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Enjoy this Page? Please Share! Prices across cities for Raspberry Ketone. Is the information useful? I agree to the terms and privacy policy. Watch related videos. Have a Question? Ask our expert. Speak your question. Raspberry ketones are claimed to cause the fat within cells to be broken down more.
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Screening FAs response promoters. Symbols are grey filled rectangle: four truncated promoters; red filled rectangle: region and region of frdA gene; yellow rightwards arrow: frdA gene. The control for these experiments was P Then we carried out genetic modification using above promoters to develop strains that were suitable for bioconversion strategy.
First, an additional copy of the AT4CL1 gene was expressed under the control of Pfrd3 and chromosomally inserted at the poxB site, resulting in new strain CR6. Raspberry ketone production by CR6 was about We attempted to increase the intracellular malonyl-CoA level by disrupting the competitive pathways. The fatty acids biosynthesis pathway is a major pathway for malonyl-CoA consumption.
Meanwile, studies have also been shown that the introduction of cyanobacteria carbon concentration mechanism CCM in E. Therefore, the bicarbonate transporter BT gene from Synechococcus sp. PCC were fused and chromosomal expressed by inserting at fabB site under the control of Pfrd3 promoter, obtained the strain CR7, and the final RK concentration reached The bioconversion conditions were further optimized to achieve higher raspberry ketone production titers.
Biomass during bioconversion was investigated at 10,20,30, and 40 O. It was shown the higher titer was obtained at 30 O. Then the speed of rotation was further optimized. Raspberry ketone titer with D biomass, bioconversion for 30 h. Based on CR8, a continual fermentation process was conducted. Then the cells were collected by centrifugation, resuspended in the MCM7 medium at 30 O. After bioconversion for additional 24 h, The above operation was repeated three times. The raspberry ketone titers were RK production by repeat bioconversion process.
Red line indicates induction and cell growth stage. The left side of the dotted line represents the cell growth and protein induction phase. Furthermore, bioconversion was carried out in a 1-L bioreactor.
RK production of strain CR8 was investigated by feeding with soybean oil as a single substrate Fig. CR8 strain was first cultivated in CD medium for 24 h. Soybean oil and glycerol were used as mixed substrates and fed according to a carbon ratio, the total carbon of the mixed feed was remained the same as the glycerol feed.
P-coumaric acid was fed in batches at 2. Phenylpropanoids, such as resveratrol and naringenin, are a class of natural products widely found in plants and microorganisms, and have important applications in medicines, cosmetics, and health products [ 6 ].
To date, no practical industrial process for the microbial production of phenylpropanoids have been developed.
Whole-cell bioconversion is a more efficient technique route for production of not only raspberry ketone and other phenylpropanoids, but also other diverse bioproducts. However, it is difficult to develop an efficient bioconversion process for raspberry ketone production previously. Raspberry ketone and other phenylpropanoids synthesis require p -coumaric acid or cinnamic acid as precursor, which are derived from aromatic amino acids.
After activation by CoA, p -coumaric acid or cinnamic acid is polymerized with different units of malonyl-CoA to form the final target product.
However, similar to previous reports, our study found that 4CL-mediated activation of p -coumaric acid was the critical rate-limiting step [ 21 ].
To our surprise, when glucose was used as the raw material, the raspberry ketone yield was low. Similar phenomenon is also reported in the production of some other phenylpropanoids [ 21 , 35 ].
The underling mechanism of the glucose inhibitory effect on bioproduction is still to be explored. There might be several reasons for this. First, the function or expression of key enzymes is limited under glucose conditions. Second, the level of some important factors or metabolites is not sufficient for the biosynthsis pathway. The precursor p -coumaric acid is significantly accumulated when glucose was used, suggesting that fine regulation of 4CL expression and CoA levels in cells was a key factor in this synthesis.
To remove this bottleneck and achieve efficient expression of enzyme 4CL in fatty acids raw materials, we systematically screened promoters that can be induced by fatty acids. The results showed that relevant promoters could be used in our strains to successfully increase the raspberry ketone production. Malonyl-CoA is among the most important metabolites, and can serve as a basic building block for fatty acids biosynthesis. Therefore, malonyl-CoA is the key precursor of diverse fatty-acid-derived compounds, including biofuels [ 36 ].
Malonyl-CoA is also a precursor for the microbial synthesis of many pharmaceutically interesting polyketides and natural products, such as phenylpropanoids [ 37 ]. As fatty acids are an easily obtained feedstock, they are ideal raw materials for novel production routes to natural products [ 28 ]. In this study, a novel biosynthesis strategy to produce raspberry ketone a representative phenylpropanoid from fatty acids raw materials was reported for the first time.
These results demonstrate a novel technical route for the production of similar compounds. Furthermore, establishing an efficient induction and fermentation process might be key to achieving a high-level fermentation strategy for phenylpropanoid production. We solved this problem through cooperative utilization of multiple carbon sources and fine-tuning of key enzyme expression. Further studies will focus on using different FA resources as raw materials.
In summary, we conducted systematic metabolic engineering of E. Then the raspberry ketone synthesis module was combined with the fatty acids utilization module to construct the engineered strain for raspberry ketone production from fatty acids. We systematically screened the fatty acid-inducible promoters.
The expression of key enzymes under Pfrd3 promoter contributed to p -coumaric acid activation, increasing the supply of precursors and NADPH. Finally, the strain CR08 produced raspberry ketone by using multiple carbon sources, and produced This study will help use cheaper raw materials to produce not only raspberry ketone, but also other important flavonoid compounds.
Nanjing, China. P -coumaric acid and raspberry ketone was purchased from Solarbio Biochemical Co. Shanghai, China. All other regular chemicals were purchased from ShengGong Biochemical Co. Beijing, China. The E. Luria—Bertani LB medium was used for all molecular construction experiments and strain culture. All strains and plasmids used in this study are shown in Table 1. All primers are listed in Additional file 2 : Table S1. Molecular cloning and genetic editing were performed using standard protocols.
For knockout genes, a single knockout library stored in the laboratory was used to achieve integration through P1 phage infection [ 41 ]. The plasmids pYB1s and pLB1a were previously constructed in our laboratory; the specific features were as follows: streptomycin and kanamycin resistance genes, araBAD promoter, multiple cloning sites, rrnB terminator, p15A, and R6k.
Promoter replacement and gene insertion replacement used the gene-editing tool plasmids constructed in our laboratory as templates for amplification to obtain homologous recombination fragments. When used glucose for one-step fermentation, the recombinant strains were grown in M9 modified medium to an OD of 0. The bioconversion medium changed based on experimental conditions. The carbon sources glucose, glycerol, or fatty acids was also added based on the experimental conditions.
In the process of optimizing the bioconversion conditions, the bioconversion starting biomass of OD was investigated at 10, 20, 30 and Excitation at nm, emission at nm, automatic gain.
Fatty acids emulsified in the medium were opaque emulsion, so OD could not be used directly to detect biomass. HPLC determined product concentration. Raspberry ketone and p -coumaric acid were detected via DAD detection. The raspberry ketone was detected at nm, p -coumaric acid was detected at nm. Erickson B. Nelson, Winters P: Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol J. Engineering microbial factories for synthesis of value-added products.
J Ind Microbiol Biotechnol. Gustavsson M, Lee SY. Prospects of microbial cell factories developed through systems metabolic engineering. Microb Biotechnol. Nielsen J, Keasling Jay D. Engineering cellular metabolism. Vogt T.
Phenylpropanoid biosynthesis. Mol Plant. Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol Biochem. Korkina LG. Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health.
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