Hartwig Schröder

From zero to hero—Design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production

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.

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.

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

ABSTRACT 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.

Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diaminopentane from hemicellulose

In the present work, the bio-based production of 1,5-diaminopentane (cadaverine), an important building block for bio-polyamides, was extended to hemicellulose a non-food raw material. For this purpose, the metabolism of 1,5-diaminopentane-producing Corynebacterium glutamicum was engineered to the use of the C(5) sugar xylose. This was realized by heterologous expression of the xylA and xylB genes from Escherichia coli, mediating the conversion of xylose into xylulose 5-phosphate (an intermediate of the pentose phosphate pathway), in a defined diaminopentane-producing C. glutamicum strain, recently obtained by systems metabolic engineering. The created mutant, C. glutamicum DAP-Xyl1, exhibited efficient production of the diamine from xylose and from mixtures of xylose and glucose. Subsequently, the novel strain was tested on industrially relevant hemicellulose fractions, mainly containing xylose and glucose as carbon source. A two-step process was developed, comprising (i) enzymatic hydrolysis of hemicellulose from dried oat spelts, and (ii) biotechnological 1,5-diaminopentane production from the obtained hydrolysates with the novel C. glutamicum strain. This now opens a future avenue towards bio-based 1,5-diaminopentane and bio-polyamides thereof from non-food raw materials.

Systems‐wide analysis and engineering of metabolic pathway fluxes in bio‐succinate producing Basfia succiniciproducens

Basfia succiniciproducens has been recently isolated as novel producer for succinate, an important platform chemical. In batch culture, the wild type exhibited a high natural yield of 0.75 mol succinate (mol glucose)−1. Systems‐wide 13C metabolic flux analysis identified undesired fluxes through pyruvate‐formate lyase (PflD) and lactate dehydrogenase (LdhA). The double deletion strain B. succiniciproducens ΔldhA ΔpflD revealed a 45% improved product yield of 1.08 mol mol−1. In addition, metabolic flux analysis unraveled the parallel in vivo activity of the oxidative and reductive branch of the TCA cycle in B. succiniciproducens, whereby the oxidative part mainly served for anabolism. The wild type re‐directed surplus NADH via a cycle involving malic enzyme or via transhydrogenase, respectively, to supply NADPH for anabolism, because the fluxes through the oxidative PPP and isocitrate dehydrogenase, that also provide this cofactor, were not sufficient. This was not observed for the deletion mutants, B. succiniciproducens ΔpflD and ΔldhA ΔpflD, where PPP and isocitrate dehydrogenase flux alone matched with the reduced anabolic NADPH demand. The integration of the production performance into the theoretical flux space, computed by elementary flux mode analysis, revealed that B. succiniciproducens ΔldhA ΔpflD reached 62% of the theoretical maximum yield. Biotechnol. Bioeng. 2013;110: 3013–3023.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Effects of feeding various tocotrienol sources on plasma lipids and aortic atherosclerotic lesions in cholesterol-fed rabbits

Abstract Tocotrienols exert hypocholesterolaemic and antioxidant effects, and may hence have anti-atherogenic properties. Therefore, the aim of this study was to investigate the cholesterol-lowering and anti-atherogenic effects of tocotrienols in cholesterol-fed rabbits. Rabbits were fed a basal diet (control) supplemented with γ-tocotrienol, γ-tocotrienyl acetate, mixed tocotrienols or α-tocotrienol for 12 weeks. All treatments resulted in significant increases in plasma tocotrienols. None of the treatments, however, had significant effects on serum lipids or size of atherosclerotic lesions. A trend towards a decrease in plasma cholesterol was observed following γ-tocotrienol treatment (−22%) after 6 weeks. The decrease was mainly attributable to a reduction in LDL cholesterol (23%), whereas HDL cholesterol increased (14%). This trend was mirrored in a non-significant reduction in lesion area (20%). Our results demonstrate that tocotrienols are absorbed, but have little effect on plasma lipids and atherosclerosis in cholesterol-fed rabbits.

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using 13C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol-1 and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71gL-1 succinate and a yield of 2.5molmol-1 from sucrose.

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13 C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamins pyrimidine ring, which is part of its isoalloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%.