Dalvie Neil C, Lorgeree Timothy R, Yang Yuchen, Rodriguez-Aponte Sergio A, Whittaker Charles A, Hinckley Joshua A, Clark John J, Del Rosario Amanda M, Love Kerry R, Love J Christopher
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 01239, USA.
Microb Cell Fact. 2024 Jul 31;23(1):217. doi: 10.1186/s12934-024-02466-2.
The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality.
We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody.
We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.
毕赤酵母广泛用于生产重组蛋白，但通过基因工程可提高重组蛋白的分泌产量。在本研究中，我们假设通过删除不需要的内源蛋白，细胞资源可从内源蛋白的生产重新导向重组蛋白的生产。然而，在诸如毕赤酵母这样的非模式微生物中，基因工程受到基因注释缺乏和基因必需性知识的限制。
我们通过质谱分析和信号肽预测鉴定了毕赤酵母中的一组内源分泌蛋白。由于必需基因的注释有限，我们破坏这些基因的努力受到阻碍。因此，为了预测必需基因，我们设计、转化并测序了一个用于CRISPR-Cas9介导的所有内源分泌蛋白敲除的向导RNA混合文库。然后，我们利用预测的基因必需性来指导对多达11个非必需基因的迭代破坏。工程菌株的人血清白蛋白产量提高了约20倍，单克隆抗体产量提高了两倍。
我们证明，破坏少至6个基因就能提高重组蛋白的产量。进一步减少毕赤酵母的内源蛋白质组可能会进一步提高菌株性能。本文报道的针对分泌蛋白组的CRISPR-Cas9向导RNA混合文库和基因必需性知识将有助于未来对毕赤酵母进行工程改造以生产其他重组蛋白和酶的研究。
