Promoters are crucial elements for engineering microbial production strains used in bioprocesses. For the increasingly popular chassis Komagataella phaffii (formerly Pichia pastoris), a limited number of well-characterized promoters constrain the data-driven engineering of production strains. Here, we present an in silico approach for condition-independent de novo identification of strong native promoters. The method relies on tRNA-codon coadaptation of coding sequences in the K. phaffii genome and is based on two complementary scores: the number of effective codons and the tRNA adaptation index. Genes with high codon bias are expected to be translated efficiently and, thus, also be under control of strong promoters. Using this approach, we identified promising strong promoter candidates and experimentally assessed their activity using fluorescent reporter assays characterizing 50 promoters spanning a 76-fold difference in expression levels in a glucose medium. Overall, we report several promoters that should be added to the molecular toolbox for engineering of K. phaffii and present an approach for identifying promoters in microbial genomes.

中文翻译：

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用于鉴定 Komagataella phaffii 中强天然启动子的密码子-tRNA 共适应偏差

启动子是生物过程中使用的微生物生产菌株工程的关键要素。对于日益流行的底盘Komagataella phaffii（以前称为毕赤酵母），有限数量的特征良好的启动子限制了生产菌株的数据驱动工程。在这里，我们提出了一种独立于条件的从头鉴定强天然启动子的计算机方法。该方法依赖于K. phaffii基因组中编码序列的 tRNA-密码子共适应，并基于两个互补的分数：有效密码子的数量和 tRNA 适应指数。具有高密码子偏倚的基因预计会被有效翻译，因此也受到强启动子的控制。使用这种方法，我们确定了有前途的强启动子候选者，并使用荧光报告分析对 50 个启动子进行实验评估，这些启动子在葡萄糖介质中的表达水平存在 76 倍的差异。总的来说，我们报告了一些应该添加到用于工程化法菲酵母的分子工具箱中的启动子，并提出了一种鉴定微生物基因组中启动子的方法。