Overexpression of succinyl-CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in engineered Escherichia coli BL21(DE3).

AIM: This study aims to increase the 3-hydroxyvalerate (3HV) fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] using succinyl-CoA synthase. METHODS AND RESULTS: Escherichia coli YH090, a polyhydroxyalkonate (PHA)-producing strain, was further engineered for overexpression of succinyl-CoA synthase genes (sucCD), and examined for P(HB-co-HV) copolymer production in the presence of various precursor molecules using mixture analysis. Glycerol, succinate and propionate were screened as important factors for controlling intracellular PHA accumulation and monomer composition. Glycerol concentrations exerted the greatest influence on the overall biomass concentration and the intracellular PHA content, while propionate concentrations in the presence of succinate influenced the 3HV content of the copolymer. Mixture analysis also demonstrated that the engineered strain has the capacity to accumulate up to 80% of its cell dry weight (CDW) as PHA with a variable fraction of 3HV monomer (maximum of 72 wt %) depending on the controlled conditions. CONCLUSIONS: Propionate is the principal precursor for 3HV monomer in P(HB-co-HV) biopolymer and its utilization requires conversion to propionyl-CoA. Engineered E. coli YHY99, overexpressing sucCD genes, leads to an increase of the succinyl-CoA pool, which enhances the conversion rate of propionate by providing a CoA supply to other acyltransferase enzymes that have a role in propionate utilization. SIGNIFICANCE AND IMPACT OF THE STUDY: Engineered E. coli YHY99 was able to utilize propionate with a 4·5-fold increase in rate, as compared to the control strain, and resulted in the synthesis of a copolymer with high 3HV monomer content.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase.

AIMS: Of the biodegradable polyhydroxyalkanoates (PHAs), poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. METHODS AND RESULTS: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P(HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). CONCLUSIONS: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.

A participatory learning module: asepsis and universal precautions.

Mastery of medical and surgical asepsis principles is mandatory for nurses in today's health care environment. The authors report on the development, implementation, and evaluation of a multimedia asepsis module incorporating universal precautions. Practical application of the content, using graphics, medical supplies, and video, occurs in the four-station participatory learning module. Student evaluations of this learning module are positive.