Bioengineering <i>Caulobacter crescentus </i>for Xylanase Applications in the Bakery Industry

This study investigated the impact of using genetic engineering strategies and a cell-free Xylanase in the baking industry. The xynA1 gene from the nonpathogenic bacterium Caulobacter crescentus (C. crescentus) (NA1000 strain) was integrated into the pAS22 vector with a xylose-inducible promoter and introduced back into the bacteria, resulting in the creation of the BS-xynA1. This construct exhibited substantial secreted xylanase activity, reaching 17.22 U/mL, and a specific activity of 278.64 U/mg after an 18-hour growth period with 0.2% (w/v) corn straw. RT-qPCR analysis confirmed that higher xylanase activity in C. crescentus cells was correlated with increased transcription of the xynA1 gene. Moreover, BS-xynA1 cells coexpress other enzymes, including cellulase, pectinase, α-amylase, β-glycosidase, β-xylosidase, and α-L-arabinosidase, at low levels (≤2 U/mL). In vitro comparison of cell-free xylanase from BS-xynA1 with three commercially available xylanase-containing mixtures commonly utilized in baking protocols revealed its superior specific activity (163.4 U/mg) across a broad temperature range (30-100 °C), with optimal performance at 50 °C. In practical baking tests, the addition of cell-free Xylanase I led to a 9% reduction in dough beat time and an impressive 18.2% increase in bread height compared to those of the control. Notably, the incorporation of Xylanase I resulted in enhanced alveolar structure formation within the bread crumb. Specifically, the following changes were observed in the mass parameters compared to those of the control: an 18.8% increase in extensibility, a 5.2% increase in elasticity, an 8.3% increase in deformation energy, and subsequent improvements in strength. Additionally, Xylanase I inclusion led to a 6.0% reduction in toughness and a 20.6% reduction in the toughness/elasticity index, indicating a reduction in the mass stiffness of the enzyme-treated bread. To date, this is the first successful application of recombinant xylanase I from C. crescentus in biotechnological processes related to baking, underscoring the potential and prospects of synthetic biology in the food industry.