Modulating Bacterial Nanocellulose Crystallinity through Post-Transcriptional Repression in Komagataeibacter xylinus

Bacterial nanocellulose (BC), a versatile and biodegradable polymer, has been extensively studied as an alternative biomaterial for various applications. For biomedical and packaging uses, where precise control over nanocellulose structure is essential, existing literature describes BC structural modification processes that involve additives or additional steps. With the aim to develop a programmable method to control the bulk microstructure, we developed sRNA-based post-transcriptional repression cassettes that allows precise regulation of the crystalline phase of BC. Before investigating the effects of post-transcriptional repression of bcsD, bcsZ, and ccpA genes, known to influence BC crystallinity, we validated the specificity of the sRNA repression cassette by targeting a genome-integrated red fluorescent protein, mRFP1. The observed growth inhibition (>80%) caused by overexpressed Hfq RNA chaperone in Komagataeibacter xylinus was alleviated ([≤] 23%) by its removal, resulting in a 43% reduction in relative mRFP1 expression. By varying the design of the repression cassette and the inducer concentrations, we successfully modulated the repression of the target genes [with relative expression reductions of 6%-34% for bcsD, 8%-24% for bcsZ, and 2%-20% for ccpA, as confirmed by qRT-PCR]. These gene repression levels led to statistically significant changes in the amorphous content of the BC microstructure, as demonstrated by X-ray diffraction and wide-angle X-ray scattering analysis.