Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

“…These recombinant C. glutamicum strains simultaneously hydrolyze biomass and produce biochemicals such as succinate, l ‐lysine, and xylonic acid using released glucose, maltose, cellobiose, d ‐xylose, and l ‐arabinose from biomass (Fig. ) . Systematic engineering of metabolic pathways in C. glutamicum has allowed the biochemical production of novel products, such as platform chemicals (ethanol, ethylene glycol, glycolate, 3‐hydroxypropionic acid, 1,2‐propanediol, 1,3 propanediol, 2,3‐butanediol, and isobutanol) and polyamide monomers (putrescine, cadaverine, gamma‐aminobutyrate, 5‐aminovalerate, succinate, and glutarate) .…”

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

“…The Gram‐positive actinobacterium, Corynebacterium glutamicum , represents an attractive expression platform for secretory protein production in industrial biotechnology due to its ability to secrete proteins at the g L −1 range into the extracellular medium (Freudl, ; Ravasi et al, ; Watanabe, Teramoto, Suzuki, Inui, & Yukawa, ; Yim, Choi, et al, ). Furthermore, it is classified as GRAS (generally regarded as safe) organism and is the major producer for food and feed amino acids (Wendisch, ).…”

“…Hence, extensive bioprocessing knowledge in an industrial environment as well as a comprehensive set of available genetic tools is available. Furthermore, C. glutamicum can be cultivated to high cell densities in fed‐batch cultivations (Knoll et al, ; Yim, An, Choi, Ryu, & Jeong, 2014; Yim, Choi, et al, ) and was shown to be very robust towards process inhomogeneities encountered in large scale cultivations (Käß et al, ; Limberg, Schulte, et al, ; Limberg, Pooth, Wiechert, & Oldiges, ).…”

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum

“…These mostly monoderm bacteria are robust in terms of cultivation conditions, low nutritional demand, and are able to secrete proteins into the extracellular medium [3, 4]. Next to Bacillus  sp., a powerful expression host for the production of technical enzymes [5], Corynebacterium glutamicum is an attractive alternative host microorganism with demonstrated protein secretion capacity in the g/L-range [6, 7]. Also, extensive bioprocess knowledge with C.   glutamicum and methods for genetic manipulation are available because this microbe is a major producer for amino acids at industrial scale for decades [8].…”