Production of the compatible solute α-D-glucosylglycerol by metabolically engineered Corynebacterium glutamicum

Abstract

Background: α-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.

Results: Plasmid-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).

Conclusions: Corynebacterium 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.

Keywords: Compatible solute; Corynebacterium glutamicum; Glycogen; Trehalose; α-D-Glucosylglycerol.

Fig. 1

Heterologous expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enable NaCl-triggerd αGG synthesis in C. glutamicum (pEKEx2-ggpSP). a Analysis of cell free extracts (CE) of C. glutamicum (pEKEx2-ggpSP) for expression of strep-tagged GgpS and his-tagged GgpP by Western immunoblot with antibodies against the tags; protein sizes (kDa) are indicated for markers (M) bands a. Growth (circles) b, substrate consumption (triangles) and αGG fromation (diamonds) c of C. glutamicum (pEKEx2-ggpSP) during cultivation in CgC-minimalmedium with initially 1% sucrose in absence (solid symbols) and presence of 750 mM NaCl (open symbols). NaCl was added to the culture after 4 h of cultivation. Data from one representative experiment of a series of three are shown

Fig. 2

Analysis of the NaCl dependency of αGG synthesis in C. glutamicum (pEKEx2-ggpSP). αGG concentrations in culture supernatants after 24 h cultivation of C. glutamicum (pEKEx2-ggpSP) in minimal medium with initially 1% sucrose as sole carbon source a. NaCl was added in indicated concentrations after 4 h of cultivation. Analysis of cell free extracts (CE) of C. glutamicum (pEKEx2-ggpSP) cultivated at different NaCl concentrations for expression of strep-tagged GgpS by Western immunoblot with antibodies against the tag b; protein sizes (kDa) are indicated for markers (M) bands

Fig. 3

αGG accumulation during cultivation of C. glutamicum IMglgA ΔotsA (pEKEx3-ggpSP) in small-scale bioreactors in CgC minimal medium with 2% sucrose. The culture was inoculated at an OD₆₀₀ of about 1, after 4 h of cultivation a hyperosmotic shock was applied by addition of NaCl (final concentration 750 mM; the timepoint of NaCl addition is indicated by a black arrow). Growth (grey triangles), sucrose concentration (white squares), and α-GG concentration (black circles) were analyzed throughout cultivation. The fermentation was performed in duplicates, for substrate and product concentrations samples from each fermentation were analyzed in triplicates. Means of substrate and product concentrations are from two independent experiments; error bars indicate standard deviations. For growth a representative curve of the two experiments is shown

Fig. 4

Effect of nitrogen limitation on α-GG production with C. glutamicum (pEKEx3-ggpSP). Growth a, substrate consumption b and αGG accumulation c were determined for C. glutamicum (pEKEx3-ggpSP) in the course of cultivation in CgC-minimal medium (open squares), or CgC-minimal medium without ammonia and 0 g L⁻¹ urea (white circles), 0.1 g L⁻¹ urea (grey squares), 0.5 g L⁻¹ urea (solid diamonds), 1.0 g L⁻¹ urea (grey circles), or 5.0 g L⁻¹ urea (solid triangles). Data from one representative experiment of a series of three are shown

Fig. 5

Effect of nitrogen limitation on αGG accumulation during cultivation of C. glutamicum IMglgA ΔotsA (pEKEx3-ggpSP) in small-scale bioreactors in CgC minimal medium with 2% sucrose. The culture was inoculated at an OD₆₀₀ of about 1, after 4 h of cultivation a hyperosmotic shock was applied by addition of NaCl (final concentration 750 mM; the timepoint of NaCl addition is indicated by a black arrow). Growth (grey triangles), sucrose concentration (open squares), and α-GG concentration (solid circles) were analyzed throughout cultivation. The fermentation was performed in duplicates, for substrate and product concentrations samples from each fermentation were analyzed in triplicates. Means of substrate and product concentrations are from two independent experiments; error bars indicate standard deviations. For growth a representative curve of the two experiments is shown