Functional cis-expression of phaCAB genes for poly(3-hydroxybutyrate) production by Escherichia coli.

AIMS: To develop a microbial strain producing poly(3-hydroxybutyrate) [P(3HB)], in the absence of antibiotic supplementation (normally required to stabilize a recombinant plasmid), by constructing a recombinant Escherichia coli strain with phaCAB and vgb integrated into the chromosome. METHODS AND RESULTS: The polyhydroxyalkanoate (PHA) synthesis operon (phaCAB) and the bacterial haemoglobin gene (vgb) were integrated downstream of nlpB (novel lipoprotein B) in E. coli K12, via homologous recombination, to form a recombinant strain, termed YH100. VHb encoded by the vgb gene was successfully expressed in YH100, as confirmed by Western blotting. P(3HB) synthesis by the YH100 strain grown in the absence of antibiotic was analysed by transmission electron microscopy. The yield of P(3HB) is 208 mg g(-1) . The thermal stability of P(3HB) produced from YH100 was similar to that of commercial P(3HB). Further, the polydispersity index (PDI) of the P(3HB) polymer derived from YH100 was 1·37, indicating that polymer uniformity was greater than that of commercial P(3HB), which had a PDI of 1·47. CONCLUSIONS: We successfully constructed a recombinant E. coli strain expressing exogenous genes, specifically phaCAB from Cupriavidus necator and vgb from Vitreoscilla stercoraria, integrated into the downstream of chromosomal dapA-nlpB locus. P(3HB) was stably produced by this strain, without any need for antibiotic supplementation to stabilize a recombinant plasmid at least for 48h. SIGNIFICANCE AND IMPACT OF THE STUDY: We report a genetic locus, the downstream of the nlpB locus in E. coli, in which the transcription of the exogenous genes is driven by the dapA-nlpB promoter without the need for the addition of inducer and antibiotic.

Genetic polymorphism of RhD-negative associated haplotypes in the Chinese.

The Rh blood group is the most polymorphic human blood group system, and is clinically significant in transfusion medicine. Individuals are classified as Rh-positive and Rh-negative depending on the presence or absence of the D antigen on the red cell surface. The RhD-negative trait could be generated by multiple genetic mechanisms, which have been shown to be ethnic group-dependent. In this study, we evaluated the status of seven RHD-specific exons (exons 3, 4, 5, 6, 7, 9, and 10) and RH intron 4 in 119 Chinese blood donors, using the sequence-specific primers polymerase chain reaction (SSP-PCR). Of the 87 individuals who were RhD-negative, 52 with the ce/ce, ce/cE, or Ce/ce genotype (60%) lacked the above seven RHD exons; 22 with the Ce/Ce or Ce/ce genotype (25%) had all the RHD exons examined; 13 with the Ce/ce genotype (15%) carried at least one RHD exon. Antigen association analysis suggested the existence of a novel class of RhD-negative associated haplotypes in the Chinese, tentatively denoted D(nf)Ce. The D(nf)Ce haplotype consisted of a normal RHCe allele and a nonfunctional RHD gene, which vary depending on the structure of the RHD gene. Among the RhD-negative Chinese, the estimated frequencies of the dce, dCe, and D(nf)Ce haplotypes were 0.7500, 0.0465, and 0.2035, respectively. No statistically significant deviation from Hardy-Weinberg equilibrium was observed using this genetic model.

Physical and biochemical characterization of a simple intermediate between fluidized and expanded bed contactors.

Physical and biochemical comparison has been made of the performance of a simple fluidized bed contactor and a commercial expanded bed contactor, characterized by identical dimensions, and operated at various settled bed heights with two anion exchange adsorbents. The contactors were tested with various feedstocks comprising bovine albumin in the absence and presence of 20 g dry cell weight biomass litre-1. Earlier classification of the simple contactor as a single-stage, well mixed fluidized bed is reviewed. The relative merits of STREAMLINE DEAE and DEAE Spherodex LS as fluidizable, anion exchange adsorbents are discussed.