A newly isolated and identified vitamin B12 producing strain: Sinorhizobium meliloti 320

Dong, Huina; Li, Sha; Fang, Huan; Xia, Miaomiao; Zheng, Ping; Zhang, Dawei; Sun, Jibin

doi:10.1007/s00449-016-1628-3

Cited by 21 publications

(17 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We conducted a series of in vitro assays to support our identification of functional CobN, CobS, and CobT enzymes for the production of CBAD. To increase the likelihood of obtaining functional versions of these three enzymes, three variants from S. meliloti 20 , B . melitensis , and R. capsulatus were tested (total of nine enzymes tested).…”

Section: Resultsmentioning

confidence: 99%

“…We successfully engineer E. coli to produce vitamin B 12 by heterologously expressing a total of 28 genes from R. capsulatus , Brucella melitensis , Sinorhizobium meliloti 320 20 , S. typhimurium , and Rhodopseudomonas palustris that are divided into six engineered modules. The enzymes of Module 1 together produce HBA from Uro III, and Module 2 produces CBAD from HBA.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

et al. 2018

Self Cite

View full text Add to dashboard Cite

The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea. Here, using genetic and metabolic engineering, we generate an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. In vitro and/or in vivo analysis of genes involved in adenosylcobinamide phosphate biosynthesis from Rhodobacter capsulatus suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increase the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg g−1 DCW via metabolic engineering and optimization of fermentation conditions. Beyond our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…The initial strain, S. meliloti 320 , was stored in our laboratory [ 22 ]. S. meliloti 320 was grown in LB medium supplemented with MgCl 2 (2.5 mM) and CaCl 2 (2.5 mM) (LB/MC medium), or minimal M9 medium supplemented with 1 μg/mL biotin and 10 ng/mL CoCl 2 , as well as 0.1% sucrose for liquid medium or 0.2% sucrose for solid medium (unless indicated otherwise), respectively, (named M9/sucrose) [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…20 μL of the final sample was injected into an Agilent 1260 HPLC with a 5C18-250A column (Agilent, 4.6 mm id × 250 mm, 5 μm) thermostated at 35 °C. The mobile phase consists of water and acetonitrile (70:30 [v/v]) at 0.8 mL/min [ 22 ]. Samples were detected at 361 nm.…”

Section: Methodsmentioning

confidence: 99%

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti

et al. 2018

Self Cite

View full text Add to dashboard Cite

BackgroundAs a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed.ResultBy the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5–2 was obtained and considered as a candidate for industrial applications. After 7 d’s cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5–2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5–2 was sequenced, and gene mutations were identified and analyzed.ConclusionTo our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0441-2) contains supplementary material, which is available to authorized users.

show abstract

“…Finally, urogen III is formed from pre-uroporphyrinogen under the action of uroporphyrinogen III synthase (UROS) [24]. First, the hemABCD genes from S. meliloti 320, a strain with a high vitamin B 12 titer [25], were heterologously expressed to supply sufficient urogen III precursor for HBA production. We designed RBS libraries for hemA, hemB, hemC, and hemD that covered a broad range of translational initiation rates (TIRs) from around 1000 to 10,000 using RBS Calculator to coordinate the expression of these four genes.…”

Section: Optimizing the Expression Ratio Of Alas Pbgs Pbgd And Urosmentioning

confidence: 99%

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

et al. 2020

Self Cite

View full text Add to dashboard Cite

Background: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B 12. The introduction of a heterologous de novo vitamin B 12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B 12 , such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B 12 production. Results: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L −1 , respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L −1 , representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. Conclusions: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L −1 in E. coli. This lays the foundation for high-yield production of vitamin B 12 in E. coli and will hopefully accelerate its industrial production.

show abstract

A newly isolated and identified vitamin B12 producing strain: Sinorhizobium meliloti 320

Cited by 21 publications

References 26 publications

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

Contact Info

Product

Resources

About