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Hepatic fibrosis is a compensatory response to chronic liver injury and inflammation, and dietary intervention is recommended as one of the fundamental prevention strategies. Raspberry ketone (RK) is an aromatic compound first isolated from raspberry and widely used to prepare food flavors. The current study investigated the hepatoprotection and potential mechanism of RK against hepatic fibrosis. In vitro, hepatic stellate cell (HSC) activation was stimulated with TGF-ß and cultured with RK, farnesoid X receptor (FXR), or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) agonist or inhibitor, respectively. In vivo, C57BL/6 mice were injected intraperitoneally with thioacetamide (TAA) at 100/200 mg/kg from the first to the fifth week. Mice were intragastrically administrated with RK or Cur once a day from the second to the fifth week. In activated HSCs, RK inhibited extracellular matrix (ECM) accumulation, inflammation, and epithelial-mesenchymal transition (EMT) process. RK both activated FXR/PGC-1α and regulated their crosstalk, which were verified by their inhibitors and agonists. Deficiency of FXR or PGC-1α also attenuated the effect of RK on the reverse of activated HSCs. RK also decreased serum ALT/AST levels, liver histopathological change, ECM accumulation, inflammation, and EMT in mice caused by TAA. Double activation of FXR/PGC-1α might be the key targets for RK against hepatic fibrosis. Above all, these discoveries supported the potential of RK as a novel candidate for the dietary intervention of hepatic fibrosis.

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SCOPE: Depression, a prevalent mental disorder, has significantly impacted the lives of 350 million people, yet it holds promise for amelioration through food-derived phenolics. Raspberries, renowned globally for their delectable flavor, harbor a phenolic compound known as raspberry ketone (RK). However, the impact of RK on depressive symptoms remains ambiguous. This study aims to investigate the impact of RK on lipopolysaccharide (LPS)-induced depressed mice and elucidates its potential mechanisms, focusing on the gut-brain axis. METHODS AND RESULTS: Through behavioral tests, RK exerts a notable preventive effect on LPS-induced depression-like behaviors in mice. RK proves capable of attenuating gut inflammation, repairing gut barrier impairment, modulating the composition of the gut microbiome (Muribaculaceae, Streptococcus, Lachnospiraceae, and Akkermansia), and promoting the production of short-chain fatty acids. Furthermore, RK alleviates neuroinflammation by suppressing the TLR-4/NF-κB pathway and bolsters synaptic function by elevating levels of neurotrophic factors and synapse-associated proteins. CONCLUSION: The current study provides compelling evidence that RK effectively inhibits the TLR-4/NF-κB pathway via the gut-brain axis, leading to the improvement of LPS-induced depression-like behaviors in mice. This study addresses the research gap in understanding the antidepressant effects of RK and illuminates the potential of utilizing RK as a functional food for preventing depression.

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Zeugodacus cucurbitae and Z. tau are two major fruit fly pests of cucurbitaceous plants in the tropical and subtropical regions. The former species has a broader host range and wider world distribution than the latter. With global climate change, Z. tau shows great potential for geographical expansion with several invasion records in recent years. Males of both species are attracted to cue lure (CL) (and raspberry ketone (RK), a deacetyl derivative of CL), a common male lure used in fruit fly population detection, monitoring and control programs. Males of both species are also known to respond to zingerone (ZN), which are produced by some rainforest orchids. Previous studies have shown that fruit fly-male lure interactions are unique to species and lure types, and significantly impact the success of a lure-based fruit fly control program. We seek to compare the attraction of Z. cucurbitae and Z. tau males to CL, RK and ZN via Probit behavioral assays. Our results showed that CL is more attractive to Z. cucurbitae and Z. tau males than RK, while ZN is a poor lure for both species. Attraction Z. tau to CL is slightly lower than Z. cucurbitae, but the former is at least 1.71 times less attractive to RK than the latter. Together with published information on species' sexual development, our current study indicates a lure-based control program via male annihilation technique for Z. tau will be more challenging than Z. cucurbitae and should incorporate other integrated pest management strategies for a desirable outcome.

