A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production.

BACKGROUND: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes. RESULTS: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM1,2-PDO mMglucose-1 which is the highest value for C. glutamicum reported so far. CONCLUSIONS: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens.

Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed-batch processes are applied to obtain high space-time yields. In this study, we established an aerobic glucose-limited fed-batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 gX L-1 . However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L-1 . Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two-fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five- to six-fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L-1 with a volumetric productivity of 8.5 and 7.7 gSuc L-1 h-1 , respectively. Both strains showed a product yield of about 1.4 molSuc molGlc -1 , which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.

Metabolic engineering of Corynebacterium glutamicum for fatty alcohol production from glucose and wheat straw hydrolysate.

BACKGROUND: Fatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable. RESULTS: To engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L-1 from 20 g glucose L-1 with a product yield of 0.054 ± 0.001 Cmol Cmol-1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h-1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(Ptac-pntAB-TrrnB) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L-1 with a product yield of 0.054 ± 0.005 Cmol Cmol-1 and a volumetric productivity of 0.109 ± 0.005 gFAL L-1 h-1 in a pulsed fed-batch cultivation using wheat straw hydrolysate. CONCLUSION: The combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock.

Complete Genome Sequence of the Carboxydotrophic Knallgas Bacterium Pseudomonas carboxydohydrogena Strain DSM 1083.

Pseudomonas carboxydohydrogena is a lithoautotrophic and obligate aerobic alphaproteobacterium, which has the unique ability to utilize CO, CO2, H2, and mixtures thereof as sole carbon and energy sources. Here, we report the complete genome sequence of type strain DSM 1083 and its close relation to Afipia carboxidovorans strain OM5.

Improving growth properties of Corynebacterium glutamicum by implementing an iron-responsive protocatechuate biosynthesis.

Corynebacterium glutamicum experiences a transient iron limitation during growth in minimal medium, which can be compensated by the external supplementation of protocatechuic acid (PCA). Although C. glutamicum is genetically equipped to form PCA from the intermediate 3-dehydroshikimate catalysed by 3-dehydroshikimate dehydratase (encoded by qsuB), PCA synthesis is not part of the native iron-responsive regulon. To obtain a strain with improved iron availability even in the absence of the expensive supplement PCA, we re-wired the transcriptional regulation of the qsuB gene and modified PCA biosynthesis and degradation. Therefore, we ushered qsuB expression into the iron-responsive DtxR regulon by replacing the native promoter of the qsuB gene by the promoter PripA and introduced a second copy of the PripA -qsuB cassette into the genome of C. glutamicum. Reduction of the degradation was achieved by mitigating expression of the pcaG and pcaH genes through a start codon exchange. The final strain C. glutamicum IRON+ showed in the absence of PCA a significantly increased intracellular Fe2+ availability, exhibited improved growth properties on glucose and acetate, retained a wild type-like biomass yield but did not accumulate PCA in the supernatant. For the cultivation in minimal medium C. glutamicum IRON+ represents a useful platform strain that reveals beneficial growth properties on different carbon sources without affecting the biomass yield and overcomes the need of PCA supplementation.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production.

BACKGROUND: Itaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy. RESULTS: In this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis. CONCLUSION: As shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Systematic analysis of the underlying genomic architecture for transcriptional-translational coupling in prokaryotes.

Transcriptional-translational coupling is accepted to be a fundamental mechanism of gene expression in prokaryotes and therefore has been analyzed in detail. However, the underlying genomic architecture of the expression machinery has not been well investigated so far. In this study, we established a bioinformatics pipeline to systematically investigated >1800 bacterial genomes for the abundance of transcriptional and translational associated genes clustered in distinct gene cassettes. We identified three highly frequent cassettes containing transcriptional and translational genes, i.e. rplk-nusG (gene cassette 1; in 553 genomes), rpoA-rplQ-rpsD-rpsK-rpsM (gene cassette 2; in 656 genomes) and nusA-infB (gene cassette 3; in 877 genomes). Interestingly, each of the three cassettes harbors a gene (nusG, rpsD and nusA) encoding a protein which links transcription and translation in bacteria. The analyses suggest an enrichment of these cassettes in pathogenic bacterial phyla with >70% for cassette 3 (i.e. Neisseria, Salmonella and Escherichia) and >50% for cassette 1 (i.e. Treponema, Prevotella, Leptospira and Fusobacterium) and cassette 2 (i.e. Helicobacter, Campylobacter, Treponema and Prevotella). These insights form the basis to analyze the transcriptional regulatory mechanisms orchestrating transcriptional-translational coupling and might open novel avenues for future biotechnological approaches.

