Combining Protein and Metabolic Engineering Strategies for High-Level Production of O-Acetylhomoserine in Escherichia coli
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O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.Keywords:
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Bacteriostatic experiment of twelve species of Chinese medicines and its compounds against escherichia coli K88 and hemoclatic escherichia coli had been made.The result showed that nine species of Chinese medicines had shown bacteriostasis against escherichia coli K88 and eleven species against hemoclastic escherichia coli. All compounds had shown bacteriostasis against escherichia coli Kgg and hemoclastic escherichia coli.The bacteriostatic effect of compuond 5 was superior to enrofloxacin's(1‰) against escherichia coli K88 (P0.05), and the bacteriostatic effect of compound 5 was superior to enrofloxacin's (1‰) against hemoclastic escherichia coli (P0.01 ).There was synergism between coptis root and enrofloxacin.
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Abstract Introduction Metabolic Pathways for PHA Biosynthesis Organization of the Genes Involved in PHA Biosynthesis Monomer‐supplying Enzymes for PHA Biosynthesis β‐Ketothiolase (PhaA) Acetoacetyl‐CoA Reductase (PhaB) ( R )‐3‐Hydroxyacyl‐ACP–CoA transferase (PhaG) ( R )‐Specific Enoyl‐CoA Hydratase (PhaJ) Metabolic Engineering and Improvements in PHA Biosynthesis Metabolic Engineering for PHA Production in Recombinant Escherichia coli Metabolic Improvement for PHA Production in Ralstonia eutropha PHB‐4 and Pseudomonads Outlook and Perspectives
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Metabolic engineering aims to balance intracellular pathways and increase the precursor supply. However, some heterologous enzymes are not evolved to support high flux. To remove the limitation, the catalytic properties of rate-limiting enzymes must be enhanced. Here, we engineered carotenoid cleavage dioxygenase 1 (CCD1), whose intrinsic promiscuity and low activity limited the production of α-ionone in Escherichia coli. Site-directed mutagenesis was carried out to mutate three structural elements of CCD1: an active site loop, η-helices, and α-helices. Furthermore, mutated CCD1 was fused with lycopene ε-cyclase to facilitate substrate channelling. Collectively, these methods improved the α-ionone concentration by >2.5-fold compared to our previously optimized strain. Lastly, the engineered enzyme was used in conjunction with the metabolic engineering strategy to further boost the α-ionone concentration by another 20%. This work deepens our understanding of CCD1 catalytic properties and proves that integrating enzyme and metabolic engineering can be synergistic for a higher microbial production yield.
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Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc.
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Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. We review advances in selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use; produce non-natural amino acids, alcohols, and carboxylic acids; and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.
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Integrating the Protein and Metabolic Engineering Toolkits for Next-Generation Chemical Biosynthesis
Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.
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