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Pinocembrin is one of the most abundant flavonoids in propolis, and it may also be widely found in a variety of plants. In addition to natural extraction, pinocembrin can be obtained by biosynthesis. Biosynthesis efficiency can be improved by a metabolic engineering strategy and a two-phase pH fermentation strategy. Pinocembrin poses an interest for its remarkable pharmacological activities, such as neuroprotection, anti-oxidation, and anti-inflammation. Studies have shown that pinocembrin works excellently in treating ischemic stroke. Pinocembrin can reduce nerve damage in the ischemic area and reduce mitochondrial dysfunction and the degree of oxidative stress. Given its significant efficacy in cerebral ischemia, pinocembrin has been approved by China Food and Drug Administration (CFDA) as a new treatment drug for ischemic stroke and is currently in progress in phase II clinical trials. Research has shown that pinocembrin can be absorbed rapidly in the body and easily cross the blood–brain barrier. In addition, the absorption/elimination process of pinocembrin occurs rapidly and shows no serious accumulation in the body. Pinocembrin has also been found to play a role in Parkinson’s disease, Alzheimer’s disease, and specific solid tumors, but its mechanisms of action require in-depth studies. In this review, we summarized the latest 10 years of studies on the biosynthesis, pharmacological activities, and pharmacokinetics of pinocembrin, focusing on its effects on certain diseases, aiming to explore its targets, explaining possible mechanisms of action, and finding potential therapeutic applications.

Pinocembrin is a natural flavonoid compound extracted from honey, propolis, ginger roots, wild marjoram, and other plants. In preclinical studies, it has shown anti-inflammatory and neuroprotective effects as well as the ability to reduce reactive oxygen species, protect the blood-brain barrier, modulate mitochondrial function, and regulate apoptosis. Considering these pharmaceutical characteristics, pinocembrin has potential as a drug to treat ischemic stroke and other clinical conditions. In this review, we summarize its pharmacologic characteristics and discuss its mechanisms of action and potential therapeutic applications.

Significance Microbes can be made to produce industrially valuable chemicals in high quantities by engineering their central metabolic pathways. This process may require evaluating billions of cells, each containing a unique pathway design, to identify the rare cells with high production phenotypes. We mutated targeted locations across the genome to modify several genes identified as key players. We used sensory proteins responsive to a number of target chemicals to couple the concentration of the target chemical in each cell to individual cell fitness. This coupling of chemical production to fitness allows us to harness evolution to progressively enrich superior pathway designs. Through iterations of genetic diversification and selection, we increased the production of naringenin and glucaric acid 36- and 22-fold, respectively. Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼10 ⁹ cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36- and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Whole-genome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.

Background 2-oxoglutarate-dependent dioxygenase (2ODD) superfamily is the second largest enzyme family in the plant genome and plays diverse roles in secondary metabolic pathways. The medicinal plant Scutellaria baicalensis Georgi contains various flavonoids, which have the potential to treat coronavirus disease 2019 (COVID-19), such as baicalein and myricetin. Flavone synthase I (FNSI) and flavanone 3-hydroxylase (F3H) from the 2ODDs of DOXC subfamily have been reported to participate in flavonoids biosynthesis. It is certainly interesting to study the 2ODD members involved in the biosynthesis of flavonoids in S. baicalensis . Results We provided a genome-wide analysis of the 2ODDs of DOXC subfamily in S. baicalensis , a total of 88 2ODD genes were identified, 82 of which were grouped into 25 distinct clades based on phylogenetic analysis of At2ODDs. We then performed a functional analysis of Sb2ODDs involved in the biosynthesis of flavones and dihydroflavonols. Sb2ODD1 and Sb2ODD2 from DOXC38 clade exhibit the activity of FNSI (Flavone synthase I), which exclusively converts pinocembrin to chrysin. Sb2ODD1 has significantly higher transcription levels in the root. While Sb2ODD7 from DOXC28 clade exhibits high expression in flowers, it encodes a F3H (flavanone 3-hydroxylase). This enzyme is responsible for catalyzing the conversion of both naringenin and pinocembrin into dihydrokaempferol and pinobanksin, kinetic analysis showed that Sb2ODD7 exhibited high catalytic efficiency towards naringenin. Conclusions Our experiment suggests that Sb2ODD1 may serve as a supplementary factor to SbFNSII-2 and play a role in flavone biosynthesis specifically in the roots of S. baicalensis . Sb2ODD7 is mainly responsible for dihydrokaempferol biosynthesis in flowers, which can be further directed into the metabolic pathways of flavonols and anthocyanins.

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p -coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae ( FjT AL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing Fj TAL combined with 4CL from Arabidopsis thaliana ( At 4CL) and CHS from Cucurbita maxima ( Cm CHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa ( Ms CHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli . Key points • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli.

