Fungal Host Cell Steroid Dehydrogenase Modification for Polypeptide Yield
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Solution Overview
Problem
Current methods for improving polypeptide production in filamentous fungal host cells are inadequate in enhancing productivity and yield, particularly due to the limitations of native putative steroid dehydrogenase activity.
Innovation Solution
Modifying, truncating, or eliminating the native putative steroid dehydrogenase in filamentous fungal host cells by reducing its expression, adding destabilization domains, or using inhibitors, thereby altering its conserved amino acid motifs such as YGAR, VPHS[W/Y]F, QC[A/V/S]RRL, LKKYTLP, and CPHYT to enhance polypeptide production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native putative steroid dehydrogenase activity is maintained in filamentous fungal host cells, then normal cellular metabolism is preserved, but polypeptide productivity and yield are limited
Solution Approach 1:
The patent removes or inactivates the native putative steroid dehydrogenase gene from the filamentous fungal host cell genome, extracting the problematic metabolic activity that limits polypeptide productivity. This gene deletion eliminates the enzyme's ability to interfere with polypeptide production while maintaining cellular viability through compensatory metabolic pathways.
Solution Approach 2:
The patent alters the metabolic state of the host cell by modifying the putative steroid dehydrogenase activity level from normal to reduced or eliminated. This parameter change in enzyme activity shifts the cellular metabolic profile to favor polypeptide production, thereby resolving the contradiction between maintaining normal metabolism and achieving high productivity.
2Productivity
If gene deletion methods are used to eliminate putative steroid dehydrogenase, then polypeptide yield is improved, but manufacturing complexity increases
Solution Approach 1:
The patent employs self-service mechanisms where the fungal host cell's own genetic recombination systems are utilized to achieve gene deletion. By introducing a linear DNA fragment with homology to the target gene region, the cell's homologous recombination machinery automatically performs the gene disruption, eliminating the need for complex external manipulation tools and reducing manufacturing complexity.
Data Source
AI summary
The present invention relates to mutated filamentous fungal host cell producing a secreted polypeptide of interest, wherein a native putative steroid dehydrogenase is modified, truncated, partly or fully inactivated, present at reduced level or eliminated compared to a non-mutated parent cell, and wherein said native putative steroid dehydrogenase comprises at least one conserved amino acid motif selected from: YGAR and/or VPHS[W/Y]F and/or QC[A/V/S]RRL and/or LKKYTLP and/or CPHYT, and methods of producing a secreted polypeptide of interest in said cells as well as methods of producing said cells.