BackgroundMalate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements.ResultsWe found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export.ConclusionsSince more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0959-y) contains supplementary material, which is available to authorized users.
Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. For this reason, finding better producer strains and optimizing their bioprocesses have been the main focus of industrial producers over the last few decades. In this review, we initially provide a historical overview of vitamin B12 research and the main biosynthetic characteristics of the two microorganism families typically used for its industrial production: several strains of Propionibacterium freudenreichii and strains related to Pseudomonas denitrificans. Later, a complete summary of the current state of vitamin B12 industrial production as well as the main advances and challenges for improving it is detailed, with a special focus on bioprocess optimization, which aims not only to increase production but also sustainability. In addition, a comprehensive list of the most important and relevant patents for the present industrial strains is provided. Finally, the potential applications of vitamin B12 in different markets are discussed.
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