Cellulosic biomass hydrolysates are rich in glucose and xylose, but most microorganisms, including Komagataella phaffii, are unable to utilize xylose effectively. To address this limitation, we engineered a K. phaffii strain optimized for xylose metabolism through the xylose oxidoreductase pathway and promoter optimization. A promoter library with varying strengths was used to fine-tune the expression levels of the XYL1, XYL2, and XYL3 genes, resulting in a strain with a strong promoter for XYL2 and weaker promoters for XYL1 and XYL3. This engineered strain exhibited superior growth, achieving 14 g cells/L and a maximal growth rate of 0.4 g cells/L-h in kenaf hydrolysate, outperforming a native strain by 17%. This study is the first to report the introduction of the xylose oxidoreductase pathway into K. phaffii, demonstrating its potential as an industrial platform for producing yeast protein and other products from cellulosic biomass.

한국식품저장유통학회지(Korean Journal of Food Preservation)

This study aimed to produce fermented cabbage powder with enhanced enzyme activity and functionality. Twenty-four strains of Bacillus spp. were isolated from two types of traditional nuruk. Four strains exhibiting superior amylase and protease activities were selected through qualitative and quantitative analyses and identified as two B. subtilis and two B. amyloliquefaciens strains using 16S rRNA sequencing. Among them, B. amyloliquefaciens S2 was ultimately selected as the most suitable strain for cabbage fermentation. A solution containing 10% cabbage powder was fermented with this strain for 24 h, followed by the addition of 5% excipient and freeze-drying; among the excipients tested, mannitol provided the highest enzyme retention. Regardless of excipient addition, the lyophilized cabbage powder maintained nearly 100% enzyme stability after heat treatment at 60°C for 12 h, but its activity decreased by approximately 50% after heat treatment at 90°C for 4 h. Furthermore, sulforaphane content exceeded 100 μg/g (based on original powder) after fermentation, representing an increase of over 100-fold compared to the unfermented sample. These results demonstrate that combining strain screening with cabbage fermentation successfully produces a fermented cabbage powder with significantly enhanced amylase and protease activities, as well as increased sulforaphane content compared to the pre-fermentation state.

The widespread use of indigo (C₁₆H₁₀N₂O₂), a historically significant blue pigment, continues across the textile, pharmaceutical, and cosmetic industries. However, conventional production routes based on petrochemical aniline or plant extraction pose serious environmental and sustainability concerns. Here, we present a simulation-guided, cell-free biomanufacturing platform for indigo synthesis that integrates enzyme kinetics modeling with scalable lysate-based catalysis. Three enzymes–tryptophanase A, flavin-containing monooxygenase, and glucose 1-dehydrogenase–were co-expressed in Escherichia coli, enabling the conversion of L-tryptophan to indigo via NADPH-dependent oxidation. Crude lysates prepared from high-cell-density fermentation retained full catalytic activity post-lyophilization, facilitating long-term storage and industrial deployment. Enzyme kinetic parameters were quantified directly in the crude lysates and incorporated into a Python-based ordinary differential equation model (ODE), enabling in silico optimization for substrate loads, cofactor regeneration dynamics, and feeding strategies. Model-optimized feeding strategies enabled cell-free titers of 21.2 g/L and 9.8 g/L from L-tryptophan and indole, respectively, the highest ever reported for FMO-based crude-lysate platforms. Compared to synthetic or whole-cell-derived indigo, the enzymatically produced dye exhibited a markedly reduced endotoxin content, negligible cytotoxicity, and enhanced anti-inflammatory and UV-protective properties. Furthermore, a preliminary raw-material cost analysis identified NADP⁺ as the dominant cost driver (~44.75% of total raw-material costs), and the kinetic model directly linked indigo yield to NADP⁺ loading and feeding strategy, providing a quantitative tool to optimize cofactor utilization in future process development. This work establishes a modular, simulation-driven framework for the sustainable and scalable biosynthesis of high-value dyes, and paves the way for the broader industrial adoption of cell-free biomanufacturing systems.