Acetate-based production of itaconic acid with Corynebacterium glutamicum using an integrated pH-coupled feeding control.

To date, most bio-based products of industrial biotechnology stem from sugar-based carbon sources originating from food and feed competing resources. Exemplary for bioproducts converted from glucose, the potential C5 platform chemical itaconic acid is presently produced by the filamentous fungus Aspergillus terreus. Here, an engineered strain of the industrial platform organism Corynebacterium glutamicum ATCC 13032 was used for acetate-based production of itaconic acid to overcome current production difficulties. For this purpose, C. glutamicum ICDR453C (pEKEx2-malEcadopt) with a mutated icd variant for reduced isocitrate dehydrogenase activity was constructed harbouring pEKEx2-malEcadopt, that includes a cis-aconitate dehydrogenase gene originating from A. terreus. Overall, a peak volumetric productivity of 1.01 gL-1h-1 was achieved resulting in an itaconate titer of 29.2 g/L, by using an integrated pH-coupled acetate feeding control in a fed-batch process without base titration. The results support the high potential of acetate as alternative substrate for bioproduction.

Exploiting Aerobic Carboxydotrophic Bacteria for Industrial Biotechnology.

Aerobic carboxydotrophic bacteria are a group of microorganisms which possess the unique trait to oxidize carbon monoxide (CO) as sole energy source with molecular oxygen (O2) to produce carbon dioxide (CO2) which subsequently is used for biomass formation via the Calvin-Benson-Bassham cycle. Moreover, most carboxydotrophs are also able to oxidize hydrogen (H2) with hydrogenases to drive the reduction of carbon dioxide in the absence of CO. As several abundant industrial off-gases contain significant amounts of CO, CO2, H2 as well as O2, these bacteria come into focus for industrial application to produce chemicals and fuels from such gases in gas fermentation approaches. Since the group of carboxydotrophic bacteria is rather unknown and not very well investigated, we will provide an overview about their lifestyle and the underlying metabolic characteristics, introduce promising members for industrial application, and give an overview of available genetic engineering tools. We will point to limitations and discuss challenges, which have to be overcome to apply metabolic engineering approaches and to utilize aerobic carboxydotrophs in the industrial environment.

Metabolic engineering of Vibrio natriegens for anaerobic succinate production.

The biotechnological production of succinate bears serious potential to fully replace existing petrochemical approaches in the future. In order to establish an economically viable bioprocess, obtaining high titre, yield and productivity is of central importance. In this study, we present a straightforward engineering approach for anaerobic succinate production with Vibrio natriegens, consisting of essential metabolic engineering and optimization of process conditions. The final producer strain V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) yielded 1.46 mol of succinate per mol of glucose under anaerobic conditions (85% of the theoretical maximum) and revealed a particularly high biomass-specific succinate production rate of 1.33 gSucc gCDW -1 h-1 compared with well-established production systems. By applying carbon and redox balancing, we determined the intracellular flux distribution and show that under the tested conditions the reductive TCA as well as the oxidative TCA/glyoxylate pathway contributed to succinate formation. In a zero-growth bioprocess using minimal medium devoid of complex additives and expensive supplements, we obtained a final titre of 60.4 gSucc l-1 with a maximum productivity of 20.8 gSucc l-1 h-1 and an overall volumetric productivity of 8.6 gSucc l-1 h-1 during the 7 h fermentation. The key performance indicators (titre, yield and productivity) of this first engineering approach in V. natriegens are encouraging and compete with costly tailored microbial production systems.

Exploiting unconventional prokaryotic hosts for industrial biotechnology.

Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic engineering ensures efficient and fast tailoring of microbial metabolism for a wide range of bioconversions. However, improving intrinsic properties such as tolerance, handling, growth, and substrate consumption rates is still challenging. At the same time, synthetic biology tools are becoming easier applicable and transferable to nonmodel organisms. These trends have resulted in the exploitation of new and unconventional microbial systems with sophisticated properties, which render them promising hosts for the bio-based industry. Here, we highlight the metabolic and cellular capabilities of representative prokaryotic newcomers and discuss the potential and drawbacks of these hosts for industrial application.

A Timed Off-Switch for Dynamic Control of Gene Expression in Corynebacterium Glutamicum.

Dynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/P vanABK * regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (P vanABK *). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/P vanABK * yielding C. glutamicum ΔP aceE ::vanR-P vanABK *. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔP aceE ::vanR-P vanABK * continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/P vanABK * system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔP aceE ::vanR-P vanABK *, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/P vanABK * system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid.

