal rates were found at 13.25 ± 4.88 mM. ADP and aspartate inhibited PCx activity with apparent Ki values of 1.5 mM and 9.3 mM, respectively. Acetyl-CoA had a weak inhibitory effect, but only at low concentrations up to 50 µM. The results presented here enable further detailed biochemical and structural studies of this enzyme.

Identification of Surf1 as an assembly factor of the cytochrome bc1-aa3 supercomplex of Actinobacteria.

Respiration in aerobic Actinobacteria involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of CuA, CuB, heme a, and heme a3. In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc1-aa3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa3 oxidase. In contrast to the wild type, a bc1-aa3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa3 oxidase.

Pyruvate Carboxylase Variants Enabling Improved Lysine Production from Glucose Identified by Biosensor-Based High-Throughput Fluorescence-Activated Cell Sorting Screening.

Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in Corynebacterium glutamicum that enable improved l-lysine production from glucose. A suitable reporter strain was transformed with a pyc gene library created by error-prone PCR and screened by fluorescence-activated cell sorting (FACS) for cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration. Two pyruvate carboxylase variants, PCxT343A,I1012S and PCxT132A were identified allowing 9% and 19% increased lysine titers upon plasmid-based expression. Chromosomal expression of PCxT132A and PCxT343A variants led to 6% and 14% higher l-lysine levels. The new PCx variants can be useful also for other microbial strains producing TCA cycle-derived metabolites. Our approach indicates that a biosensor such as pSenLys enables directed evolution of many enzymes involved in converting a carbon source into the target metabolite.

Communities of Niche-Optimized Strains: Small-Genome Organism Consortia in Bioproduction.

Bacterial genome reduction is a common phenomenon in nature that has also inspired several projects with biotechnologically relevant organisms. In many cases, however, no performance improvements with the streamlined chassis were obtained. A comparative analysis of natural and synthetic reduced genomes and their corresponding ecological niches reveals that: (i) deleting expressed genes can lead to noticeable energy conservation usable for improved biomass or product synthesis; and (ii) the simplified strains depend on the environment (e.g., another community member) to complement deleted functions. Based on this analysis, we introduce the concept of Community of Niche-optimized Strains (CoNoS) as an alternative strategy to generate superior biotechnological production processes.

NADPH-related processes studied with a SoxR-based biosensor in Escherichia coli.

NADPH plays a crucial role in cellular metabolism for biosynthesis and oxidative stress responses. We previously developed the genetically encoded NADPH biosensor pSenSox based on the transcriptional regulator SoxR of Escherichia coli, its target promoter PsoxS and eYFP as fluorescent reporter. Here, we used pSenSox to study the influence of various parameters on the sensor output in E. coliduring reductive biotransformation of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by the strictly NADPH-dependent alcohol dehydrogenase of Lactobacillus brevis (LbAdh). Redox-cycling drugs such as paraquat and menadione strongly activated the NADPH biosensor and mechanisms responsible for this effect are discussed. Absence of the RsxABCDGE complex and/or RseC caused an enhanced biosensor response, supporting a function as SoxR-reducing system. Absence of the membrane-bound transhydrogenase PntAB caused an increased biosensor response, whereas the lack of the soluble transhydrogenase SthA or of SthA and PntAB was associated with a strongly decreased response. These data support the opposing functions of PntAB in NADP+ reduction and of SthA in NADPH oxidation. In summary, the NADPH biosensor pSenSox proved to be a useful tool to study NADPH-related processes in E. coli.