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The natural occurrence, distribution (within a plant) and roles of four phenylbutanoid compounds (anisyl acetone, cue-lure, raspberry ketone and zingerone) are elucidated for the Asia-Pacific and Oceania regions. These phenylbutanoids may act individually or in combination to attract true fruit fly males belonging to a tribe Dacini of subfamily Dacinae (Diptera: Tepritidae). Of special interest are the mutualistic interactions between the Dacini fruit fly males and the tropical daciniphilous (attracting exclusively Dacini fruit flies) orchids - leading to cross pollination for the orchids and enchanced mating success for the flies. When offered to male flies, anisyl acetone and cue-lure are generally converted to raspberry ketone. Upon consumption, raspberry ketone and zingerone are individually sequestered in the male rectal (pheromonal) gland unchanged. Attracted male flies readily imbibe the phenylbutanoid(s) in the floral synomone to compliment the endogenously synthesized male sex pheromonal components - to enhance attraction of conspecific females during courtship as well as attract conspecific males to form 'leks'. The phenylbutanoid(s) may also act as an allomone to deter vertebrate predators, especially geckos, besides possessing antimicrobial and antioxidant activities. Cue-lure, raspberry ketone and zingerone are important attractants/lures used in pest surveillance and mass trapping under the integrated pest management (IPM) program against quarantine Dacini fruit fly pest species, particularly Bactrocera tryoni and Zeugodacus cucurbitae.

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Raspberry ketone has generated interest in recent years both as a flavor agent and as a health promoting supplement. Raspberry ketone can be synthesized chemically, but the value of a natural nonsynthetic product is among the most valuable flavor compounds on the market. Coumaroyl-coenzyme A (CoA) is the direct precursor for raspberry ketone but also an essential precursor for flavonoid and lignin biosynthesis in plants and therefore highly regulated. The synthetic fusion of 4-coumaric acid ligase (4CL) and benzalacetone synthase (BAS) enables the channeling of coumaroyl-CoA from the ligase to the synthase, proving to be a powerful tool in the production of raspberry ketone in both N. benthamiana and S. cerevisiae. To the best of our knowledge, the key pathway genes for raspberry ketone formation are transiently expressed in N. benthamiana for the first time in this study, producing over 30 µg/g of the compound. Our raspberry ketone producing yeast strains yielded up to 60 mg/L, which is the highest ever reported in yeast.

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The current study aimed to investigate the effect of orally administered raspberry ketone (RK) on ameliorating nonalcoholic fatty liver disease (NAFLD) induced in rats by high-fat high-fructose diet (HFFD) in comparison to calorie restriction (CR) regimen. Thirty male Wistar rats were divided into two experimental groups; one was fed normal chow diet (NCD, n = 6) for 15 weeks to serve as normal control group and the other group was fed HFFD (n = 24) for 7 weeks to induce NAFLD. After induction, rats in the HFFD group were randomly allocated into four groups (n = 6 rats each). One group continued on HFFD feeding for 8 weeks (NAFLD control group). The remaining 3 groups received NCD, calorie-restricted diet, or NCD along with RK (55 mg/kg/day, orally) for 8 weeks. Like CR, RK effectively attenuated NAFLD and ameliorated the changes attained by HFFD. RK upregulated the expression of the phosphorylated AMP-activated protein kinase (P-AMPK) and fatty acid oxidation factors; peroxisome proliferator-activated receptor alpha (PPAR-α) and carnitine palmitoyltransferase-1 (CPT-1) and downregulated lipogenic factors; sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) in the hepatic tissue. Also, RK improved lipid profile parameters, liver enzymes and both body and liver tissue weights. Altogether, these findings suggest that oral administration of RK, along with normal diet, ameliorated NAFLD in a way similar to CR. This approach could be an alternative to CR in the management of NAFLD, overcoming the poor compliance to long term CR regimen.

