Abstract
Pipecolic acid or L-PA is a cyclic amino acid derived from L-lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L-PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L-lysine biosynthetic pathway. L-PA is a six-membered ring homolog of the five-membered ring amino acid L-proline, which serves as compatible solute in C. glutamicum.Here, we show that de novo synthesized or externally added L-PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L-PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L-PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L-PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L-PA production. Under hyper-osmotic conditions, a ΔproP strain showed similar growth behavior than the parent strain when L-proline was added externally. By contrast, the growth impairment of the ΔproP strain under hyper-osmotic conditions could not be alleviated by addition of L-PA unless proP was expressed from a plasmid. This is commensurate with the view that L-proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L-PA uptake under hyper-osmotic stress conditions.
Highlights
In nature, all living organisms must respond to environmental fluctuations to survive
We provide evidence that YggB may be involved in export of L-PA and ProP in its import into the C. glutamicum cell
When L-PA production was induced, C. glutamicum GSL(pVWEx1-lysine dehydrogenase gene (lysDH)-pyrroline-5-carboxylate reductase gene (proC)) grew faster (Figure 1A) and to higher biomass concentrations (Figure 1B) in the presence of NaCl than the parent strain GSL(pVWEx1) (Figure 1). This indicated that biosynthesis of L-PA helps C. glutamicum to withstand hyperosmolar conditions
Summary
All living organisms must respond to environmental fluctuations to survive. Bacteria have developed defense mechanisms for hyper- and hypo-osmotic external conditions to maintain cell viability including the de novo synthesis or uptake of osmo compatible solutes such as betaines. Pipecolic Acid in Corynebacterium glutamicum and to avoid cell lysis, MSCs are immediately activated by membrane turgor pressure to release solutes and to decrease in the internal osmolality (Ruffert et al, 1997; Morbach and Krämer, 2003). Proline is the major de novo synthetized compatible solute in C. glutamicum (Skjerdal et al, 1996; Wolf et al, 2003). Added proline can be taken up into the C. glutamicum cell by the import systems EctP, ProP, and PutP (Peter et al, 1998). The import of proline by the carriers EctP and ProP is osmoregulated (Peter et al, 1998), while the import of proline by PutP is not (Peter et al, 1997)
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