J Hoffmann1, J Altenbuchner. 1. Institute of Industrial Genetics, University of Stuttgart, Stuttgart, Germany.
Abstract
AIMS: Corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (HA). METHODS AND RESULTS: A set of expression vectors containing hasA, encoding HA synthase from Streptococcus equi subsp. zooepidemicus, alone or in combination with genes encoding enzymes for HA precursor production (hasB, hasC, glmU from Pseudomonas putida KT2440) or bacterial haemoglobin (vgb from Vitreoscilla sp.) was constructed. Recombinant Coryne. glutamicum strains were cultivated in two different minimal media, CGXII and MEK700. HA was isolated from the culture broth by ethanol precipitation or ultrafiltration. Analyses of the isolated HA revealed that overall production was higher in CGXII medium (1241 mg l(-1)) than in MEK700 medium (363 mg l(-1)), but molecular weight of the product was higher in MEK700 (>1·4 MDa) than in CGXII (<270 kDa). Coexpression of hasB, hasC or glmU had no effect on HA yield and did not improve molecular weight of the product. Coexpression of vgb lowered HA yield about 1·5-fold and did not affect molecular weight of the product. Microscopy of negative-stained cultures revealed that Coryne. glutamicum produces no distinct HA capsule. CONCLUSIONS: Regulation of cell growth and gene expression level of hasA are reasonable starting points for controlling the molecular weight of HA produced by Coryne. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Corynebacterium glutamicum has a great potential as an alternative production host for HA. The fact that Coryne. glutamicum produces no distinct HA capsule facilitates HA isolation and improves overall yield.
AIMS: Corynebacterium glutamicum was tested as an alternative host for heterologous production of hyaluronic acid (HA). METHODS AND RESULTS: A set of expression vectors containing hasA, encoding HA synthase from Streptococcus equi subsp. zooepidemicus, alone or in combination with genes encoding enzymes for HA precursor production (hasB, hasC, glmU from Pseudomonas putida KT2440) or bacterial haemoglobin (vgb from Vitreoscilla sp.) was constructed. Recombinant Coryne. glutamicum strains were cultivated in two different minimal media, CGXII and MEK700. HA was isolated from the culture broth by ethanol precipitation or ultrafiltration. Analyses of the isolated HA revealed that overall production was higher in CGXII medium (1241 mg l(-1)) than in MEK700 medium (363 mg l(-1)), but molecular weight of the product was higher in MEK700 (>1·4 MDa) than in CGXII (<270 kDa). Coexpression of hasB, hasC or glmU had no effect on HA yield and did not improve molecular weight of the product. Coexpression of vgb lowered HA yield about 1·5-fold and did not affect molecular weight of the product. Microscopy of negative-stained cultures revealed that Coryne. glutamicum produces no distinct HA capsule. CONCLUSIONS: Regulation of cell growth and gene expression level of hasA are reasonable starting points for controlling the molecular weight of HA produced by Coryne. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Corynebacterium glutamicum has a great potential as an alternative production host for HA. The fact that Coryne. glutamicum produces no distinct HA capsule facilitates HA isolation and improves overall yield.
Authors: Sebastián Cerminati; Mélanie Leroux; Pablo Anselmi; Salvador Peirú; Juan C Alonso; Bernard Priem; Hugo G Menzella Journal: Appl Microbiol Biotechnol Date: 2021-04-05 Impact factor: 4.813