Enhanced Hyaluronic Acid Production in Bacillus subtilis by Coexpressing Bacterial Hemoglobin

Abstract

Bacillus subtilis strains that can produce hyaluronic acid (HA) were constructed by integrating the HA synthase gene (hasA) and the UDP-glucose dehydrogenase gene of group C Streptococcus (hasB) or of B. subtilis itself (tauD) into the amyE locus of the B. subtilis chromosome. All of the inserted genes were under the control of a strong constitutive vegII promoter of B. subtilis. Although HA production could be achieved by expressing hasA alone, coexpressing hasB or tauD with hasA could enhance HA production at least 2-fold. To replenish the energy consumed for HA biosynthesis, Vitreoscilla hemoglobin (VHb) was coexpressed with the HA-expressing genes. With the expression of VHb, not only the cell concentration was enhanced 25%, but also HA production was further increased by 100%. About 1.8 g/L of HA was obtained by the recombinant strain B. subtilis carrying VHb, hasA, and tauD genes in the expression cassette after 30 h cultivation.

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

“…The molecular mass observed during cultivation on either carbon source compares favorably to that from a previous report of HA production in B. subtilis (41), and both the HA titer and molecular mass were similar to those obtained with our HA-producing strains developed through conventional cloning. The titers obtained with AW005-2 also compared favorably to the titer reported for a similar strain of B. subtilis over the same cultivation period (57). A significantly longer cultivation was required to achieve a similar titer, and molecular mass was not assessed in the aforementioned study.…”

“…The culture was then used to inoculate 20 ml prewarmed nonselect cultivation medium (4%, vol/vol) with the following composition: (NH 4 (20 g/liter), and the cultures were grown at 37°C and 280 rpm in triplicate. Samples were diluted 2-fold in phosphate-buffered saline, and HA was purified with cetylpyridinium chloride as previously described (57). The HA titer was determined using the modified carbazole assay (58), and the molecular mass was analyzed via agarose gel electrophoresis as described previously (59) with slight modifications.…”

Biotechnological Production and Application of Hyaluronan

“…In particular, Widner and co-workers (2005) overexpressed in a Bacillus subtilis strain the hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase along with the endogenous tuaD gene encodes for UDP-Glc dehydrogenase resulting in the production of HA in the 1 MDa range in 3L fermentation experiments. Successively, also Chien and Lee (2007) succeeded in producing hyaluronan from a B. subtlis strain. The recombinant B. subtilis strain developed contained VHb ( Vitreoscilla haemoglobin) gene, S. zooepidemicus hasA, and endogenous tauD genes in the expression cassette, by cultivation of these recombinant strains in 250 mL shaked flasks (30 h) they obtained about 1.8 g/L of HA.…”

Hyaluronic Acid in the 3^rd Millennium

“…Since the natural hyaluronan-producing organisms are mostly pathogenic, metabolic engineering is currently representing an interesting opportunity to obtain HA from nonpathogenic, GRAS microorganism. Endotoxin-free HA has already been synthetized by recombinant hosts including Lactococcus lactis [128], Bacillus subtilis [129], Escherichia coli [130] and Corynebacterium glutamicum [131]. However, up to now, there has been no heterologous bacterial host producing as much HA as the natural ones.…”