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BACKGROUND: Queensland fruit fly (Qfly) males exhibit accelerated sexual maturation when their diet is supplemented with raspberry ketone (RK) for 48 h following emergence, which is beneficial for sterile insect technique operation. The present study tests whether RK supplementation makes Qfly more vulnerable to starvation or desiccation. RESULTS: Flies were fed for 48 h with a yeast hydrolysate and sugar diet (1:3) that contained 0% RK (control), 1.25% RK (low dose) or 5% RK (high dose) to test subsequent vulnerability to starvation and desiccation. RK feeding decreased body weight and water content in males and increased lipid levels in both sexes before exposure to any stress treatment. Under nutritional stress, flies fed the low RK dose, but not the high RK dose, had higher survival than controls. Under desiccation stress, flies fed both the low and high RK doses had lower survival than the controls. Body weight, water content and lipid reserves at death were all affected by RK dose when under nutritional stress, but not when under desiccation stress. In the absence of stress, body weight at death was higher than controls in flies provided with the high RK dose and lipids were lower than controls in flies provided with the low RK dose. CONCLUSION: Feeding with RK makes Qflies more vulnerable to desiccation but not starvation. In most conditions, it is expected that the disadvantage of increased desiccation vulnerability would be outweighed by the benefits of accelerated sexual maturation in RK-fed young adult Qflies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Tyrosinase is the key enzyme for melanin biosynthesis. Overproduction and deposition of this pigment cause different problems in various industries including agriculture and food. Finding safe tyrosinase inhibitors thus attracts great research interest. The goal of this study is evaluation of inhibitory potencies of some novel synthetic derivatives of tyrosol and raspberry ketone on diphenolase activity of mushroom tyrosinase. The ligands inhibited enzyme activity and compound 4-(2-(4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl)ethyl)phenol (1d) exhibited the most inhibitory potency (77% inhibition, IC50 = 0.32 µmol L-1) via the mixed inhibition mode. This compound was also safe according to the results of in vitro analyses. The enzyme-ligands interactions were theoretically and experimentally investigated using molecular docking and fluorescence quenching approaches, respectively. Modes of quenching and related parameters were also determined and molecular docking data showed that the ligands bind to important sites of the enzyme. These compounds, especially 1d, can be suggested as efficient candidates for further investigations.

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Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

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OBJECTIVES: To investigate the therapeutic role of calorie-restricted diet (CR) and raspberry ketone (RK) in non-alcoholic fatty liver disease (NAFLD) and the implication of sphingosine kinase-1 (SphK1)/sphingosine-1-phosphate (S1P) and toll-like receptor 4 (TLR4) signalling. METHODS: NAFLD was induced by feeding rats high-fat-fructose-diet (HFFD) for 6 weeks. Rats were then randomly assigned to three groups (n = 6 each); NAFLD group continued on HFFD for another 8 weeks. CR group was switched to CR diet (25% calorie restriction) for 8 weeks and RK group was switched to normal diet and received RK (55 mg/kg/day; orally) for 8 weeks. Another six rats were used as normal control. KEY FINDINGS: HFFD induced a state of NAFLD indicated by increased fat deposition in liver tissue along with dyslipidemia, elevated liver enzymes, oxidative stress and inflammation. Either CR diet or RK reversed these changes and decreased HFFD-induced elevation of hepatic SphK1, S1P, S1PR1 and TLR4. Of notice, RK along with a normal calorie diet was even better than CR alone in most studied parameters. CONCLUSIONS: SphK1/S1P and TLR4 are interconnected and related to the establishment of HFFD-induced NAFLD and can be modulated by RK. Supplementation of RK without calorie restriction to patients with NAFLD unable to follow CR diet to achieve their treatment goals would be a promising therapeutic modality.

