A one‐pot biocascade of two enzymatic steps catalyzed by an l‐lactate oxidase and a tyrosine phenol‐lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l‐tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio‐based l‐lactate catalyzed by a highly active l‐lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol‐lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l‐tyrosine derivatives synthesis.

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Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

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Reaction coupling separation systems using calcium fumarate as a substrate can break the reaction equilibrium and promote the production of l-malate. However, the low reusability and stability of fumarase limit its further application. In this study, partially purified fumarase of Thermus thermophilus (87.0 U mg−1) was immobilized within konjac-κ-carrageenan beads. An amalgamation of konjac and carrageenan gum (2%) was used to form the beads, and polyethylene polyamine (0.2%) and glutaraldehyde (0.1%) were used as the cross-linking agents. The pH and temperature profiles of free and immobilized fumarases were remarkably similar. The diffusion limit of immobilized fumarase caused a decline in the maximal velocity (Vmax), whereas the kinetic constant (Km) value increased. Optimization of the parameters for biotransformation by immobilized fumarase showed that 88.3% conversion of 200 mM calcium fumarate could be achieved at 55 °C within 8 h. The beads were stored for 30 days at 4 °C with minimal loss in activity and were reusable for up to 20 cycles with 78.1% relative activity. By recycling the reaction supernatant, a total amount of 3.98 M calcium fumarate was obtained with a conversion of 99.5%, which is the highest value ever reported.