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The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol -1 . Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol -1 , representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L -1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production., Results: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl 2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 P tac pedACD Cg provided 66 mg L -1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain P tuf pedACD Cg allowed successful production without the need for IPTG., Conclusions: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production., (© 2023. The Author(s).)

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis-muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p- hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated., Competing Interests: Declaration of competing interest The authors have filed patent applications on the use of lignin for bio-production., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing., Results: Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectD PST achieved a titre of 74 g L -1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABC opt , was successfully achieved without intermediate purification., Conclusions: C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar., (© 2022. The Author(s).)

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. Denovo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L -1 AVA in a fed-batch process and achieved a high yield of 0.21 g g -1 . Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L -1 AVA at a yield of 0.34 g g -1 and a maximum productivity of 1.52 g L -1 h -1 , outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L -1 l-lysine from mannitol at a yield of 0.26 mol mol -1 and a maximum productivity of 2.1 g L -1 h -1 . Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol -1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol -1 . Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity., (© 2021 The Author(s).)

Ectoine is formed in various bacteria as cell protectant against all kinds of stress. Its preservative and protective effects have enabled various applications in medicine, cosmetics, and biotechnology, and ectoine therefore has high commercial value. Industrially, ectoine is produced in a complex high-salt process, which imposes constraints on the costs, design, and durability of the fermentation system. Here, Corynebacterium glutamicum is upgraded for the heterologous production of ectoine from sugar and molasses. To overcome previous limitations, the ectoine pathway taken from Pseudomonas stutzeri is engineered using transcriptional balancing. An expression library with 185,193 variants is created, randomly combining 19 synthetic promoters and three linker elements. Strain screening discovers several high-titer mutants with an improvement of almost fivefold over the initial strain. High production thereby particularly relies on a specifically balanced ectoine pathway. In an optimized fermentation process, the new top producer C. glutamicum ectABC opt achieves an ectoine titer of 65 g L -1 and a specific productivity of 120 mg g -1  h -1 . This process is the first reported example of a simple fermentation process under low-salt conditions using well-established feedstocks to produce ectoine with industrial efficiency. There is a compelling case for more intensive implementation of transcriptional balancing in future metabolic engineering of C. glutamicum., (© 2019 The Authors. Biotechnology Journal Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Corynebacterium glutamicum is a major workhorse in industrial biotechnology. For the past several decades, this soil bacterium has been used to produce the amino acids l-glutamate and l-lysine at a level of 6 million tons per year. Utilizing novel genome editing methods and advanced tools of systems and synthetic biology, the portfolio of C. glutamicum has exploded from classical food and feed products to the chemical industry, cosmetics and healthcare applications. Within the past five years, the number of industrial products has almost doubled to approximately 70 natural and non-natural compounds. This review summarizes the state-of-the-art regarding metabolically engineered cell factories of C. glutamicum, illustrates recent key technological developments and provides examples of the major industrial applications of this important microbe., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Cis, cis-muconic acid (MA) is a dicarboxylic acid of recognized industrial value. It provides direct access to adipic acid and terephthalic acid, prominent monomers of commercial plastics., Results: In the present work, we engineered the soil bacterium Corynebacterium glutamicum into a stable genome-based cell factory for high-level production of bio-based MA from aromatics and lignin hydrolysates. The elimination of muconate cycloisomerase (catB) in the catechol branch of the β-ketoadipate pathway provided a mutant, which accumulated MA at 100% molar yield from catechol, phenol, and benzoic acid, using glucose as additional growth substrate. The production of MA was optimized by constitutive overexpression of catA, which increased the activity of the encoded catechol 1,2-dioxygenase, forming MA from catechol, tenfold. Intracellular levels of catechol were more than 30-fold lower than extracellular levels, minimizing toxicity, but still saturating the high affinity CatA enzyme. In a fed-batch process, the created strain C. glutamicum MA-2 accumulated 85 g L -1 MA from catechol in 60 h and achieved a maximum volumetric productivity of 2.4 g L -1  h -1 . The strain was furthermore used to demonstrate the production of MA from lignin in a cascade process. Following hydrothermal depolymerization of softwood lignin into small aromatics, the MA-2 strain accumulated 1.8 g L -1 MA from the obtained hydrolysate., Conclusions: Our findings open the door to valorize lignin, the second most abundant polymer on earth, by metabolically engineered C. glutamicum for industrial production of MA and potentially other chemicals.

