ABSTRACT
Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.
ABSTRACT
The rapid emergence of multidrug-resistant pathogens has invoked concerns of our current limitations in controlling the spread of infectious disease. To resolve this, we have applied synthetic biology principles to engineer human commensal microbe that can specifically sense and kill an antibiotic-resistant strain of P. aeruginosa. In this chapter, we describe the methods used to assemble, characterize, and evaluate the effectiveness of our engineered microbe in multicellular systems.
Subject(s)
Escherichia coli/physiology , Genetic Engineering/methods , Pseudomonas Infections/microbiology , Pseudomonas Infections/therapy , Pseudomonas aeruginosa/physiology , Coculture Techniques/methods , Drug Resistance, Bacterial , Escherichia coli/genetics , Humans , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Quorum Sensing , Synthetic Biology/methodsSubject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/metabolism , Pyocins/metabolism , Amino Acid Sequence , Genes, Bacterial , Humans , Molecular Sequence Data , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/genetics , Pyocins/chemistry , Sequence AlignmentABSTRACT
The nucleic acid sequence at the positions 1067817-1066321 of Pseudomonas aeruginosa PAO1 genome was predicted to encode a novel S-type pyocin, designated S5, based on the genome sequence. However, its antimicrobial spectrum, activity and mechanism have not been investigated. Herein, we report that pyocin S5 has an antimicrobial activity against seven clinical P. aeruginosa isolates (DWW3, InA, InB, In3, In4, In7, and In8). Among them, DWW3 is most sensitive with a minimum inhibitory concentration of 12.6 microg/ml and a killing percentage of 95.7 at 225 microg/ml. Further, we demonstrated that the antimicrobial mechanism of pyocin S5 is membrane damage, evidenced by the leakage of intracellular materials, the increase of membrane permeability, and cell surface disruption.