Background Cytochrome P450 monooxygenase constitutes a significant group of oxidative enzymes that can introduce an oxygen atom in a high regio- and stereo-selectivity mode. We used the Bacillus megaterium cytochrome P450 BM3 (CYP450 BM3) and its variants namely mutant 13 (M13) and mutant 15 (M15) for the hydroxylation of diverse class of flavonoids. Results Among 20 flavonoids, maximum seven flavonoids were hydroxylated by the variants while none of these molecules were accepted by CYP450 BM3 in in vitro reaction. Moreover, M13 exhibited higher conversion of substrates than M15 and CYP450 BM3 enzymes. We found that M13 carried out regiospecific 3ʹ-hydroxylation reaction of naringenin with the highest conversion among all the tested flavonoids. The apparent K m and k cat values of M13 for naringenin were 446 µM and 1.955 s −1 , respectively. In whole-cell biotransformation experiment with 100 µM of naringenin in M9 minimal medium with 2 % glucose in shake flask culture, M13 showed 2.14- and 13.96-folds higher conversion yield in comparison with M15 (16.11 %) and wild type (2.47 %). The yield of eriodictyol was 46.95 µM [~40.7 mg (13.5 mg/L)] in a 3-L volume lab scale fermentor at 48 h in the same medium exhibiting approximately 49.81 % conversion of the substrate. In addition, eriodictyol exhibited higher antibacterial and anticancer potential than naringenin, flavanone and hesperetin. Conclusions We elucidated that eriodictyol being produced from naringenin using recombinant CYP450 BM3 and its variants from B. megaterium , which shows an approach for the production of important hydroxylated compounds of various polyphenols that may span pharmaceutical industries.

The 1092 bp F3H gene from Trapa bispinosa Roxb., which was named TbF3H, was cloned and it encodes 363 amino acids. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbF3H with flavanone 3-hydroxylase from other plants. A functional analysis showed that TbF3H of Trapa bispinosa Roxb. encoded a functional flavanone 3-hydroxylase; it catalyzed the formation of dihydrokaempferol (DHK) from naringenin in S. cerevisiae. The promoter strengths were compared by fluorescence microscopy and flow cytometry detection of the fluorescence intensity of the reporter genes initiated by each constitutive promoter (FITC), and DHK production reached 216.7 mg/L by the promoter adjustment strategy and the optimization of fermentation conditions. The results presented in this study will contribute to elucidating DHK biosynthesis in Trapa bispinosa Roxb.

Baicalein is a unique flavonoid compound with important pharmacological activities, derived from Scutellaria baicalensis Georgi. Baicalein, as the aglycone of baicalin, is a key form for exerting pharmacological activity in vivo. β-glucuronidases (GUSs) are the enzymes involved in the conversion of baicalin to baicalein. In this study, the content of baicalein in S. baicalensis was significantly increased by 20.44% after treatment with 5% PEG6000. Seven GUSs from the glycoside hydrolase 79 family were identified through comparative transcriptome analysis. Among them, GUS1 and GUS2 were confirmed to have catalytic activity in converting baicalin to baicalein in prokaryotic and eukaryotic systems. The correlation analysis further revealed a significant positive correlation of 0.962 (p < 0.01) between the expression of GUS2 and baicalein content in six different sources of S. baicalensis. Interestingly, the presence of variable sites in the GUS1 and GUS2 genes significantly affected their catalytic efficiency in the S. baicalensis samples from the six geographic origins. These findings also provide valuable GUS biological enzyme resources for the effective synthesis of baicalein and offer new insights into the accumulation pattern of baicalein in S. baicalensis.

Background Flavonoids are a structurally diverse group of secondary metabolites, predominantly produced by plants, which include a range of compounds with pharmacological importance. Pinocembrin is a key branch point intermediate in the biosynthesis of a wide range of flavonoid subclasses. However, replicating the biosynthesis of these structurally diverse molecules in heterologous microbial cell factories has encountered challenges, in particular the modest pinocembrin titres achieved to date. In this study, we combined genome engineering and enzyme candidate screening to significantly enhance the production of pinocembrin and its derivatives, including chrysin, pinostrobin, pinobanksin, and galangin, in Escherichia coli . Results By implementing a combination of established strain engineering strategies aimed at enhancing the supply of the building blocks phenylalanine and malonyl-CoA, we constructed an E. coli chassis capable of accumulating 353 ± 19 mg/L pinocembrin from glycerol, without the need for precursor supplementation or the fatty acid biosynthesis inhibitor cerulenin. This chassis was subsequently employed for the production of chrysin, pinostrobin, pinobanksin, and galangin. Through an enzyme candidate screening process involving eight type-1 and five type-2 flavone synthases (FNS), we identified Petroselinum crispum FNSI as the top candidate, producing 82 ± 5 mg/L chrysin. Similarly, from a panel of five flavonoid 7- O -methyltransferases (7-OMT), we found pinocembrin 7-OMT from Eucalyptus nitida to yield 153 ± 10 mg/L pinostrobin. To produce pinobanksin, we screened seven enzyme candidates exhibiting flavanone 3-hydroxylase (F3H) or F3H/flavonol synthase (FLS) activity, with the bifunctional F3H/FLS enzyme from Glycine max being the top performer, achieving a pinobanksin titre of 12.6 ± 1.8 mg/L. Lastly, by utilising a combinatorial library of plasmids encoding G. max F3H and Citrus unshiu FLS, we obtained a maximum galangin titre of 18.2 ± 5.3 mg/L. Conclusion Through the integration of microbial chassis engineering and screening of enzyme candidates, we considerably increased the production levels of microbially synthesised pinocembrin, chrysin, pinostrobin, pinobanksin, and galangin. With the introduction of additional chassis modifications geared towards improving cofactor supply and regeneration, as well as alleviating potential toxic effects of intermediates and end products, we anticipate further enhancements in the yields of these pinocembrin derivatives, potentially enabling greater diversification in microbial hosts.

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.