The amino acid lysine is among the world's most important biotechnological products, and enabling its manufacture from the most attractive new materials is an ever-present challenge. In this study, we describe a cell factory of Corynebacterium glutamicum, which produces lysine from mannitol. A preliminary mutant C. glutamicum SEA-1 obtained by the deletion of the mannitol repressor MtlR in the glucose-based, lysine-producing strain C. glutamicum LYS-12 produced only small amounts of lysine. This limitation was due to a significant accumulation of fructose and a limited NADPH supply, which caused a low flux of only 6% into the oxidative pentose phosphate (PP) pathway. Subsequent expression of fructokinase slightly increased production but failed to substantially redirect the flux from the Emden-Meyerhof-Parnas (EMP) pathway to the PP pathway. This suggested the design of C. glutamicum SEA-3, which overexpressed the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase GapN from Streptococcus mutans and coupled the EMP pathway flux to NADPH formation. When grown on mannitol, the SEA-3 strain had a lysine yield of 0.24 mol mol -1 and a specific productivity of 1.3 mmol g -1 h -1 , approximately 60% and 75% higher, respectively, than those of the basic producer SEA-1. A computational pathway analysis revealed that this design would potentially enable a lysine yield of 0.9 mol mol -1 , providing room for further development. Our findings open new avenues for lysine production from marine macroalgae, which is farmed globally as an attractive third-generation renewable resource. Mannitol is a major constituent of these algae (up to 30% and higher) and can be easily extracted from their biomass with hot water., (Copyright © 2018. Published by Elsevier Inc.)

Since its discovery 60 years ago, Corynebacterium glutamicum has evolved into a workhorse for industrial biotechnology. Traditionally well known for its remarkable capacity to produce amino acids, this Gram-positive soil bacterium, has become a flexible, efficient production platform for various bulk and fine chemicals, materials, and biofuels. The central turnstile of all these achievements is our excellent understanding of its metabolism and physiology. This knowledge base, together with innovative systems metabolic engineering concepts, which integrate systems and synthetic biology into strain engineering, has upgraded C. glutamicum into one of the most successful industrial microorganisms in the world.

Background: The steadily growing world population and our ever luxurious life style, along with the simultaneously decreasing fossil resources has confronted modern society with the issue and need of finding renewable routes to accommodate for our demands. Shifting the production pipeline from raw oil to biomass requires efficient processes for numerous platform chemicals being produced with high yield, high titer and high productivity., Results: In the present work, we established a de novo bio-based production process for the two carbon-5 platform chemicals 5-aminovalerate and glutarate on basis of the lysine-hyperproducing strain Corynebacterium glutamicum LYS-12. Upon heterologous implementation of the Pseudomonas putida genes davA, encoding 5-aminovaleramidase and davB, encoding lysine monooxygenase, 5-aminovalerate production was established. Related to the presence of endogenous genes coding for 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase, 5-aminovalerate was partially converted to glutarate. Moreover, residual L-lysine was secreted as by-product. The issue of by-product formation was then addressed by deletion of the lysE gene, encoding the L-lysine exporter. Additionally, a putative gabT gene was deleted to enhance 5-aminovalerate production. To fully exploit the performance of the optimized strain, fed-batch fermentation was carried out producing 28 g L(-1) 5-aminovalerate with a maximal space-time yield of 0.9 g L(-1) h(-1)., Conclusions: The present study describes the construction of a recombinant microbial cell factory for the production of carbon-5 platform chemicals. Beyond a basic proof-of-concept, we were able to specifically increase the production flux of 5-aminovalerate thereby generating a strain with excellent production performance. Additional improvement can be expected by removal of remaining by-product formation and bottlenecks, associated to the terminal pathway, to generate a strain being applicable as centerpiece for a bio-based production of 5-aminovalerate.

Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae in particular, have become established as important industrial workhorses in biotechnology. Recent years have seen tremendous progress in their advance into tailor-made producers, driven by the upcoming demand for sustainable processes and renewable raw materials. Here, the diversity and complexity of nature is simultaneously a challenge and a benefit. Harnessing biodiversity in the right manner through synergistic progress in systems metabolic engineering and chemical synthesis promises a future innovative bio-economy., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2gL(-1)h(-1), and a final titer of 88gL(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems., Results: Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media., Conclusions: The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

Dissolved oxygen plays an essential role in aerobic cultivation especially due to its low solubility. Under unfavorable conditions of mixing and vessel geometry it can become limiting. This, however, is difficult to predict and thus the right choice for an optimal experimental set-up is challenging. To overcome this, we developed a method which allows a robust prediction of the dissolved oxygen concentration during aerobic growth. This integrates newly established mathematical correlations for the determination of the volumetric gas-liquid mass transfer coefficient (kLa) in disposable shake-flasks from the filling volume, the vessel size and the agitation speed. Tested for the industrial production organism Corynebacterium glutamicum, this enabled a reliable design of culture conditions and allowed to predict the maximum possible cell concentration without oxygen limitation.

The sustainable production of industrial platform chemicals is one of the great challenges facing the biotechnology field. Ideally, fermentation feedstocks would rather rely on industrial waste streams than on food-based raw materials. Corynebacterium glutamicum was metabolically engineered to produce the bio-nylon precursor 1,5-diaminopentane from the hemicellulose sugar xylose. Comparison of a basic diaminopentane producer strain on xylose and glucose feedstocks revealed a 30% reduction in diaminopentane yield and productivity on the pentose sugar. The integration of in vivo and in silico metabolic flux analysis by (13) C and elementary modes identified bottlenecks in the pentose phosphate pathway and the tricarboxylic acid cycle that limited performance on xylose. By the integration of global transcriptome profiling, this could be specifically targeted to the tkt operon, genes that encode for fructose bisphosphatase (fbp) and isocitrate dehydrogenase (icd), and to genes involved in formation of lysine (lysE) and N-acetyl diaminopentane (act). This was used to create the C. glutamicum strain DAP-Xyl1 icd(GTG) Peftu fbp Psod tkt Δact ΔlysE. The novel producer, designated DAP-Xyl2, exhibited a 54% increase in product yield to 233 mmol mol(-1) and a 100% increase in productivity to 1 mmol g(-1) h(-1) on the xylose substrate. In a fed-batch process, the strain achieved 103 g L(-1) of diaminopentane from xylose with a product yield of 32%. Xylose utilization is currently one of the most relevant metabolic engineering subjects. In this regard, the current work is a milestone in industrial strain engineering of C. glutamicum. See accompanying commentary by Hiroshi Shimizu DOI: 10.1002/biot.201300097., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Since their discovery almost 60 years ago, Corynebacterium glutamicum and related subspecies are writing a remarkable success story in industrial biotechnology. Today, these gram-positive soil bacteria, traditionally well-known as excellent producers of L-amino acids are becoming flexible, efficient production platforms for various chemicals, materials and fuels. This development is intensively driven by systems metabolic engineering concepts integrating systems biology and synthetic biology into strain engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Here, we describe the development of a genetically defined strain of l-lysine hyperproducing Corynebacterium glutamicum by systems metabolic engineering of the wild type. Implementation of only 12 defined genome-based changes in genes encoding central metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L(-1) lysine and a productivity of 4.0 g L(-1) h(-1) in fed-batch culture. The specific glucose uptake rate of the wild type could be completely maintained during the engineering process, providing a highly viable producer. For these key criteria, the genetically defined strain created in this study lies at the maximum limit of classically derived producers developed over the last fifty years. This is the first report of a rationally derived lysine production strain that may be competitive with industrial applications. The design-based strategy for metabolic engineering reported here could serve as general concept for the rational development of microorganisms as efficient cellular factories for bio-production., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.

