Production of vitamin B12 in recombinant Escherichia coli: an important step for heterologous production of structurally complex small molecules.

Abstract

See accompanying article by Yeounjoo Ko et al. DOI 10.1002/biot.201400221 Vitamin B12 is one of the most complex small molecules acting as a co-factor in processes that are crucial to many living organisms. Vitamin B12 can be produced by fermentation of native bacterial producers with up to 10000 kilogram commercial production of vitamin B12 annually [1]. Because native bacterial producers are not the usual industrial producer strains, many attempts have been made to heterologously produce vitamin B12 in non-native microbial hosts. Since the de novo biosynthesis of vitamin B12 involves 30 enzyme-mediated reactions, those efforts have been unsuccessful until the recent study made by Ko et al. [2], which is published in this issue of Biotechnology Journal. The structure of vitamin B12 is so complex that Dorothy Crowfoot Hodgkin, who resolved the three-dimensional structure of vitamin B12 (cyanocobalamin) [3] and other important biochemical substances, was awarded the Nobel Prize in Chemistry in 1964. Chemical synthesis of vitamin B12 is a highly complicated process involving at least 60 steps, which is technically challenging and economically unviable. In nature, vitamin B12 is unique in the sense that its de novo biosynthesis appears to be limited to some bacteria and archaea [1]. This includes aerobi biosynthesis in Rhodobacter sphaeroides and Pseudomonas denitrificans, and anaerobic biosynthesis in Bacillus megaterium, Propionibacterium shermanii, Salmonella typhimurium and Lactobacillus reuteri [4]. Transferring a metabolic pathway from its native producer into heterologous microbial hosts has become a common practice in metabolic engineering. Nevertheless, assembling a functional metabolic pathway comprising more than 30 biochemical reactions remains a great challenge. Successful assembly of such a complex metabolic pathway requires that all of the genes be cloned accurately and expressed correctly, and that all of the introduced enzymes be functional so that the carbon flux can be transferred to the final target product. Very often, a single nucleotide mutation would result in failure of the entire pathway, and trouble-shooting would be very difficult and time-consuming. Ko et al. [2] use three compatible plasmids to clone the Pseudomonas denitrificans vitamin B12 pathway, which is encoded by 25 genes located in six different operons [2]. This strategy successfully resulted in the production of vitamin B12 in Escherichia coli, demonstrating that vitamin B12, which has the most complex structure amongst all vitamins, can be produced heterologously. Interestingly, although the synthesis of vitamin B12 in P. denitrificans is strictly oxygen-dependent, Ko et al. [2] show that the recombinant E. coli can produce vitamin B12 under anaerobic conditions. This suggests that the biosynthetic route towards vitamin B12 may not be dependent on molecular oxygen; the oxygen-dependant vitamin B12 biosynthesis in P. denitrificans might be due to some regulatory mechanisms in those native hosts, which is only effective under aerobic conditions. Biosynthesis of vitamin B12 appears to be restricted to a few representative bacteria and archaea, while vitamin B12-dependent enzymes are widespread throughout all domains of life [5]. Generally, vitamin B12-dependent reactions are described to catalyze methyl transfers and carbon backbone rearrangements. Besides the well-known diol and glycerol dehydratase, some additional examples of enzymes that require vitamin B12 include methionine synthase, methylmalonyl-CoA mutase, glutamate mutase, isobutyryl-CoA mutase, and reductive dehalogenases. For those organisms that do not possess vitamin B12 biosynthetic ability, addition of vitamin B12 to the medium is a necessity to initiate vitamin B12-dependent reactions. Production of vitamin B12 in recombinant E. coli enables scientists to consider vitamin B12-dependent biochemical reactions when designing new pathways, thus expanding the availability of enzymes. Previously, production of vitamin B12 in its native producers can be improved by optimization of the culture medium and process, mutagenesis of the producing strain, overexpression of the gene cluster involved in biosynthesis of vitamin B12, or optimizing promoters, ribosomal-binding sites and terminators [1]. Production of vitamin B12 in recombinant E. coli opens new possibilities for further improvement. Recently, engineered E. coli strains which are capable of producing some amino acids, organic acids, and alcohols have outcompeted their native producers. This demonstrates the power of applying synthetic biology approaches in strain improvement. With this rapid progress in E. coli cell factories, it can be optimistically predicted that production of vitamin B12 by engineered E. coli will have a bright future and lead to many more complex applications of metabolic engineering and synthetic biology.