Targeting metabolic driving and minimization of by‐products synthesis for high‐yield production of D‐pantothenate in Escherichia coli

Abstract

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

Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

“…The targeting gene and vector backbones were ligated using ClonExpress. 52 The primers used in this study are shown in Table S1.…”

“…The culture conditions were implemented as described in our previous study. 52 For fedbatch fermentation, the fermentation medium contained 20 g/ L glucose, 16 g/L (NH 4 ) 2 SO 4 , 2 g/L yeast extract, 2 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 , 0.5 g/L betaine, and 1 mL/L essential metal salt solution. In the process of fed-batch fermentation, DO was maintained at 20%.…”

“…The detection methods for cell growth, glucose concentration, β-alanine production, and organic acids used in this study are described in a previous study. 52 Statistical Analysis. The data in this study are presented as mean ± SD of the independent experiments performed in triplicate.…”

“…The figures were created using the Origin software version 8.0 (OriginLab Corp., Northampton, MA, USA), GraphPad Prism 8 (GraphPad, San Diego, California, USA), and ChemDraw 17.0 (Cambridge Soft, Massachusetts, USA). 52 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…Amplification of genes and the vector backbone was performed by a polymerase chain reaction. The targeting gene and vector backbones were ligated using ClonExpress . The primers used in this study are shown in Table S1.…”