Improving Production of Coniferyl Alcohol in Escherichia coli by Regulating the Multiple Cofactors and Efflux of Intermediates

Coniferyl alcohol, a fundamental component of lignin, serves as a precursor for the biosynthesis of lignan compounds. However, the low content of free coniferyl alcohol limits its production via plant extraction. Besides, the demand for multiple cofactors, complex and inefficient pathways, and metabolite toxicity constrained coniferyl alcohol production using microorganisms. To overcome these shortcomings, flexible linkers and efflux proteins encoded by srpB were used to optimize coniferyl alcohol biosynthesis in Escherichia coli. After the optimization of the biosynthetic pathway, NADPH regeneration and byproduct elimination were employed to increase coniferyl alcohol titer. The efflux protein encoded by yddG, mtn, and luxS involved in the S-adenosyl-l-methionine cycle promoted the production of the precursor ferulic acid. Subsequently, the coniferyl alcohol biosynthetic pathway was optimized using flexible linker (GGGGS)2 between Pc4CL1 and ZmCCR. As excessive accumulation of ferulic acid significantly reduced coniferyl alcohol biosynthesis, the heterologous efflux protein SrpB was used to verify the possibility of increasing cell tolerance to ferulic acid. Followed by integration of heterologous srpB into the curA loci on the genome, the coniferyl alcohol titer increased from 659.5 to 742.8 mg/L in shake flasks and reached 1315.3 mg/L in a 5-L fermenter. This study emphasizes the importance of precursor control in the biosynthesis of phenylpropanoid compounds.