Protein engineering for natural product biosynthesis:expanding diversity for therapeutic applications

In this dispensation of the fourth industrial revolution,protein engineering has become a popular approach for increasing enzymatic activity,stability,and titer in the biosynthesis of natural products.This is attributed to its numerous advantages(over direct isolation from plants or via chemical synthesis),including decreasing or eliminating reaction byproducts,high precision,moderate handling of intricate and chemically unstable chemicals,overall reusability,and cost efficiency.Recently,protein engineering tools have advanced to redesign and enhance natural product biosynthesis.These methods include direct evolution,substrate engineering,medium engineering,enzyme engineering and immobilization,structure-assisted protein engineering,and advanced computational.Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article.Also,the advantages,limitations,and applications in industrial and medical biotechnology were discussed.Last,future research directions and potential were also highlighted.

Cytochrome P450s in algae:Bioactive natural product biosynthesis and light-driven bioproduction

Algae are a large group of photo synthetic organisms responsible for approximately half of the earth’s total photosynthesis.In addition to their fundamental ecological roles as oxygen producers and as the food base for almost all aquatic life,algae are also a rich source of bioactive natural products,including several clinical drugs.Cytochrome P450 enzymes(P450s) are a superfamily of biocatalysts that are extensively involved in natural product biosynthesis by mediating various types of reactions.In the post-genome era,a growing number of P450 genes have been discovered from algae,indicating their important roles in algal life-cycle.However,the functional studies of algal P450s remain limited.Benefitting from the recent technical advances in algae cultivation and genetic manipulation,the researches on P450s in algal natural product biosynthesis have been approaching to a new stage.Moreover,some photoauto trophic algae have been developed into "photo-bioreactors" for heterologous P450s to produce high-value added pharmaceuticals and chemicals in a carbon-neutral or carbon-negative manner.Here,we comprehensively review these advances of P450 studies in algae from 2000 to 2021.

Investigation of carbonyl amidation and O-methylation during biosynthesis of the pharmacophore pyridyl of antitumor piericidins

Piericidins are a large family of bacterialα-pyridone antibiotics with antitumor activities such as their anti-renal carcinoma activity exhibited recently in nude mice.The backbones of piericidins are derived fromβ,δ-diketo carboxylic acids,which are offloaded from a modular polyketide synthase(PKS)and putatively undergo a carbonyl amidation beforeα-pyridone ring formation.The tailoring modifications to theα-pyridone structure mainly include the verified hydroxylation and O-methylation of the C-4′position and an unidentified C-5′O-methylation.Here,we describe a piericidin producer,terrestrial Streptomyces conglobatus,which contains a piericidin biosynthetic gene cluster in two different loci.Deletion of the amidotransferase gene pieD resulted in the accumulation of two fatty acids that should be degraded from the nascent carboxylic acid released by the PKS,supporting the carbonyl amidation function of PieD duringα-pyridone ring formation.Deletion of the O-methyltransferase gene pieB1 led to the production of three piericidin analogues lacking C-5′O-methylation,therefore confirming that PieB1 specifically catalyses the tailoring modification.Moreover,bioactivity analysis of the mutant-derived products provided clues regarding the structure-function relationship for antitumor activity.The work addresses two previously unidentified steps involved in pyridyl pharmacophore formation during piericidin biosynthesis,facilitating the rational bioengineering of the biosynthetic pathway towards valuable antitumor agents.