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Summary: The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of 'Ser‐Hyp‐Hyp‐Hyp‐Hyp' motif or (SP4)18 and an arabinogalactan protein module consisting of 32 tandem repeats of 'Ser‐Hyp' motif or (SP)32. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)18 and (SP)32 modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)18‐EGFP and (SP)32‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)18 module and AGP‐styled (SP)32 module is proposed. [ABSTRACT FROM AUTHOR]

Whole grain rice is a rich source of fiber, nutrients, and phytochemicals that may promote gastrointestinal health, but such beneficial components are typically removed with the bran during polishing. Soluble feruloylated arabinoxylan oligosaccharides (FAXO) and polyphenols (RBPP) isolated from rice bran are hypothesized to have positive impacts on human gut microbiota through a prebiotic function. Using an in vitro human fecal fermentation bioassay, FAXO and RBPP treatments were assessed for short-chain fatty acids (SCFA) production patterns and by evaluating their impacts on the phylogentic composition of human gut microbiota by 16S rRNA gene sequencing. Fresh fecal samples collected from healthy adults (n = 10, 5 males, 5 females) were diluted with anaerobic medium. Each sample received five treatments: CTRL (no substrates), FOS (fructooligosaccharides), FAXO, RBPP, and MIX (FAXO with RBPP). Samples were incubated at 37 °C and an aliquot was withdrawn at 0, 4, 8, 12, and 24 h Results showed that SCFA production was significantly increased with FAXO and was comparable to fermentation with FOS, a well-established prebiotic. RBPP did not increase SCFA productions, and no significant differences in total SCFA production were observed between FAXO and MIX, indicating that RBPP does not modify FAXO fermentation. Changes in microbiota population were found in FAXO treatment, especially in Bacteroides, Prevotella, and Dorea populations, indicating that FAXO might modulate microbiota profiles. RBPP and MIX increased Faecalibacterium, specifically F. prausnitzii. Combined FAXO and RBPP fermentation increased abundance of butyrogenic bacteria, Coprococcus and Roseburia, suggesting some interactive activity. Results from this study support the potential for FAXO and RBPP from rice bran to promote colon health through a prebiotic function. [ABSTRACT FROM AUTHOR]

Whole grain rice is a rich source of fiber, nutrients, and phytochemicals that may promote gastrointestinal health, but such beneficial components are typically removed with the bran during polishing. Soluble feruloylated arabinoxylan oligosaccharides (FAXO) and polyphenols (RBPP) isolated from rice bran are hypothesized to have positive impacts on human gut microbiota through a prebiotic function. Using an in vitro human fecal fermentation bioassay, FAXO and RBPP treatments were assessed for short-chain fatty acids (SCFA) production patterns and by evaluating their impacts on the phylogentic composition of human gut microbiota by 16S rRNA gene sequencing. Fresh fecal samples collected from healthy adults ( n = 10, 5 males, 5 females) were diluted with anaerobic medium. Each sample received five treatments: CTRL (no substrates), FOS (fructooligosaccharides), FAXO, RBPP, and MIX (FAXO with RBPP). Samples were incubated at 37 °C and an aliquot was withdrawn at 0, 4, 8, 12, and 24 h Results showed that SCFA production was significantly increased with FAXO and was comparable to fermentation with FOS, a well-established prebiotic. RBPP did not increase SCFA productions, and no significant differences in total SCFA production were observed between FAXO and MIX, indicating that RBPP does not modify FAXO fermentation. Changes in microbiota population were found in FAXO treatment, especially in Bacteroides , Prevotella , and Dorea populations, indicating that FAXO might modulate microbiota profiles. RBPP and MIX increased Faecalibacterium , specifically F. prausnitzii . Combined FAXO and RBPP fermentation increased abundance of butyrogenic bacteria, Coprococcus and Roseburia , suggesting some interactive activity. Results from this study support the potential for FAXO and RBPP from rice bran to promote colon health through a prebiotic function., Competing Interests: The authors declare no conflicts of interest.