CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

Abstract

The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage.

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

“…S7B), and CvFAP cassette from p13001 were amplified with overlapping homologous overhangs (Additional file 1: Tables S5, S6). For the assembly of both parts, SLiCE method was used, described by [25] including minor deviations described in [26]. This resulted in vector p13012.…”

Agrobacterium strains and strain improvement: Present and outlook

“…Further optimization may be possible by modifying, for example, the promoter (Someya et al, 2013), the ribosome-binding site (RBS), the copy number, or by codon improvement and other methods increasing transgenic protein expression (Bonde et al, 2016;Nadler et al, 2019;Saito et al, 2019). The utilization of pathogen-derived effectors is also an intriguing possibility.…”

Biyofarmasötik Keşif, Geliştirme ve Üretimin Güncel Paradigması Olarak Mikroorganizmaların Metabolik Mühendisliği: Sentetik Biyolojinin Katkıları

“…Bazı genler birçok gen kopyasından en iyi şekilde ifade edilirken, diğer genler için orta veya hatta tek kopya sayıları, inklüzyon cisimciklerinin, toksik gen dozaj etkilerinden kaçınmanın veya metabolik mühendisliği için istenen seviyelere ulaşmanın tek yoludur [70]. Plazmidler, konak genomundan bağımsız olarak çoğalan bakterilerdeki dairesel DNA'nın ekstra kromozomal elemanlarıdır.…”