Model‐driven design of synthetic N‐terminal coding sequences for regulating gene expression in yeast and bacteria

Abstract

N‐terminal coding sequences (NCSs) are key regulatory elements for fine‐tuning gene expression during translation initiation—the rate‐limiting step of translation. However, owing to the complex combinatory effects of NCS biophysical factors and endogenous regulation, designing NCSs remains challenging. In this study, a multi‐view learning strategy for model‐driven generation of synthetic NCSs for Saccharomyces cerevisiae and Bacillus subtilis are implemented, which are widely used in laboratories and industries. NCS libraries for S. cerevisiae and B. subtilis with nearly 150,000 cells were sorted. Next, model training was performed with NCS deep features extracted from DNA, codon, and amino acid sequences, as well as calculated features from the minimum free energy (MFE) and tRNA adaption index. Two models were separately developed for generating synthetic NCSs for both up‐ and down‐regulating gene expression with accuracies higher than 65% for S. cerevisiae and B. subtilis. Synthetic NCSs were then applied to enhance bioproduction, yielding 1.48‐ and 1.71‐fold production improvements of D‐limonene by S. cerevisiae and ovalbumin by B. subtilis, respectively. This work provides model‐driven design of synthetic NCSs as a toolbox for regulating gene expression in S. cerevisiae and B. subtilis. The machine learning‐based modeling approach can be used for NCS design in other microorganisms.

The amino acid sequence determines protein abundance through its conformational stability and reduced synthesis cost

“…By inspecting MGEM mutants, we discovered that in terms of sequence position, the N-terminus was the most important, with the majority of amino acids remaining unchanged in this region (Figure 2E,F). This suggested that the N-terminus is generally evolutionarily optimized for expression efficiency, which also supports why it is widely used for protein expression optimization [86][87][88] . A short hotspot at the very last position in the C-terminus was frequently mutated, which is known as a signal involved in protein degradation 5,6 .…”

Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

“…The N-terminal coding sequence (NCS) is a rate-limiting step in translation and an important element in gene regulation. Wang et al [ 137 ] applied a multiview learning strategy for NCS synthesis in S. cerevisiae . Two models were developed through model training and used to upregulate and downregulate gene expression.…”

Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica

“…52−54 In summary, as a crucial component for functional expression, the application of direct evolution for modifying proteins is a prospective technological advancement in the field of molecular biology, which holds immense potential for advancing the rapid development of monoterpenoids biosynthesis. 55 3.3. Downregulating the Competitive Pathway Flux.…”

“…One example is that Jiang et al deduced the microscopic mechanism underlying the high activity of geraniol synthase from Catharanthus roseus ( CrGES ) through structural prediction. Subsequently, they fused ERG20 F96W–N127W with tCrGES (the randomly truncated geraniol synthase) to obtain the optimal pairing, eventually achieving 1.68 g/L geraniol titer in S. cerevisiae . − In summary, as a crucial component for functional expression, the application of direct evolution for modifying proteins is a prospective technological advancement in the field of molecular biology, which holds immense potential for advancing the rapid development of monoterpenoids biosynthesis …”