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Raspberry Ketone (RK) and Resveratrol (RSV) are natural phenolic antioxidants and anti-inflammatory agents. However, its combined pharmacokinetic and pharmacodynamics potentials are not reported. The study aims to assess the combined effect of RK with RSV to protect rats from carbon-tetrachloride (CCl4) induced oxidative stress and NASH. The toxicant CCl4 was employed at a concentration of 1 mL/kg as a 1:1 (v/v) mixture with olive oil twice weekly for 6 weeks to induce liver toxicity. Animal treatment was followed for 2 weeks. Silymarin was used as a standard control drug to compare the hepatoprotective effect of RK and RSV. Hepatic histology, oxidative stress, MMP, reduced glutathione (GSH), plasma levels of SGOT, SGPT, and lipid profile (total cholesterol and triglycerides) were measured. Anti-inflammation genes (IL-10), and fibrotic genes (TGF-ß) were also examined in liver tissue. Oral administration of combined RK with RSV (50 + 50 mg/kg for 2 weeks) showed significantly more hepatoprotection by significantly decreasing elevated plasma markers and lipid profile than alone RK and RSV (100 mg/kg daily for 2 weeks). It also significantly alleviated the hepatic lipid peroxidation, restoring the activities of GSH levels in the liver. RT-PCR and Immunoblotting studies confirmed that significantly upregulation of anti-inflammation genes and protein expression (MMP-9) ameliorated the disease. Pharmacokinetic studies confirmed more synergistic stability in simulated gastric-intestinal fluids (FaSSGF, FaSSIF) and rat liver microsomes (CYP-450, NADPH oxidation & glucuronidation. Moreover, coadministration of drugs augmented the relative bioavailability, Vd/ F (L/Kg), and MRT0-∞( h), which leads to more efficacy. This pharmacokinetic and pharmacodynamic reveals a new adjuvant therapy for the treatment of steatohepatitis.

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Salmonella enterica is an important foodborne pathogen that causes gastroenteritis and systemic infection in humans and livestock. Salmonella biofilms consist of two major components-amyloid curli and cellulose-which contribute to the prolonged persistence of Salmonella inside the host. Effective agents for inhibiting the formation of biofilms are urgently needed. We investigated the antibiofilm effect of Raspberry Ketone (RK) and its mechanism of action against Salmonella Typhimurium 14028 using the Congo red agar method, Calcofluor staining, crystal violet method, pellicle assay, and the TMT-labeled quantitative proteomic approach. RK suppressed the formation of different types of Salmonella biofilms, including pellicle formation, even at low concentrations (200 µg/mL). Furthermore, at higher concentrations (2 mg/mL), RK exhibited bacteriostatic effects. RK repressed cellulose deposition in Salmonella biofilm through an unknown mechanism. Swimming and swarming motility analyses demonstrated reduced motility in RK-treated S. typhimurium. Proteomics analysis revealed that pathways involved in amyloid curli production, bacterial invasion, flagellar motility, arginine biosynthesis, and carbohydrate metabolism, were targeted by RK to facilitate biofilm inhibition. Consistent with the proteomics data, the expressions of csgB and csgD genes were strongly down-regulated in RK-treated S. typhimurium. These findings clearly demonstrated the Salmonella biofilm inhibition capability of RK, justifying its further study for its efficacy assessment in clinical and industrial settings.

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ETHNOPHARMACOLOGICAL RELEVANCE: Rubus idaeus Linnaeus (RI) is a Chinese herbal medicine that has been widely used in China for a long time to reinforce the kidney, nourish the liver, improve vision, and arrest polyuria. AIM OF THE STUDY: This work aims to evaluate the recent progress of the chemical composition, pharmacological activity, pharmacokinetics, metabolism, and quality control and of Rubus idaeus, which focuses on the insufficiency of existing research and will shed light on future studies of Rubus idaeus. METHODS: Literatures about "Rubus idaeus","Red raspberry" and "Fupenzi"are retrieved by browsing the database, such as Web of Science (http://www.webofknowledge.com/wos), Pubmed (https://pubmed.ncbi.nlm.nih.gov/), CNKI (http://www.cnki.net/), and Wanfang Data (http://www.wanfangdata.com.cn). In addition, related textbooks and digital documents are interrogated to provide a holistic and critical review of the topic. The period of the literature covered from 1981 to 2022. RESULTS: Approximately 194 compounds have been isolated from Rubus idaeus, which is rich in phenols, terpenoids, alkaloids, steroids, and fatty acids. Numerous investigations have demonstrated that Rubus idaeus exhibits many pharmacological activities, including hypoglycemic and hypolipidemic, anti-Alzheimer effect, anti-osteoporosis, hepatoprotective, anti-cancer, neuroprotective, anti-bacteria and skin care, etc. However, it is worth noting that most of the research is not associated with the conventional effect, such as reducing urination and treating opacity of the cornea. CONCLUSION: The effectiveness of Rubus idaeus has been proved by its long-term clinical application. The research on the pharmacological activity of Rubus idaeus has flourished. In many pharmacological experiments, only the high-dose group can achieve the corresponding efficacy, so the efficacy of Rubus idaeus needs to be further interrogated. Meanwhile, the relationship between pharmacological activity and specific compounds of Rubus idaeus has not been clarified yet. Last but not least, studies involving toxicology and pharmacokinetics are very limited. Knowledge of bioavailability and toxicological behavior of Rubus idaeus can help understand the herb's pharmacodynamic and safety profile.

