Background: D-Pantothenate (DPA) is an important functional chemical that has been widely applied in healthcare, cosmetics, animal food, and feed industries. Methods and Results:In this study, a high-yield DPA-producing strain was constructed by metabolic engineering strategies with targeting metabolic driving and by-products minimization. The metabolic driving force of push and pull was firstly obtained to improve the production of DPA via enrichment of precursor pool and synthetic pathway, accumulating 4.29 g L −1 DPA in shake flask fermentation. To eliminate the metabolic pressure on DPA production, an amino throttling system was proposed and successfully attenuated the synthesis of four competitive amino acids by a single-step regulation of gdhA. Further minimization of acetate was carried out by pta deletion, and utilization of β-alanine was improved via enhancing its uptake system with producing 5.78 g L −1 DPA. Finally, the engineered strain produced 66.39 g L −1 DPA with β-alanine addition in fermentor under fed-batch fermentation. Conclusion:This study paved a foundation for the industrial production of DPA. K E Y W O R D Sβ-alanine, by-products, D-pantothenate, metabolic driving force, metabolic engineering INTRODUCTIOND-Pantothenate (DPA), also known as vitamin B5 (C 9 H 17 O 5 N), is a water-soluble vitamin that was discovered and extracted from animal liver to cure chicken pellagra, and was firstly synthesized in 1940. [1][2][3] DPA, the bioactive dextrorotatory (D) isomer, [4] is a key precursor used for biosynthesizing coenzyme A (CoA) and acyl carrier protein. [5,6] DPA, therefore, is widely used in healthcare, cosmetic, animal food, and feed industries. [7] DPA can be synthesized themselves in bacteria, fungi, archaea, and some plants, but animals can only obtain DPA from diet because of the absence of de novo biosynthetic pathway. [8,9] Abbreviations: AHAS, acetohydroxy acid synthase; CoA, coenzyme A; DPA, D-pantothenate;IPTG, isopropyl β-D-1-thiogalactopyranoside; PEP, phosphoenolpyruvate Consequently, the biosynthesis pathway of DPA in microorganisms and plants is worth noting for the development of antimicrobial, fungicidal, and herbicidal compounds. [10] In addition, DPA as a coenzyme participates in some enzyme reactions involved in protein metabolism and lipid metabolism. [11] Currently, DPA used for industrial application is mainly produced by chemical method via condensation of D-pantolactone and β-alanine in methanol or ethanol. D-Pantolactone is performed by chemical resolution or enzymatic catalysis from DL-pantolactone. However, chemical resolution, in addition to the usage of toxic chemical reagents, is laborious and expensive. Although enzymatic catalysis avoids the above disadvantages, the substrate DL-pantolactone is synthesized from the condensation of isobutyraldehyde and formaldehyde which are also not eco-friendly reagents. With the development of genetic
Ezetimibe is a top-selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)-ET-5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET-4 concentration of 200 g/L. Using a substrate fed-batch strategy, under the optimal conditions, the substrate ET-4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space-time yield of 494.5 g L −1 d −1 and the space-time yield per gram biocatalyst of 24.7 g L −1 d −1 g −1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate.
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