De novo biosynthesis of 8‑prenylnaringenin in Saccharomyces cerevisiae improved by screening and engineering of prenyltransferases and precursor pathway

8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrate precursor levels in microbial chassis.First,six PTs from different sources and their truncated cognates were expressed in a(2S)-naringenin producing strain.Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate,tSfN8DT-1,could synthe-size 8-PN.Second,tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1^(Q12E)with greater catalytic activity was obtained.Third,key genes,tHMGR and IDI1,of the mevalonate(MVA)pathway were overexpressed using a copy number combinatorial strategy,which greatly improved 8-PN titer by 368.75%.Fourth,a predicted structure of tSfN8DT-1^(Q12E)was used for molecular docking and virtual saturation mutagenesis.Two key residues,P229 and N305,were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M.The best-performing mutant,tSfN8DT-1^(Q12EN305M),produced 49.35±0.05 mg/L(5.57±0.01 mg/g DCW)8-PN in a shaking flask.Finally,101.40±2.55 mg/L of 8-PN was obtained in a 5-L bioreactor,which is the greatest titer reported to date for 8-PN.This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1.The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods,which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.

Biocatalytic access to diverse prenylflavonoids by combining a regiospecific C-prenyltransferase and a stereospecific chalcone isomerase

Prenylflavonoids are valuable natural products that have diverse biological properties, and are usually generated biologically by multiple metabolic enzymes in nature. In this study, structurally diverse prenylflavonoids were conveniently synthesized by enzymatic catalysis by combining GuILDT, a regiospecific chalcone prenyltransferase, and Gu CHI, a stereospecific chalcone isomerase that has promiscuous activity for both chalcones and prenylchalcones as substrates. Our findings provided a new approach for the synthesis of natural/unnatural bioactive prenylflavonoids, including prenylchalcones and optical prenylflavanones with chalcone origins.

A Prenyltransferase Gene Confirmed to Be a Carotenogenic CRTE Gene from Sweetpotato

Sweetpotato （Ipomoea batatas L. Lain.） ranks fifth among the most important food crops, after rice, wheat, maize and cas- sava, on a fresh-weight basis in developing countries （Scott and Maldonado, 1999）. Sweetpotato cultivars have white, yellow, purple or orange flesh, but only orange-fleshed sweetpotato cultivars are the rich sources of β-carotene, which is the precursor of vitamin A （Failla et al., 2009）. Accordingly, the orange-fleshed sweetpotato is the main source of 6-carotene for the people in underdeveloped coun- tries in Africa and Southeast Asia. Unfortunately, theβ-caro- tene content in most sweetpotato cultivars is rather low and insufficient in meeting the normal demand of human physi- ology （Liao et al., 2008）. Hence, developing sweetpotato cultivars rich inβ-carotene will be most desirable in crop development.

Microbial soluble aromatic prenyltransferases for engineered biosynthesis

Prenyltransferase(PTase)enzymes play crucial roles in natural product biosynthesis by transferring isoprene unit(s)to target substrates,thereby generating prenylated compounds.The prenylation step leads to a diverse group of natural products with improved membrane affinity and enhanced bioactivity,as compared to the nonprenylated forms.The last two decades have witnessed increasing studies on the identification,characterization,enzyme engineering,and synthetic biology of microbial PTase family enzymes.We herein summarize several examples of microbial soluble aromatic PTases for chemoenzymatic syntheses of unnatural novel prenylated compounds.

Structural Analyses of Short-Chain Prenyltransfemses Identify an Evolutionarily Conserved GFPPS Clade in Brassicaceae Plants

Terpenoids are the largest and most diverse class of plant-specialized metabolites, which function in diverse physiological processes during plant development. In the biosynthesis of plant terpenoids, short-chain prenyltransferases （SC-PTs）, together with terpene synthases （TPSs）, play critical roles in determining terpenoid diversity. SC-PTs biosynthesize prenyl pyrophosphates with different chain lengths, and these compounds are the direct precursors of terpenoids. Arabidopsis thaliana possesses a subgroup of SC-PTs whose functions are not clearly known. In this study, we focus on 10 geranylgeranyl pyro- phosphate synthase-like [GGPPSL] proteins, which are commonly thought to produce GGPP [C20]. We found that a subset of members of the Arabidopsis GGPPSL gene family have undergone neo- functionalization： GGPPSL6, 7, 9, and 10 mainly have geranylfarnesyl pyrophosphate synthase activity （C25; renamed AtGFPPS1, 2, 3, and 4）, and GGPPSL8 produces even longer chain prenyl pyrophosphate （〉C30; renamed polyprenyl pyrophosphate synthase 2, AtPPPS2）. By solving the crystal structures of AtGFPPS2, AtPPPS2, and AtGGPPS11, we reveal the product chain-length determination mechanism of SC-PTs and interpret it as a ＂three floors＂ model. Using this model, we identified a novel GFPPS clade distributed in Brassicaceae plants and found that the GFPPS gene typically occurs in tandem with a gene encoding a TPS, forming a GFPPS-TPS gene cluster.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action,high potency and reduced toxicity compared to today's standard drugs.However,production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism.Previously,we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum.Four brasilicardin congeners,intermediates of the BraA biosynthesis,were produced.Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization.Therefore,we used a metabolic engineering approach to increase heterologous production of brasilicardin in A.japonicum.Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate(IPP)and to channel the precursor into the direction of diterpenoid biosynthesis.Both approaches contributed to an elevated heterologous production of the brasilicardin backbone,which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Targeting the biological activity and biosynthesis of hyperforin:amini-review

Hyperforin is a representative polycyclic polyprenylated acylphloroglucinols(PPAPs)that exerts a variety of pharmacological activities.The complete biosynthesis pathway of hyperforin has not been elucidated due to its complex structure and unclear genetic background of its source plants.This mini-review focuses on the bioactivity and biosynthesis of hyperforin.These analyses can provide useful insights into the biosynthesis investigations of hyperforin and other PPAPs with complex structures.