In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme.

The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfbr. The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

In the present work, we established the bio-based production of glutarate, a carbon-5 dicarboxylic acid with recognized value for commercial plastics and other applications, using metabolically engineered Corynebacterium glutamicum. The mutant C. glutamicum AVA-2 served as a starting point for strain development, because it secreted small amounts of glutarate as a consequence of its engineered 5-aminovalerate pathway. Starting from AVA-2, we overexpressed 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase (gabD) under the control of the constitutive tuf promoter to convert 5-aminovalerate further to glutarate. The created strain GTA-1 formed glutarate as a major product, but still secreted 5-aminovalerate as well. This bottleneck was tackled at the level of 5-aminovalerate re-import. The advanced strain GTA-4 overexpressed the newly discovered 5-aminovalerate importer NCgl0464 and formed glutarate from glucose in a yield of 0.27 mol mol−1. In a fed-batch process, GTA-4 produced more than 90 g L−1 glutarate from glucose and molasses based sugars in a yield of up to 0.70 mol mol−1 and a maximum productivity of 1.8 g L−1 h−1, while 5-aminovalerate was no longer secreted. The bio-based glutaric acid was purified to ＞99.9% purity. Interfacial polymerization and melt polymerization with hexamethylenediamine yielded bionylon-6,5, a polyamide with a unique structure. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae in particular, have become established as important industrial workhorses in biotechnology. Recent years have seen tremendous progress in their advance into tailor-made producers, driven by the upcoming demand for sustainable processes and renewable raw materials. Here, the diversity and complexity of nature is simultaneously a challenge and a benefit. Harnessing biodiversity in the right manner through synergistic progress in systems metabolic engineering and chemical synthesis promises a future innovative bio-economy. [ABSTRACT FROM AUTHOR]

Background: Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results: In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion: The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering. [ABSTRACT FROM AUTHOR]

Lignin is one of the most abundant renewable resources on earth and is readily produced as a sidestream during biomass fractioning. So far, these large quantities of lignin have been severely underutilized, thereby wasting this valuable renewable. Recent technological advances in lignin recovery, breakdown, and conversion have now started forming the first sustainable value chains to take advantage of lignin. Microbial cell factories, inspired by nature's miscellaneous set of lignin-degrading microbes, are at the heart of these novel processes. Recent success stories in which the enzymes and pathways of these microbes were harnessed for biobased production from lignin hold great promise for a sustainable upgrading of this renewable polymer into value-added compounds. [ABSTRACT FROM AUTHOR]

Abstract: Bio-based production promises a sustainable route to myriads of chemicals, materials and fuels. With regard to eco-efficiency, its future success strongly depends on a next level of bio-processes using raw materials beyond glucose. Such renewables, i.e., polymers, complex substrate mixtures and diluted waste streams, often cannot be metabolized naturally by the producing organisms. This particularly holds for well-known microorganisms from the traditional sugar-based biotechnology, including Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae which have been engineered successfully to produce a broad range of products from glucose. In order to make full use of their production potential within the bio-refinery value chain, they have to be adapted to various feed-stocks of interest. This review focuses on the strategies to be applied for this purpose which combine rational and evolutive approaches. Hereby, the three industrial platform microorganisms, E. coli, C. glutamicum and S. cerevisiae are highlighted due to their particular importance. [Copyright &y& Elsevier]