Biofabrication of a Hyaluronan/Bacterial Cellulose Composite Nanofibril by Secretion from EngineeredGluconacetobacter

Abstract

Naturally occurring polysaccharides, such as cellulose, hemicellulose, and chitin, have roles in plant skeletons and/or related properties in living organisms. Their hierarchically regulated production systems show potential for designing nanocomposite fabrication using engineered microorganisms. This study has demonstrated that genetically engineered Gluconacetobacter hansenii (G. hansenii) individual cells can fabricate naturally composited nanofibrils by simultaneous production of hyaluronan (HA) and bacterial cellulose (BC). The cells were manipulated to contain hyaluronan synthase and UDP-glucose dehydrogenase genes, which are essential for HA biosynthesis. Fluorescence microscopic observations indicated the production of composited nanofibrils and suggested that HA secretion was associated with the cellulose secretory pathway in G. hansenii. The gel-like nanocomposite materials produced by the engineered G. hansenii exhibited superior properties compared with conventional in situ nanocomposites. This genetic engineering approach facilitates the use of G. hansenii for designing integrated cellulose-based nanomaterials.

Advances in the treatment of inflammatory bowel disease: Focus on polysaccharide nanoparticulate drug delivery systems

“…Viruses and bacteria also produced HA. 273 HA is a linear heteropolysaccharide composed of repeating -1,4 bond disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked together through -1,3 glycosidic bonds. HA is a polyanionic polysaccharide.…”

Physical characteristics and cell-adhesive properties of in vivo fabricated bacterial cellulose/hyaluronan nanocomposites

“…All procedures were according to our previous report (Takahama et al 2021). Brie y, plasmids were introduced into Gluconacetobacter hansenii ATCC23769 (Gh) by electroporation (Hall et al 1992) to obtain Gh(HA), an HA-producing transformant, and Gh(pTI99), a negative control (Sunagawa et al 2012;Fang et al 2015) carrying a plasmid vector without genes for HA synthesis.…”

“…According to our previous report on nanocomposites production (Takahama et al 2021), Gh transformants (Gh(HA) and Gh(pTI99)) were grown in two culture steps, namely, pre-culture and main culture. Brie y, the seed culture was inoculated to HS medium supplemented with cellulase (0.1%, w/v) and Amp, and then grown under shaking conditions (125 rpm) (pre-culture).…”

“…The pellicles were puri ed with a combination method of enzymatic treatments using lysozyme and NaOH/sodium dodecyl sulfate (SDS), as previously described (Takahama et al 2021).…”

“…In previous reports, we developed an in vivo fabrication technique for the production of a polysaccharide nanocomposite of curdlan and BC (Fang et al 2015), and hyaluronan (HA) and BC (Takahama et al 2021), using genetically engineered G. hansenii with introduction of minimum and essential genes for these biosyntheses. Related to the present study, the engineered G. hansenii (hereafter referred to as Gh(HA)) successfully synthesized HA and, more importantly, extruded from one side of the bacterial cell surface.…”