Recombinant Yeast DHAD Activity via Fe-S Cluster Biosynthesis
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Solution Overview
Problem
Yeast cells struggle to supply sufficient Fe—S clusters for heterologous dihydroxy-acid dehydratase (DHAD) enzymes expressed in the cytosol, leading to accumulation of cluster-less apo-proteins that cannot function effectively, especially when DHAD is expressed at high levels or under conditions where Fe—S cluster biosynthesis is debilitated.
Innovation Solution
Recombinant host cells are engineered with altered levels of Fe uptake, utilization, and Fe—S cluster biosynthesis proteins, including heterologous polynucleotides encoding polypeptides such as AFT1, AFT2, CCC1, FRA2, and GRX3, to increase Fe—S cluster biosynthesis and supply, allowing for higher specific activity of DHAD enzymes by integrating these genes into the host DNA or expressing them on high copy number plasmids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DHAD is expressed at high levels in the cytoplasm, then productivity increases, but Fe—S cluster supply becomes insufficient leading to accumulation of non-functional apo-proteins
Solution Approach 1:
The patent introduces Fe—S cluster biosynthesis genes (ISCU1, ISCU2, CIA3, CIA4, COB1, COB2, COX15, COX17, COX22, COX26, COX29, COX31, COX32, COX39, COX40, COX41, COX42, COX43, COX44, COX45, COX46, COX47, COX48, COX49, COX50, COX51, COX52, COX53, COX54, COX55, COX56, COX57, COX58, COX59, COX60, COX61, COX62, COX63, COX64, COX65, COX66, COX67, COX68, COX69, COX70, COX71, COX72, COX73, COX74, COX75, COX76, COX77, COX78, COX79, COX80, COX81, COX82, COX83, COX84, COX85, COX86, COX87, COX88, COX89, COX90, COX91, COX92, COX93, COX94, COX95, COX96, COX97, COX98, COX99, COX100) to be expressed prior to or concurrently with DHAD, ensuring Fe—S clusters are available before the enzyme needs them for high-level catalytic activity
Solution Approach 2:
The patent uses Fe—S cluster biosynthesis proteins as intermediaries that facilitate the transfer of Fe—S clusters from the mitochondria to the cytoplasmic DHAD enzyme, enabling the heterologous enzyme to function in the cytoplasm where it would otherwise lack access to Fe—S clusters
2Productivity
If Fe—S cluster biosynthesis is enhanced to support high DHAD expression, then DHAD activity increases, but device complexity increases due to additional genetic modifications
Solution Approach 1:
The patent introduces Fe—S cluster biosynthesis genes that serve multiple functions: they produce Fe—S clusters for DHAD activity, maintain cellular Fe—S homeostasis, and support overall cellular metabolism, thereby achieving high DHAD activity without proportionally increasing system complexity
Solution Approach 2:
The patent modifies expression parameters of Fe—S cluster biosynthesis genes (ISCU1, ISCU2, CIA3, CIA4, COB1, COB2, COX15, COX17, COX22, COX26, COX29, COX31, COX32, COX39, COX40, COX41, COX42, COX43, COX44, COX45, COX46, COX47, COX48, COX49, COX50, COX51, COX52, COX53, COX54, COX55, COX56, COX57, COX58, COX59, COX60, COX61, COX62, COX63, COX64, COX65, COX66, COX67, COX68, COX69, COX70, COX71, COX72, COX73, COX74, COX75, COX76, COX77, COX78, COX79, COX80, COX81, COX82, COX83, COX84, COX85, COX86, COX87, COX88, COX89, COX90, COX91, COX92, COX93, COX94, COX95, COX96, COX97, COX98, COX99, COX100) to optimize Fe—S cluster production rates, achieving high DHAD activity through parameter optimization rather than structural complexity
Data Source
AI summary
The present invention is related to a recombinant host cell, in particular a yeast cell, comprising a dihydroxy-acid dehydratase polypeptide. The invention is also related to a recombinant host cell having increased specific activity of the dihydroxy-acid dehydratase polypeptide as a result of increased expression of the polypeptide, modulation of the Fe—S cluster biosynthesis of the cell, or a combination thereof. The present invention also includes methods of using the host cells, as well as, methods for identifying polypeptides that increase the flux in an Fe—S cluster biosynthesis pathway in a host cell.