Metabolic engineering of Escherichia coli for acetaldehyde overproduction using pyruvate decarboxylase from Zymomonas mobilis.

For the sustainable production of acetaldehyde, a key raw-material for a large number of chemical products, microbial production is a promising alternative. We have engineered an Escherichia coli strain for acetaldehyde production from glucose by introducing the pyruvate decarboxylase (Pdc) from Zymomonas mobilis and NADH oxidase (Nox) from Lactococcus lactis. Acetaldehyde production was systematically improved by knocking out the competing metabolic pathways. Multiple knockout strains were created and a final acetaldehyde titre of 0.73g/L was achieved using a quadruple knockout strain E. coli MC4100 ΔadhE ΔldhA ΔfrdC ΔackA-pta. In addition to acetaldehyde, about 0.37g/L acetoin was produced by these strains due to the additional carboligase activity exhibited by pyruvate decarboxylase resulting in a total carbon yield of 0.27g/g glucose.

Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis.

BACKGROUND: Fermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes. RESULTS: We present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation. CONCLUSION: The genome-scale metabolic model developed for Scheffersomyces stipitis successfully predicted substrate utilization and anaerobic growth requirements. Useful insights were drawn on xylose metabolism, cofactor recycling and mechanism of mitochondrial respiration from model simulations. These insights can be applied for efficient xylose utilization and cofactor recycling in other industrial microorganisms. The developed model forms a basis for rational analysis and design of Scheffersomyces stipitis metabolic network for the production of fuels and chemicals from lignocellulosic biomass.

Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification.

Saccharomyces cerevisiae was transformed for higher ethanol tolerance by error-prone whole genome amplification. The resulting PCR products were transformed back to the parental strain for homologous recombination to create a library of mutants with the perturbed genomic networks. A few rounds of transformation led to the isolation of mutants that grew in 9% (v/v) ethanol and 100 g glucose l(-1) compared to untransformed yeast which grew only at 6% (v/v) ethanol and 100 g glucose l(-1).

Myozenin 2 is a novel gene for human hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in sarcomeric proteins (excluding phenocopy). The causal genes in approximately one-third of the cases remain unknown. We identified a family comprised of 6 clinically affected members. The phenotype was characterized by early onset of symptoms, pronounced cardiac hypertrophy, and cardiac arrhythmias. We excluded MYH7, MYBPC3, TNNT2, and ACTC1 as the causal gene either by direct sequencing or by haplotype analysis. To map the putative candidate sarcomeric gene, we perforbold locus-specific haplotyping to detect cosegregation of the locus haplotype with the phenotype, followed by mutation screening. We genotyped 5 short-tandem-repeat markers that spanned a 4.4-centimorgan region on 4q26-q27 locus and encompassed myozenin 2 (MYOZ2), a Z-disk protein. The maximum logarithm of odds score was 2.03 (P=0.005). All affected members shared a common haplotype, implicating MYOZ2 as the causal gene. To detect the causal mutation, we sequenced all exons and exon-intron boundaries of MYOZ2 in 10 family members and identified a T-->C missense mutation corresponding to S48P substitution, which cosegregated with inheritance of HCM (N=6). It was absent in 4 clinically normal family members and in 658 additional normal individuals. To determine frequency of the MYOZ2 mutations in HCM, we sequenced MYOZ2 in 516 HCM probands and detected another missense mutation (I246M). It was absent in 2 normal family members and 517 controls. Both mutations affect highly conserved amino acids. We conclude MYOZ2 is a novel causal gene for human HCM.

Pharmacokinetics of ionized versus total magnesium in subjects with preterm labor and preeclampsia.

OBJECTIVE: Intravenous magnesium sulfate is widely used in obstetrics for the treatment of both preterm labor and preeclampsia. Although therapeutic levels of total magnesium have been proposed, the levels remain controversial. Because the active form of magnesium is the free or ionized form, it is essential to determine whether ionized magnesium and total magnesium levels are highly correlated in vivo. We sought to examine the correlation between ionized magnesium and total magnesium under basal and therapeutic conditions and to define the initiation and elimination pharmacokinetics of both forms during intravenous magnesium sulfate infusion. STUDY DESIGN: Twenty-four singleton pregnant patients who were candidates for magnesium sulfate were studied (preterm labor, 15; preeclampsia, 9). Serial blood samples were taken before the magnesium sulfate infusion, during the first 4 hours after the initiation of magnesium sulfate infusion and for 4 hours after the discontinuation of the infusion. RESULTS: Baseline levels of total magnesium and ionized magnesium were not different between patients with preterm labor and with preeclampsia. Among patients with preeclampsia, although not patients with preterm labor, the initial apparent volume of distribution was significantly smaller for total magnesium than for ionized magnesium (16,397 +/- 1441 vs 23,856 +/- 2745 mL, respectively; P =.03), and the elimination half-life was greater for total magnesium as compared to ionized magnesium (707 +/- 160 vs 313 +/- 29 minutes;P <.05). Linear regression analysis demonstrated a lack of correlation between ionized magnesium and total magnesium during the pretreatment period and during the steady state infusion for both preterm labor and preeclampsia. CONCLUSION: The measurement of total magnesium may not be adequate for the titration of therapeutic magnesium infusions in patients with preeclampsia or preterm labor because of the lack of correlation between total magnesium and the physiologically active ionized magnesium. Further studies may determine whether the measurement of ionized magnesium is a superior method for following the adequacy and safety of the treatment of preeclampsia and preterm labor.