Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing α-amylase and lysine decarboxylase

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ؎ 28 mM (21.8 ؎ 3.2 g liter ؊1 ) 2-ketoisovalerate and showed a product yield of about 0.47 ؎ 0.05 mol per mol (0.3 ؎ 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ؎ 0.6 mM (0.53 ؎ 0.07 g liter ؊1 ) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

mentioning

confidence: 99%

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

Krause

Blombach

Eikmanns

2010

“…Recent studies also showed the successful employment of C. glutamicum for the production of the diamines putrescine and cadaverine (5)(6)(7)(8)(9)(10), the organic acids D-lactate, succinate, 2-ketoisovalerate, and pyruvate (11)(12)(13)(14)(15), the biofuels ethanol and isobutanol (16)(17)(18), xylitol (19), and heterologous proteins (20,21).…”

mentioning

confidence: 99%

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l -Lysine, l -Valine, and 2-Ketoisovalerate

Buchholz

Schwentner

Brunnenkan

et al. 2013

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the L-valine biosynthetic genes ilvBNCE, all strains produced L-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 ⌬pqo ⌬ppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) L-valine with an overall yield (Y P/S ) of 0.36 mol per mol of glucose and a volumetric productivity (Q P ) of 13.6 mM per h [1.6 g/(liter ؋ h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the L-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y P/S of 0.24 mol per mol of glucose and a Q P of 6.9 mM per h [0.8 g/(liter ؋ h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum L-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

“…From a metabolic viewpoint, diaminopentane is formed directly from lysine by decarboxylation, meaning that Corynebacterium glutamicum, which produces more than 1,000,000 metric tons of L-lysine per year, is a promising production organism. In a first proof of principle, a C. glutamicum wild type was modified by replacing homoserine dehydrogenase with heterologous lysine decarboxylase (cadA) from Escherichia coli (19,24). The yield of diaminopentane was, however, rather low, underlining that this modification can only be a first step toward a competitive industrial strain.…”

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confidence: 99%

Identification and Elimination of the Competing N -Acetyldiaminopentane Pathway for Improved Production of Diaminopentane by Corynebacterium glutamicum

Kind

Jeong

Schröder

et al. 2010

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.Polyamides are polymers containing monomers joined by peptide bonds, with examples being nylons, aramids, and polyaspartates. They are commonly used in textiles, automotives, carpet, and sportswear due to their extreme durability and strength. The most prominent products, polyamides PA 6 and PA 6.6, have an annual market volume of about 6 million tons. Currently, polyamides are derived via chemical routes from fossil raw materials. Due to the shortage of these resources and problems of escalating CO 2 production and global warming linked to the underlying processes, bio-based production using renewable resources arises as a promising alternative (11,23,27). In this context, fermentative production of diaminopentane (cadaverine) as a monomer building block for polyamides has recently come into focus (19). Using diaminopentane derived from microbial biosynthesis, polymerization with appropriate bioblocks, such as succinate (9, 22), provides completely bio-based products. Moreover, polyamides based on diaminopentane reveal excellent material properties (7). The experience of the past clearly shows that a superior production strain with a high yield, level of productivity, and titer requires substantial modification at different key points of the metabolism, which have to be identified by careful investigation of the underlying metabolism (28). The major targets typically compri...