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We reported that raspberry ketone (RK) is produced from rhododendrol (RD) in excised mouse skin. We confirmed that RK is also produced from RD in human skin homogenates. We also observed more conversion of RD to RK when the oxidized form of nicotinamide adenine dinucleotide (NAD+), a coenzyme of alcohol dehydrogenase (ADH), was added to human skin homogenates. Chiral column analysis of the consumption of RD enantiomers in human skin homogenates also showed that more of the R enantiomers of RD remained than the S enantiomers of RD. This suggests that the S-enantiomer of RD is more easily oxidized in human skin. We confirmed that RD is partially metabolized to RK in human skin, thus suggesting that ADH in the skin may be the main cause of the appearance of this oxidation product.

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Obesity is a global health problem and a risk factor for cardiovascular diseases and cancers. Exercise is an effective intervention to combat obesity. Fibronectin type III domain containing protein 5 (FNDC5)/irisin, a myokine, can stimulate the browning of white adipose tissue by increasing uncoupling protein 1 (UCP1) expression, and therefore may represent a link between the beneficial effects of exercise and improvement in metabolic diseases. Thus, upregulating the endogenous expression of FNDC5/irisin by administering medication would be a good approach for treating obesity. Herein, we evaluated the efficacy of raspberry ketone (RK) in inducing FNDC5/irisin expression and the underlying mechanisms. The expression of brown fat-specific proteins (PR domain containing 16 (PRDM16), CD137, and UCP1), heme oxygenase-1 (HO-1), FNDC5, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) in differentiated 3T3-L1 adipocyte was analyzed by western blotting or immunofluorescence. The level of irisin in the culture medium was also assayed using an enzyme-linked immunosorbent assay kit. Results showed that RK (50 µM) significantly induced the upregulation of FNDC5 protein in differentiated 3T3-L1 adipocytes; however, the irisin level in the culture media was unaffected. Moreover, RK significantly increased the levels of PGC1α, brown adipocyte markers (PRDM16, CD137, and UCP1), and HO-1. Furthermore, the upregulation of PGC1α and FNDC5 and the browning effect induced by RK were significantly reduced by SnPP or FNDC5 siRNA, respectively. In conclusion, RK can induce FNDC5 protein expression via the HO-1 signaling pathway, and this study provides new evidence for the potential use of RK in the treatment of obesity.

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The demand for raspberry ketone (RK) as a plant-based natural flavoring agent is high, but natural RK is one of the most expensive flavor compounds due to its limited content in plants. Here, we produced RK de novo from simple carbon sources in Escherichia coli. We genetically engineered E. coli metabolism to overproduce the metabolic precursors tyrosine and p-coumaric acid and increase RK production. The engineered E. coli produced 19.3- and 1.9 g/L of tyrosine and p-coumaric acid from glucose, respectively. The p-coumaric acid CoA ligase from Agrobacterium tumefaciens and amino acid substituted benzalacetone synthase of Rhemu palmatum (Chinese rhubarb) were overexpressed in E. coli overproducing p-coumaric acid. The overexpression of fabF, encoding ß-ketoacyl-acyl carrier protein synthetase II increased intracellular malonyl-CoA, the precursor of benzalacetone synthase for RK biosynthesis, and improved RK production. Fed-batch cultures given glucose as a carbon source produced 62 mg/L of RK under optimized conditions. Our production system is inexpensive and does not rely on plant extraction; thus, it should significantly contribute to the flavor and fragrance industries.

