Explore or exploit? A model-based screening strategy for PETase secretion by Corynebacterium glutamicum.

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide, and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization, and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision-making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.

Variations in the multiple tbp genes in different Halobacterium salinarum strains and their expression during growth.

The presence and expression of the multiple tbp genes encoding TATA-box binding proteins (TBPs) was investigated in various strains and mutants of the archaeon Halobacterium salinarum. Six genes, tbpA through tbpF, are present in the genome of Hbt. salinarum NRC-1 and also in the gas vesicle negative mutant strain R1. The only tbp gene located in the chromosome is tbpE, whereas all others are found in the plasmid DNA. Due to the dynamic nature of the plasmids in the Halobacterium strains, the copy numbers of the alternative tbp genes vary significantly. Five tbp genes (tbpA through tbpE) were present in the wild-type strain Hbt. salinarum PHH1. The tbpC gene of Hbt. salinarum PHH1 carried an ISH27-2 insertion element at the start of the reading frame that prevented the expression. All other tbp genes of PHH1 were expressed under aerobic and anaerobic growth conditions and quantitative RT-PCR yielded tbpE as dominant tbp transcript during the exponential growth phase. The plasmid deletion variant Hbt. salinarum PHH4 lacked all of the tbp genes except for tbpE and showed an altered growth behaviour compared to PHH1 wild-type in the stationary growth phase under anaerobic growth conditions.