Construction of a novel anaerobic pathway in Escherichia coli for propionate production.

BACKGROUND: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application. RESULTS: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose. CONCLUSIONS: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Correction of methylmalonic aciduria in vivo using a codon-optimized lentiviral vector.

Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector-mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut(-/-)MUT(h2)) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut(-/-)MUT(h2) and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder.

Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin.

The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B12, the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

Effects of dietary supplements of folic acid and vitamin B12 on metabolism of dairy cows in early lactation.

The present experiment was undertaken to determine the effects of dietary supplements of folic acid and vitamin B12 given from 3 wk before to 8 wk after calving on lactational performance and metabolism of 24 multiparous Holstein cows assigned to 6 blocks of 4 cows each according to their previous milk production. Supplementary folic acid at 0 or 2.6 g/d and vitamin B12 at 0 or 0.5 g/d were used in a 2 x 2 factorial arrangement. Supplementary folic acid increased milk production from 38.0 +/- 0.9 to 41.4 +/- 1.0 kg/d and milk crude protein yield from 1.17 +/- 0.02 to 1.25 +/- 0.03 kg/d. It also increased plasma Gly, Ser, Thr, and total sulfur AA, decreased Asp, and tended to increase plasma Met. Supplementary B12 decreased milk urea N, plasma Ile, and Leu and tended to decrease Val but increased homocysteine, Cys, and total sulfur AA. Liver concentration of phospholipids was higher in cows fed supplementary B12. Plasma and liver concentrations of folates and B12 were increased by their respective supplements, but the increase in plasma folates and plasma and liver B12 was smaller for cows fed the 2 vitamins together. In cows fed folic acid supplements, supplementary B12 increased plasma glucose and alanine, tended to decrease plasma biotin, and decreased Km of the methylmalonyl-coenzyme A mutase in hepatic tissues following addition of deoxyadenosylcobalamin, whereas it had no effect when cows were not fed folic acid supplements. There was no treatment effect on plasma nonesterified fatty acids as well as specific activity and gene expression of Met synthase and methylmalonyl-coenzyme A mutase in the liver. Ingestion of folic acid supplements by cows fed no supplementary B12 increased total lipid and triacylglycerols in liver, whereas these supplements had no effect in cows supplemented with B12. The increases in milk and milk protein yields due to folic acid supplements did not seem to be dependent on the vitamin B12 supply. However, when vitamin B12 was given in combination with folic acid, utilization of the 2 vitamins seems to be increased, probably more so in extrahepatic tissues. Metabolic efficiency seems also to be improved as suggested by similar lactational performance and dry matter intake for cows fed supplementary folic acid but increased plasma glucose and decreased hepatic lipids in cows fed folic acid and vitamin B12 together.

Purification and characterization of homodimeric methylmalonyl-CoA mutase from Sinorhizobium meliloti.

High activity (>60 munit/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000+/-5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0+/-2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH(2)-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.

Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.

Methylmalonic acidemia resulting from genetic deficiency of methylmalonyl CoA mutase (MCM) is an often fatal metabolic disease. Somatic gene therapy for this disorder may require gene replacement in the liver. We describe overexpression of MCM in the liver of mice after in vivo gene delivery using asialoglycoprotein/polylysine/DNA (ASO/PL/DNA) targeted delivery to the liver of plasmids expressing recombinant MCM. After intravenous administration of the ASO/PL/DNA complex, the vector sequences are cleared from the blood with t1/2 = 2.5 min and > 95% of the vector is taken up by the liver. Vector sequences are cleared from the liver with t1/2 = 1.0-1.3 hr. MCM enzyme activity in the liver increases to levels 30-40% over baseline 6-24 hr after injection. No acute or chronic toxicity was observed. This net level of expression is likely to be therapeutic for MCM if the complex could be administered repetitively to treat acute episodes of life-threatening acidosis or establish a steady-state level of MCM activity. Repetitive administration of the ASO/PL/DNA complexes in mice was associated with formation of antibodies against asialo-orosomucoid and the asialo-orosomucoid complex but not against DNA.

Identification of two novel mutations in the methylmalonyl-CoA mutase gene with decreased levels of mutant mRNA in methylmalonic acidemia.

Genetic defects in the methylmalonyl-CoA mutase (MCM) gene result in methylmalonic acidemia which is inherited as an autosomal recessive disease. We investigated fibroblast cultures obtained from two Japanese patients with MCM deficiency. MCM mRNA was not detected by Northern blot analysis, suggesting that MCM mRNA was markedly decreased. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of MCM mRNA in these fibroblasts was less than 1% of normal controls. This minute amount of MCM mRNA was successfully amplified by nested RT-PCR and subjected to primary structure analysis. Sequence analysis revealed two novel mutations: a G-to-T substitution at nucleotide position 425 and a 2 bp deletion at nucleotide positions 769 and 770. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The second mutation (769 delta CA) resulted in a frame shift which created a premature termination codon 508 amino acid upstream of the C-terminus of the protein. Patient 1 was homozygous for G425T and patient 2 was a compound heterozygote for G425T and 769 delta CA. Our report is the first to identify MCM mutations that affect the stability of MCM mRNA. An analysis of 16 Japanese patients revealed the presence of G425T in six patients, suggesting a relatively high incidence of the mutation among Japanese patients. This is in sharp contrast to a previous report describing diverse heterogeneity of MCM mutations among Caucasians.

Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.