Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome

Whole genome codon compression-the reassignment of all instances of a specific codon to synonymous codons-can generate organisms capable of tolerating knockout of otherwise essential transfer RNAs (tRNAs). As a result, such knockout strains enable numerous unique applications, such as high-efficiency production of DNA encoding extremely toxic genes or non-canonical proteins. However, achieving stringent control over protein expression in these organisms remains challenging, particularly with proteins where incomplete repression results in deleterious phenotypes. One platform enjoying increasing popularity utilizes serine TCA codon compression, relying on the prevailing understanding that deletion of tRNASer(UGA) (serT) would render the serine codon compressed organism incapable of translating any genes containing TCA codons. Here, we report evidence that tRNASer(CGA) (serU) can, surprisingly, also decode TCA, thereby precluding complete control over expression of TCA-containing genes in organisms with serT deletion. We then demonstrate the conditions necessary, including the precise modifications to the GRO and codon usage within the transgene, to overcome this interaction and achieve exceptionally stringent control over protein expression. Our findings provide critical insights and corresponding methods for guiding future use of serine codon compression for absolute control over protein expression, as well as a general strategy for optimizing repression via compression of other codons.