Abstract
Backgroundα-d-Glucosylglycerol (αGG) has beneficial functions as a moisturizing agent in cosmetics and potential as a health food material, and therapeutic agent. αGG serves as compatible solute in various halotolerant cyanobacteria such as Synechocystis sp. PCC 6803, which synthesizes αGG in a two-step reaction: The enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GGPS) is followed by the dephosphorylation of the intermediate by the GG-phosphate phosphatase (GGPP). The Gram-positive Corynebacterium glutamicum, an industrial workhorse for amino acid production, does not utilize αGG as a substrate and was therefore chosen for the development of a heterologous microbial production platform for αGG.ResultsPlasmid-bound expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enabled αGG synthesis exclusively in osmotically stressed cells of C. glutamicum (pEKEx2-ggpSP), which is probably due to the unique intrinsic control mechanism of GGPS activity in response to intracellular ion concentrations. C. glutamicum was then engineered to optimize precursor supply for αGG production: The precursor for αGG synthesis ADP-glucose gets metabolized by both the glgA encoded glycogen synthase and the otsA encoded trehalose-6-phosphate synthase. Upon deletion of both genes the αGG concentration in culture supernatants was increased from 0.5 mM in C. glutamicum (pEKEx3-ggpSP) to 2.9 mM in C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP). Upon nitrogen limitation, which inhibits synthesis of amino acids as compatible solutes, C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP) produced more than 10 mM αGG (about 2 g L−1).ConclusionsCorynebacterium glutamicum can be engineered as efficient platform for the production of the compatible solute αGG. Redirection of carbon flux towards αGG synthesis by elimination of the competing pathways for glycogen and trehalose synthesis as well as optimization of nitrogen supply is an efficient strategy to further optimize production of αGG.
Highlights
Accumulation of compatible solutes is required for the acclimation of organisms to adverse environmental conditions with increasing external osmolality, to salt and drought stress [1, 2]
NaCl‐triggerd αGG synthesis in C. glutamicum The compatible solute αGG is synthesized in Synechocystis sp
Presence of the plasmid encoded enzymes in extracts of C. glutamicum was shown by western blot analysis using antibodies raised against the C-terminal His-Tag of GgpP and the C-terminal Strep-Tag of GgpS (Fig. 1a), and a GgpS activty of 9.52 ± 2.06 μmol min−1−1 was observed in cell free extracts of C. glutamicum
Summary
NaCl‐triggerd αGG synthesis in C. glutamicum (pEKEx2‐ggpSP) The compatible solute αGG is synthesized in Synechocystis sp. Accumulation of αGG in the culture broth and αGG productivity of C. glutamicum ΔotsAΔtreYΔtreS (pEKEx3-ggpSP) in cultivations in CgC-minimal medium as well as N-limited medium were significantly lower (Table 5) than the parameters determined for C. glutamicum ΔotsA IMglgA (pEKEx3ggpSP) and quite similar to the parameters determined for C. glutamicum ΔotsA (pEKEx3-ggpSP) (Tables 3, 4), which is still able to synthesize trehalose [37] Taken together these results show that the strategy to increase availability of the precursor ADP-glucose by inactivation of glgA and deletion of otsA was responsible for the improved αGG by C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP) and not the concomitantly obtained trehalose-synthesis deficiency
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