SummaryBecause of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h−1, followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h−1 and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased.Funding Information No funding information provided.
Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the L-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more L-glutamate and L-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM L-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM L-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.
A triphasic process was developed for the production of  dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE al ) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter ؊1 citrate, pH 6.5, and 37°C are ideal parameters for CphE al production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter ؊1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter ؊1 induced CphE al production with only about 30% efficiency in comparison to that with CGP. CphE al was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE al turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to -aspartate-arginine and -aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE al preparation. Optimum degradation parameters were 100 g liter ؊1 CGP, 10 g liter ؊1 crude CphE al powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE al powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.Cyanophycin granule polypeptide (CGP), or multi-L-arginylpoly(L-aspartic acid), was discovered in cyanobacteria about 130 years ago (6). The branched polymer consists of a poly(aspartic acid) backbone with arginine moieties linked to the -carboxyl group of each aspartic acid by its ␣-amino group (34, 42) and accumulates during the transition from the exponential to the stationary growth phase (23, 40) and under limiting conditions, including low temperature, low light intensity, and phosphorus or sulfur limitation (44). CGP functions as a temporary nitrogen, energy, and possibly also carbon reserve (10, 21). Because CGP contains five nitrogen atoms in every building block, it is an ideal intracellular nitrogen reserve (43). Most genera of cyanobacteria (5,22,23,43,48) and some heterotrophic bacteria (12, 16, 51) harbor a cyanophycin synthetase gene (cphA) and synthesize CGP. The polymer is insoluble at neutral pH as well as at physiological ionic strength (4). In cyanobacteria, CGP has a molecular mass of 25 to 100 kDa (41), while in recombinant strains it exhibits a molecular mass of 25 to 30 kDa and a lower polydispersity and contains lysine, which partially replaces arginine (2, 50).CGP degradation (intra-or extracellularly) leads mainly to the release of dipeptides. Intracellular degradation of CGP is catalyzed b...
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