Abstract
High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.
Highlights
Cell factories have become an established role player in the sustainable production of chemicals and biological products proven with hundreds of billions of USD/year value on global markets (Davy et al, 2017)
The optimized gene expression cassette architecture proposed by Nielsen et al was evaluated in P. taiwanensis VLB120 for the expression of fluorescent reporter genes and a 2-step acetoin biosynthesis operon (Nielsen et al, 2013)
The highest fold improvement was observed once the RNase III site was removed and evaluated on a single genomic integration basis under the control of the isopropylβ-D-1 thiogalactopyranoside (IPTG) inducible PTrc promoter
Summary
Cell factories have become an established role player in the sustainable production of chemicals and biological products proven with hundreds of billions of USD/year value on global markets (Davy et al, 2017). A commonality in the development of such cell factories is the continuous pursuit of increased productivities through directed or selection-based genetic engineering methods. With both approaches, increasing activity of the partaking pathways commonly leads to the desired rise in productivity. High enzyme activity can be achieved by optimization of transcription, translation, post-translational modifications, and the process conditions (Liu et al, 2013). A common strategy is to employ strong promoters to overexpress product biosynthesis genes.
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