Presentation Information
[P01-088]Study on lignin depolymerization for preparing p-coumaric acid and heterologous biosynthesis of cardamonin
○Yue Bai1 (1. China Medical University (China))
Keywords:
lignin depolymerization,tungstic acid catalysis,cardamonin,biosynthesis,heterologous expression
[Purpose]As an abundant renewable aromatic resource, lignin’s high-value directional conversion is a focus in biomass utilization. p-Coumaric acid, a high-value aromatic product from lignin depolymerization, is widely used in pharmaceuticals, food, and chemical engineering. Cardamonin, a natural flavonoid with diverse biological activities, has low natural extraction yield and complex chemical synthesis, making heterologous biosynthesis critical for large-scale production. This study aimed to use bagasse to explore the catalytic mechanism and optimize processes for p-coumaric acid production via tungstic acid-catalyzed lignin depolymerization, identify key enzymes in cardamonin biosynthesis, construct efficient heterologous expression systems, and optimize fermentation conditions, providing a novel technical route for agricultural waste lignin utilization and cardamonin scale-up.
[Method]Bagasse was pretreated, followed by tungstic acid-catalyzed alcoholysis and alkaline hydrolysis to prepare p-coumaric acid; HPLC quantified products, and reaction time, temperature, and particle size were optimized. Core candidate enzymes for cardamonin biosynthesis were screened via bioinformatics analysis of database sequences. Optimized CHS and CHI genes were synthesized, cloned into vectors, transformed into E. coli BL21(DE3), induced by IPTG, purified by Ni-NTA chromatography, and verified by SDS-PAGE. Four cardamonin biosynthesis systems were established, and the optimal system was screened by HPLC. Single-factor experiments and response surface methodology optimized fermentation conditions; p-coumaric acid toxicity to host strains was evaluated. Fed-batch scale-up fermentation was conducted in a 5 L bioreactor, with cell growth, substrate consumption, and product accumulation monitored by HPLC.
[Results]Tungstic acid showed high catalytic activity for bagasse lignin alcoholysis, with optimal conditions (200 °C, 6 h) yielding 32.852 mg/g methyl p-coumarate; raw material particle size had no significant effect. Subsequent hydrolysis (5% NaOH, 80 °C, 4 h) achieved 90.3% methyl p-coumarate conversion, with a final p-coumaric acid yield of 168.7 mg/g (based on lignin content). Bioinformatics identified conserved functional sites in wheat CHS and Arabidopsis thaliana CHI; their yields in E. coli were 66.31±2.3 mg/L and 56.35±3.2 mg/L, respectively. The single-strain 4CL-CHS-CHI co-transformation system was the most efficient. Optimal fermentation conditions were 30 °C, pH 7.0, and 0.3 mM IPTG; the safe p-coumaric acid concentration was 4 mM. 5 L fed-batch fermentation achieved a final cardamonin titer of 6.44±0.83 mg/L, confirming scalability.
[Consideration]This study has limitations, including relatively low product yield and unvalidated pathway mechanisms. Future research will focus on key enzyme catalytic mechanisms and system optimization.
[Conclusion](1) A process route for p-coumaric acid from bagasse lignin via tungstic acid-catalyzed alcoholysis followed by alkaline hydrolysis was established, with key parameters determined. (2) CHS and CHI (cardamonin biosynthesis candidates) were screened, expressed, and incorporated into multiple 4CL/FCS-containing expression systems. (3) Fermentation conditions of the superior system were optimized via single-factor experiments and response surface methodology, with preliminary 5 L bioreactor scale-up verification completed.
[Method]Bagasse was pretreated, followed by tungstic acid-catalyzed alcoholysis and alkaline hydrolysis to prepare p-coumaric acid; HPLC quantified products, and reaction time, temperature, and particle size were optimized. Core candidate enzymes for cardamonin biosynthesis were screened via bioinformatics analysis of database sequences. Optimized CHS and CHI genes were synthesized, cloned into vectors, transformed into E. coli BL21(DE3), induced by IPTG, purified by Ni-NTA chromatography, and verified by SDS-PAGE. Four cardamonin biosynthesis systems were established, and the optimal system was screened by HPLC. Single-factor experiments and response surface methodology optimized fermentation conditions; p-coumaric acid toxicity to host strains was evaluated. Fed-batch scale-up fermentation was conducted in a 5 L bioreactor, with cell growth, substrate consumption, and product accumulation monitored by HPLC.
[Results]Tungstic acid showed high catalytic activity for bagasse lignin alcoholysis, with optimal conditions (200 °C, 6 h) yielding 32.852 mg/g methyl p-coumarate; raw material particle size had no significant effect. Subsequent hydrolysis (5% NaOH, 80 °C, 4 h) achieved 90.3% methyl p-coumarate conversion, with a final p-coumaric acid yield of 168.7 mg/g (based on lignin content). Bioinformatics identified conserved functional sites in wheat CHS and Arabidopsis thaliana CHI; their yields in E. coli were 66.31±2.3 mg/L and 56.35±3.2 mg/L, respectively. The single-strain 4CL-CHS-CHI co-transformation system was the most efficient. Optimal fermentation conditions were 30 °C, pH 7.0, and 0.3 mM IPTG; the safe p-coumaric acid concentration was 4 mM. 5 L fed-batch fermentation achieved a final cardamonin titer of 6.44±0.83 mg/L, confirming scalability.
[Consideration]This study has limitations, including relatively low product yield and unvalidated pathway mechanisms. Future research will focus on key enzyme catalytic mechanisms and system optimization.
[Conclusion](1) A process route for p-coumaric acid from bagasse lignin via tungstic acid-catalyzed alcoholysis followed by alkaline hydrolysis was established, with key parameters determined. (2) CHS and CHI (cardamonin biosynthesis candidates) were screened, expressed, and incorporated into multiple 4CL/FCS-containing expression systems. (3) Fermentation conditions of the superior system were optimized via single-factor experiments and response surface methodology, with preliminary 5 L bioreactor scale-up verification completed.
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