Abstract A current challenge in high‐throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid‐agar plate‐based HTS assay for screening ortho ‐specific hydroxylation of daidzein by sensing formaldehyde generated from the O‐dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde‐specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho ‐carbon position to COH correlates with a linear relationship to O‐dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4′,7‐dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B‐ring, was examined for O‐dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O‐dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A‐ring inactivated in order to find enhanced 3′‐regioselectiviy). The whole‐cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L −1 production titer and greatly increased 3′‐regioselectiviy (3′/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.
The increasing amount of plastic waste has become a considerable environmental issue worldwide, but traditional methods of plastic waste disposal, such as by incineration or in landfills, have negative impacts on the environment and human health. Therefore, sustainable solutions are needed to address this problem. This study proposes an innovative approach to upcycle mixed plastic wastes into useful chemicals through chemo-biological processes. The upcycle process involves the pyrolysis of polyvinyl chloride-containing mixed plastic waste to produce crude pyrolysis oil containing hydrocarbons with broad chain lengths (C7 to C32). To enable efficient biotransformation of hydrocarbons, crude oil was processed through distillation and hydrogenation. The upgraded oil was then biotransformed into the corresponding α,ω-diacids, industrially useful and high-value platform chemicals, by using β-oxidation-blocked Candida tropicalis as a whole-cell biocatalyst, and 94.3% of the produced α,ω-diacids were medium-chain length (C7 to C14). These results suggest a new method for the use of plastic waste as feedstock for value-added chemical production through combined chemical and biological processes to meet sustainability challenges.
Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.
'%3e%3cg id='b'%3e%3cpath id='a' stroke='%23000' stroke-width='2' d='M-6-25H6m-12 3H6m-12 3H6'/%3e%3cuse xlink:href='%23a' y='44'/%3e%3c/g%3e%3cpath stroke='%23fff' d='M0 17v10'/%3e%3ccircle r='12' fill='%23cd2e3a'/%3e%3cpath fill='%230047a0' d='M0-12A6 6 0 0 0 0 0a6 6 0 0 1 0 12 12 12 0 0 1 0-24z'/%3e%3c/g%3e%3cg transform='rotate(-123.69)'%3e%3cuse xlink:href='%23b'/%3e%3cpath stroke='%23fff' d='M0-23.5v3M0 17v3.5m0 3v3'/%3e%3c/g%3e%3c/svg%3e)
'%3e%3ccircle r='20' fill='%23008'/%3e%3ccircle r='17.5' fill='%23fff'/%3e%3ccircle r='3.5' fill='%23008'/%3e%3cg id='d'%3e%3cg id='c'%3e%3cg id='b'%3e%3cg id='a' fill='%23008'%3e%3ccircle r='.875' transform='rotate(7.5 -8.75 133.5)'/%3e%3cpath d='M0 17.5.6 7 0 2l-.6 5L0 17.5z'/%3e%3c/g%3e%3cuse xlink:href='%23a' transform='rotate(15)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(30)'/%3e%3c/g%3e%3cuse xlink:href='%23c' transform='rotate(60)'/%3e%3c/g%3e%3cuse xlink:href='%23d' transform='rotate(120)'/%3e%3cuse xlink:href='%23d' transform='rotate(-120)'/%3e%3c/g%3e%3c/svg%3e)