Auxotrophic Selection Strategy for Improved Production of Coenzyme B12 in Escherichia coli

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology

“…The whole-genome sequence-based metabolic reconstructions have been performed for the microbial species from soil, gut, marine environments, including algal and human microbial communities, to elucidate the mechanism of auxothrophy to amino acids and cofactors [33,[60][61][62][63][64]. Genome functional annotations from Enzyme Commission (EC) numbers (http://www.sbcs.qmul.ac.uk/iubmb/enzyme/, accessed on 8 April 2021), the Pfam protein families database (http://pfam.xfam.org; Pfam 34.0 (accessed on 15 March 2021, 19,179 families), the TIGRFAM database of protein family definitions (http://tigrfams.…”

“…The whole-genome sequence-based metabolic reconstructions have been performed for the microbial species from soil, gut, marine environments, including algal and human microbial communities, to elucidate the mechanism of auxothrophy to amino acids and cofactors [ 33 , 60 , 61 , 62 , 63 , 64 ]. Genome functional annotations from Enzyme Commission (EC) numbers ( , accessed on 8 April 2021), the Pfam protein families database ( ; Pfam 34.0 (accessed on 15 March 2021, 19,179 families), the TIGRFAM database of protein family definitions ( ; Current Release: 15.0, 4488 families, accessed on 16 September 2014), Clusters of Orthologous Groups (COG) ( , accessed on 15 January 2021), The Integrated Microbial Genomes (IMG) system ( , accessed on 9 April 2021) for Cba biosynthesis, Cba-dependent and -independent enzymes are used for identification of both Cba biosynthesis and Cba-dependence in genomes [ 7 ].…”

“…The additional knockdown of the heme biosynthetic gene expression, combined with optimal fermentation conditions, could increase the vitamin B12 productivity of the recombinant E. coli strains from 1–2 to 307.00 µg g −1 , which is still much less than the industrial strains of P. denitrificans (214.3 mg L −1 ) and P. freudenreichii (206.0 mg L −1 ), but is comparable with the wild-type strain of B. megaterium and its engineered strain (0.26 and 8.51 µg L −1 , respectively), and the wild-type P. denitrificans (2.75 µg g −1 ) [ 105 ]. Recently, an auxotrophic selection strategy for improved production of coenzyme B12 was successfully developed for E. coli [ 64 ]. Given that the expression of coenzyme B12-independent methionine synthase gene metE is repressed by the presence of vitamin B12, the prolonged repression by a reliable source of the vitamin could lead to accumulation of mutations and eventually gene loss [ 81 ].…”

“…Given that the expression of coenzyme B12-independent methionine synthase gene metE is repressed by the presence of vitamin B12, the prolonged repression by a reliable source of the vitamin could lead to accumulation of mutations and eventually gene loss [ 81 ]. To select a highly effective producer, the gene metE was deleted in E. coli , thus limiting its methionine synthesis to only that via coenzyme B12-dependent synthase encoded by metH [ 64 ].…”

Control strategies to manage trade-offs during microbial production

“…Instead of developing a new sensor, which can be challenging by itself, an alternative approach is to use a metabolite-dependent enzyme for auxotrophic selection. For example, a coenzyme B 12 -dependent methionine synthase, which links the end product B 12 to cell growth, has been used to increase production [ 61 ]. Second, the cross-membrane transport of the product cannot be faster than the response time of sensing-selection.…”