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The current investigation was accomplished to evaluate the hepatoprotective effect of White tea and Raspberry Ketone against toxicity induced by acrylamide in rats. Sixty adult male rats were divided randomly into group (I) control; group (II) rats received RK with dose (6 mg/kg/day); Group III: rats received 5 ml of WT extract/kg/day; Group IV rats received AA (5 mg/kg/day); Group V: rats administrated with both AA (5 mg/kg/day) and RK (6 mg/kg/day) and Group VI: rats administrated AA (5 mg/kg/day) and 5 ml of WT extract/kg/day. The biochemical assays exhibited a significant increase in serum levels of Adiponectin, AST, ALT, ALP of the group treated with acrylamide if compared to the control group and an improvement in their levels of groups V and VI. The histopathological and immunohistochemical findings confirm the biochemical observations. In conclusion, the present investigation proved that the supplementation of WT and RK enhanced the liver histology, immunohistochemistry and biochemistry against the oxidative stress induced by acrylamide.

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Novel food and food compounds interventions have attracted a lot of attention nowadays for the prevention and treatment of metabolic diseases. Raspberry ketone (RK) is aromatic compound found within red fruits and berries, has been used as an over-the-counter product for weight loss. However, actually, the effect of RK on weight loss is still controversial, and the mechanism is largely unknown. Besides, in vivo and in vitro studies have demonstrated the beneficial effect of RK on the development of other metabolic diseases. In this review, we comprehensively highlighted the synthesis, bioavailability, and metabolism of RK, and summarized the progress made in our understanding of the potential biological activities of RK, including antiobesity, antidiabetes, cardioprotection, and hepatoprotection, as well as their underlying mechanisms. This paper provides a critical overview about the current findings and proposes the future studies in the area of RK on human health. PRACTICAL APPLICATIONS: Raspberry ketone (RK) has been used for weight control for years, but this effect is controversial considering food intake. Additionally, RK is beneficial for T2DM, liver and heart injury. The underlying mechanisms of the protective effect of RK including accelerating fatty acid oxidation, balancing serum glucose level, anti-inflammation, antioxidant process, and so on. In this context, we provide a comprehensive analysis of the benefits of RK against many metabolic diseases and discuss the underlying molecular mechanisms. We hope our work will be helpful for further researches on RK and improve its public recognition.

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Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1-4 mg kg-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L-1. Herein, we report an efficient cell-free strategy to probe into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalyzed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a 'one-pot' cell-free reaction to produce raspberry ketone at high-yield (61 mg L-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.

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Raspberry ketone (RK) is a major flavor compound in red raspberries, and it has been marketed as a popular weight-loss dietary supplement with high potential in accumulating in fatty tissues. However, challenges in extracting and characterizing RK and its associated phenolic compounds in fatty tissues persist due to the complex matrix effect. In this work, we reported a high-throughput sample preparation method for RK and 25 related phenolic compounds in white adipose tissues using an improved micro-scale QuEChERS (quick, efficient, cheap, easy, rugged and safe) approach with enhanced matrix removal (EMR)-lipid cleanup in 96-well plates, followed by UHPLC-QqQ-MS/MS analysis. The absolute recovery was 73-105% at the extraction step, and achieved 71-96% at the EMR cleanup step. The EMR cleanup removed around 66% of total lipids in the acetonitrile extract as profiled by UHPLC-QTOF-MS/MS. The innovative introduction of a reversed-phase C18 sorbent into the extract significantly improved the analytes' recovery during SpeedVac drying. The final accuracy achieved 80-120% for most analytes. Overall, this newly developed and validated method could serve as a powerful tool for analyzing RK and related phenolic compounds in fatty tissues.

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