Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.

Bioreactor technology for clonal propagation of plants and metabolite production

Plant cell culture in bioreactors is an enabling tool for large scale production of clonal elite plants in agriculture, horticulture, forestry, pharmaceutical sectors, and for biofuel production. Advantages of bioreactors for plant cell culture have resulted in various types of bioreactors differing in design, operating technologies, instrumentations, and construction of culture vessels. In this review, different types of bioreactors for clonal propagation of plants and secondary metabolites production are discussed. Mechanical and biochemical parameters associated with bioreactor design, such as aeration, flow rate, mixing, dissolved oxygen, composition of built-up gas in the headspace, and pH of the medium, are pivotal for cell morphology, growth, and development of cells within tissues, embryos, and organs. The differences in such parameters for different bioreactor designs are described here, and correlated to the plant materials that have been successfully cultured in different types of bioreactors.