Lactobacillus plantarum constitutes a well-recognized food-grade system for the expression of recombinant proteins in the field of industrial and medical biotechnology. For applications in vivo or in biotechnological processes, the level of expression of e.g. antigens or enzymes is often critical, as expression levels should be of a certain effectiveness, yet, without putting too much strain to the overall system. The key factors that control gene expression are promoter strength, gene copy number and translation efficiency. In order to estimate the impact of these adjusting screws in L. plantarum CD033, we have tested several constitutive promoters in combination with high and low copy number plasmid backbones and varying space between the Shine-Dalgarno sequence and the start-codon.
By combining strong promoters, such as transcription elongation factor promoters, isolated from L. plantarum CD033 and L. buchneri CD034, a synthetic promoter, originally derived from L. plantarum WCSF1 and a heterologous promoter derived from L. buchneri CD034 with a high and a low copy number origin of replication we demonstrated various expression levels of the model protein mCherry. All promoters were feasible for protein expression and in all cases, the high copy number origin of replication increased expression twofold. We found that the optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides and elongation as well as shortening this sequence gradually down-regulates gene expression.
We have evaluated the effects of a set of gene regulatory tools to fine tune recombinant gene expression in L. plantarum CD033. We have thus, provided potential expression vectors useful for constitutive protein expression in lactic acid bacteria ranging from moderate to strong production levels.