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

Abstract

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

Keywords: auxotrophy; biosynthesis pathways and regulation; cobalamin biotechnology; cobamide-producing strains; coenzyme B12 family cofactors; genetic diversity.

Figure 1

Scheme of the aerobic, anaerobic and salvage cobalamin biosynthetic pathways: the aerobic pathway is shown for P. denitrificans or Sinorhizobium meliloti (the intermediates are outlined on the right), and the anaerobic pathway and its intermediates are outlined on the left for well-studied bacteria S. typhimurium, P. shermanii and B. megaterium; the structure of vitamin B12 with the lower ligand 5,6-dimethylbenzimidazole (DMB) and the upper ligand consisting of either 5′-deoxyadenosyl, methyl, hydroxyl or cyan group, with their respective names being adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl); the gene names are in blue, red, and black and used throughout this work. The anaerobic and aerobic cobalamin pathways are characterized by the early and late cobalt insertions, respectively. In the anaerobic pathway, CysG is a common enzyme for siroheme and cobalamin synthesis. Whilst decarboxylation of UroIII leads to the biosynthesis of hemes and chlorophylls, methylation of UroIII at C-2 and C-7 results in the synthesis of precorrin-2, that is also the last common intermediate in the synthesis of coenzyme F430 and siroheme [7,9,20,24].

Figure 2

Genetic organization of B12 biosynthesis in different bacteria. The genes are indicated by colorful arrows; fine arrows denote confirmed promoters; hairpins symbols denote confirmed terminators [26,32,34,35,52,53].

Figure 3

Schematic representation of coenzyme B12 biosynthetic genes cluster I and its riboswitches (RS1, RS2 and RS3) of the P. denitrifians ATCC 13867 genome, according to Nguyen-Vo et al. [26]: biosynthetic genes-thick colored arrows; operons-Operon 1, Opn2, Opn3, Opn4, Opn5, Opn6, Opn7; putative promoters-curved arrows; transcripts-straight black arrows above operons. A coenzyme B12-responsive riboswitch (RS3) structure and schematic mechanism of regulation in mRNA is shown below, according to Zhu et al. [76]: CCCC–P3 aptamer domain forming a pseudoknot under a high concentration of coenzyme B12; antisequestor–sequence complementary to RBS and forming a hairpin to attenuate translation; RBS–ribosome-binding site; ATG–strart-codon; cbtBAcobEM–sequences of the structural genes of the transcript from Opn7.

Figure 4

Distribution of microbial cobalamins (vitamin B12 family cofactors) biosynthesis capability in various ecosystems: (A) general distribution of the basic B12-related pathway variants among publicly available human gut microbial (HGM) genomes, according to Rodionov et al. [12]: P1 and P2-de novo anaerobic and aerobic Cbl biosynthesis (protothrophy), respectively; P1&S and P2&S-de novo anaerobic and aerobic biosynthesis and salvage; Aba&S-cobyrinate auxotrophy and salvage; Acbr&S-cobyrinate diamide auxotrophy and salvage; Acbi&S-cobinamide auxotrophy and salvage; A&S-cobalamin/B12 auxotrophy and salvage; (B) distribution of the basic B12-related pathway variants at the microbial genera level (number of genus); (D) distribution of the basic B12-related pathway variants at the species level (number of genomes from five and more strains of a species); (C) distribution of cobamides in human, ground water and rumen, according to Degnan et al. [66]; (E) Distribution of vitamin B12 availability and ribonucleotide reductase activity in the P. aeruginosa biofilm-forming cells according to the experimental data of Crespo et al. [58]: vitamin B12 is indicated by red color; oxygen concentration gradient is in blue; ribonucleotide reductase (RNR) activity is indicated by green color: oxygen-dependent class Ia RNR (NrdAB) and oxygen-sensitive III RNA (RnrdJ) are B12-independent, while oxygen-independent class II RNR (NrdJ) is highly B12-dependent and important for providing the cells with deoxyribonucleotide triphosphates (dNTP) during the biofilm growth.

Figure 5

General flowsheet of the cobalamin industrial production applicable for aerobic process in P. denitrificans.