Micro-aerobic production of isobutanol with engineered Pseudomonas putida.

Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with P. putida from shake flask to fed-batch cultivation in a 30 L bioreactor. The design of a two-stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L-1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2-ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro-aerobic conditions during production doubled the integral glucose-to-isobutanol conversion yield to 60 mgisobutanol gglucose -1 while preventing undesired carbon loss as 2-ketogluconic acid.

Metabolic engineering of Vibrio natriegens.

Vibrio natriegens is emerging as a promising host for biotechnology which is basically due to the remarkable intrinsic properties such as the exceptionally high growth and substrate consumption rates. The facultatively anaerobic marine bacterium possesses a versatile metabolism, is able to utilize a variety of substrates as carbon and energy sources and is easy to handle in the lab. These features initiated the rapid development of genetic tools and resulted in extensive engineering of production strains in the past years. Although recent examples illustrate the potential of V. natriegens for biotechnology, a comprehensive understanding of the metabolism and its regulation is still lacking but essential to exploit the full potential of this bacterium. In this review, we summarize the current knowledge on the physiological traits and the genomic organization, provide an overview of the available genetic engineering tools and recent advances in metabolic engineering of V. natriegens. Finally, we discuss the obstacles which have to be overcome in order to establish V. natriegens as industrial production host.

Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host.

Today's possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicuml-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.

Engineering Pseudomonas putida KT2440 for the production of isobutanol.

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback-resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions.

CO2/HCO3- Accelerates Iron Reduction through Phenolic Compounds.

Iron is a vital mineral for almost all living organisms and has a pivotal role in central metabolism. Despite its great abundance on earth, the accessibility for microorganisms is often limited, because poorly soluble ferric iron (Fe3+) is the predominant oxidation state in an aerobic environment. Hence, the reduction of Fe3+ is of essential importance to meet the cellular demand of ferrous iron (Fe2+) but might become detrimental as excessive amounts of intracellular Fe2+ tend to undergo the cytotoxic Fenton reaction in the presence of hydrogen peroxide. We demonstrate that the complex formation rate of Fe3+ and phenolic compounds like protocatechuic acid was increased by 46% in the presence of HCO3- and thus accelerated the subsequent redox reaction, yielding reduced Fe2+ Consequently, elevated CO2/HCO3- levels increased the intracellular Fe2+ availability, which resulted in at least 50% higher biomass-specific fluorescence of a DtxR-based Corynebacterium glutamicum reporter strain, and stimulated growth. Since the increased Fe2+ availability was attributed to the interaction of HCO3- and chemical iron reduction, the abiotic effect postulated in this study is of general relevance in geochemical and biological environments.IMPORTANCE In an oxygenic environment, poorly soluble Fe3+ must be reduced to meet the cellular Fe2+ demand. This study demonstrates that elevated CO2/HCO3- levels accelerate chemical Fe3+ reduction through phenolic compounds, thus increasing intracellular Fe2+ availability. A number of biological environments are characterized by the presence of phenolic compounds and elevated HCO3- levels and include soil habitats and the human body. Fe2+ availability is of particular interest in the latter, as it controls the infectiousness of pathogens. Since the effect postulated here is abiotic, it generally affects the Fe2+ distribution in nature.

A synthetic glycerol assimilation pathway demonstrates biochemical constraints of cellular metabolism.

The engineering of synthetic metabolic routes can provide valuable lessons on the roles of different biochemical constraints in shaping pathway activity. In this study, we designed and engineered a novel glycerol assimilation pathway in Escherichia coli. While the synthetic pathway was based only on well-characterized endogenous reactions, we were not able to establish robust growth using standard concentrations of glycerol. Long-term evolution failed to improve growth via the pathway, indicating that this limitation was not regulatory but rather relates to fundamental aspects of cellular metabolism. We show that the activity of the synthetic pathway is fully controlled by three key physicochemical constraints: thermodynamics, kinetics and metabolite toxicity. Overcoming a thermodynamic barrier at the beginning of the pathway requires high glycerol concentrations. A kinetic barrier leads to a Monod-like growth dependency on substrate concentration, but with a very high substrate saturation constant. Finally, the flat thermodynamic profile of the pathway enforces a pseudoequilibrium between glycerol and the reactive intermediate dihydroxyacetone, which inhibits growth when the feedstock concentration surpasses 1000 mm. Overall, this study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism.

Comprehensive Analysis of C. glutamicum Anaplerotic Deletion Mutants Under Defined d-Glucose Conditions.

Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate.

Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h-1 in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (PCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h-1 and 1.03 gGlc gCDW -1 h-1 in minimal medium without abovementioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes.