A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

Production of 17α-hydroxyprogesterone using an engineered biocatalyst with efficient electron transfer and improved 5-aminolevulinic acid synthesis coupled with a P450 hydroxylase.

17α-Hydroxyprogesterone (17α-OH-PROG) is an important intermediate with a wide range of applications in the pharmaceutical industry. Strategies based on efficient electron transfer and cofactor regeneration were used for the production of 17α-OH-PROG. Here, CYP260A1, Fpr and Adx were expressed using a double plasmid system, resulting in higher biotransformation efficiency. Further optimization of reaction conditions and addition of polymyxin B increased the production of 17α-OH-PROG from 12.52 mg/L to 102.37 mg/L after 12 h of biotransformation. To avoid the addition of external 5-aminolevulinic acid (ALA) as a heme precursor for the P450 enzyme, a modified C5 pathway was introduced into the engineered strain, further reducing the overall process cost. The resulting whole-cell biocatalyst achieved the highest biotransformation yield of 17α-OH-PROG reported to date, offering a promising strategy for commercial application of P450 enzymes in industrial production of hydroxylated intermediates.

[Biosynthesis of calcifediol and calcitriol: a review].

VD3 is a crucial vitamin for human health, as it enhances calcium absorption in the intestines and prevent rickets. Calcifediol (25(OH)VD3) and calcitriol (1α,25(OH)2VD3) are two derivatives of vitamin D3 that play an important role in preventing and treating osteoporosis, as well as regulating human physiological functions. Currently, the production of calcifediol, and calcitriol primarily relies on chemical synthesis, which has disadvantages such as low product yield, numerous by-products, and environmental unfriendliness. Therefore, developing a green, safe, and environmentally friendly biocatalytic synthesis pathway is of utmost importance. This article mainly reviews the biocatalytic synthesis pathways of calcifediol, and calcitriol. The P450 enzymes, including P450 monooxygenases (cytochrome P450 monooxygenases, CYPs) and P450 peroxygenases (unspecific peroxygenases, UPOs), are crucial for the production of calcifediol and calcitriol. The catalytic mechanism of the extensively studied P450 monooxygenases, the selection of suitable redox partners, and the key residues involved in the enzyme's catalytic activity are analyzed. In addition, the review explores H2O2-driven UPOs, including their catalytic mechanism, strategies for high heterologous expression, and in situ regeneration of H2O2. UPOs are regarded as highly promising biocatalysts because they can facilitate reactions without the need for expensive cofactors and redox partners. This review offers insights into the engineering of P450 for the efficient production of vitamin D3 derivatives.

Customized exogenous ferredoxin functions as an efficient electron carrier.

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe-2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe-2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD (4-androstene-3,17-dione) conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF + Fdx + KshA (KshA, Rieske-type oxygenase of 3-ketosteroid-9-hydroxylase) in the reaction system rather than KshAB complex system was proposed based on analysis of protein-protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier.