Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-1 h-1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc.

From zero to hero - production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum.

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.

Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum.

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.

Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum.

The present work describes the development of a superior strain of Corynebacterium glutamicum for diaminopentane (cadaverine) production via metabolic engineering of cellular transport processes. In C. glutamicum DAP-3c, a tailor-made producer, the diaminopentane forming enzyme, lysine decarboxylase, was inhibited in vivo by its end-product, suggesting a potential bottleneck at the level of the export. The previously proposed lysine exporter lysE was shown not to be involved in diaminopentane export. Its deletion did not reduce diaminopentane secretion and could therefore be exploited to completely eliminate the export of lysine, an undesired by-product. Genome-wide transcription profiling revealed the up-regulation of 35 candidate genes as response to diaminopentane overproduction, including several transporters. The highest expression increase (2.6-fold) was observed for a permease, encoded by cg2893. Targeted gene deletion in the producer resulted in a 90% reduced diaminopentane secretion. Genome-based overexpression of the exporter, however, revealed a 20% increased yield, a 75% reduced formation of the undesired by-product N-acetyl-diaminopentane and a substantially higher viability, reflected by increased specific rates for growth, glucose uptake and product formation. Similarly, deletion of cg2894, TetR type repressor neighboring the permease gene, resulted in improved production properties. The discovery and amplification of the permease, as presented here, displays a key contribution towards superior C. glutamicum strains for production of the platform chemical diaminopentane. The exact function of the permease remained unclear. Its genetic modification had pronounced effects on various intracellular pools of the biosynthetic pathway, which did not allow a final conclusion on its physiological role, although a direct contribution to diaminopentane export appears possible.

Bio-based production of the platform chemical 1,5-diaminopentane.

In the rising era of bio-economy, the five carbon compound 1,5-diaminopentane receives increasing interest as platform chemical, especially as innovative building block for bio-based polymers. The vital interest in bio-based supply of 1,5-diaminopentane has strongly stimulated research on the development of engineered producer strains. Based on the state-of-art knowledge on the pathways and reactions linked to microbial 1,5-diaminopentane metabolism, the review covers novel systems metabolic engineering approaches towards hyper-producing cell factories of Corynebacterium glutamicum or Escherichia coli. This is integrated into the whole value chain from renewable feedstocks via 1,5-diaminopentane to innovative biopolymers involving bioprocess engineering considerations for economic supply.

Identification and elimination of the competing N-acetyldiaminopentane pathway for improved production of diaminopentane by Corynebacterium glutamicum.

The present work describes the development of a superior strain of Corynebacterium glutamicum for diaminopentane (cadaverine) production aimed at the identification and deletion of the underlying unknown N-acetyldiaminopentane pathway. This acetylated product variant, recently discovered, is a highly undesired by-product with respect to carbon yield and product purity. Initial studies with C. glutamicum DAP-3c, a previously derived tailor-made diaminopentane producer, showed that up to 20% of the product occurs in the unfavorable acetylated form. The strain revealed enzymatic activity for diaminopentane acetylation, requiring acetyl-coenzyme A (CoA) as a donor. Comparative transcriptome analysis of DAP-3c and its parent strain did not reveal significant differences in the expression levels of 17 potential candidates annotated as N-acetyltransferases. Targeted single deletion of several of the candidate genes showed NCgl1469 to be the responsible enzyme. NCgl1469 was functionally assigned as diaminopentane acetyltransferase. The deletion strain, designated C. glutamicum DAP-4, exhibited a complete lack of N-acetyldiaminopentane accumulation in medium. Hereby, the yield for diaminopentane increased by 11%. The mutant strain allowed the production of diaminopentane as the sole product. The deletion did not cause any negative growth effects, since the specific growth rate and glucose uptake rate remained unchanged. The identification and elimination of the responsible acetyltransferase gene, as presented here, display key contributions of a superior C. glutamicum strain producing diaminopentane as a future building block for bio-based polyamides.

Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an acetylated variant, N-acetyl-diaminopentane, which reached levels of more than 25% of that of the desired product.