Abstract
BackgroundThe 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.ResultsIn 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.ConclusionsThe 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.
Highlights
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
The metabolic pathway from l-lysine towards 5-aminovalerate was reconstituted by stable genome-based implementation of the Pseudomonas putida KT2440 genes encoding gene of lysine monooxygenase (davB), encoding lysine monooxygenase, and encoding gene of aminovaleramide amidase (davA), encoding 5-aminovaleramide amidase (Fig. 1)—a strategy previously established for E. coli [19, 21]
In E. coli, production of 5-aminovalerate and glutarate relied on heterologous expression of the davAB cluster (5-aminovalerate) or a combined expression of the davBA-davDT clusters as, in contrast to C. glutamicum, endogenous genes for davDT are obviously missing [19, 20, 22]
Summary
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. As 5-aminovalerate and glutarate are degradation products of the proteinogenic amino acid l-lysine, we choose the non-pathogenic Gram-positive soil bacterium Corynebacterium glutamicum, a well-established industrial l-lysine producer [1], as metabolic chassis for the production of these carbon-5 platform chemicals. Beyond the excellent availability of genetic tools [23], and knowledge of its physiology and large-scale fermentation [24, 25], engineered C. glutamicum has only recently been established as centerpiece for the production of l-lysine-derived cadaverine within a pipeline towards the manufacturing of the fully bio-based polyamide PA5.10 [13]. The metabolic pathway from l-lysine towards 5-aminovalerate was reconstituted by stable genome-based implementation of the Pseudomonas putida KT2440 genes davB, encoding lysine monooxygenase, and davA, encoding 5-aminovaleramide amidase (Fig. 1)—a strategy previously established for E. coli [19, 21].
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