Microalgae Expression Vector for Stoichiometric Protein Co-Expression
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Solution Overview
Problem
Current methods for co-expressing multiple proteins in microalgae face challenges such as random genome insertion, transgene silencing, and uneven expression levels, which can result in insoluble, inactive, or unstable protein complexes when subunits are expressed separately.
Innovation Solution
A microalgae expression vector with different 5′ and 3′ cis regulatory regions is developed to co-express proteins in a stoichiometric manner, using sequences like the HSP70A/RBCS2 chimeric promoter, RPL23 gene promoter, and FDX gene promoter, along with their respective UTRs, to ensure simultaneous and efficient expression of protein subunits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple vectors with different selection markers are used for co-expression, then multiple genes can be expressed, but random genome insertion results in very different expression patterns and requires large screenings
Solution Approach 1:
The patent combines multiple expression cassettes into a single vector construct, allowing co-expression of multiple genes without requiring multiple separate vectors. This merging approach eliminates the need for large screenings while maintaining the ability to express multiple genes simultaneously.
Solution Approach 2:
The vector is segmented into multiple independent expression cassettes, each capable of expressing a specific gene. This segmentation allows each gene to be expressed under its own promoter and regulatory elements, ensuring independent and controlled expression while avoiding the randomness of multiple vector integrations.
2Device complexity
If a single vector with duplicate strong promoter/UTR is used, then multiple genes can be expressed from one vector, but this introduces the risk of silencing and recombination
Solution Approach 1:
Each expression cassette in the vector has its own unique promoter and 3'UTR sequences, creating local diversity in regulatory elements. This prevents the silencing and recombination issues that arise from duplicate sequences while maintaining a single vector structure for co-expression.
3Productivity
If subunits of protein complex are expressed separately, then individual proteins can be produced, but they may not be soluble, active or stable
Solution Approach 1:
The vector is designed to simultaneously express all subunits of a protein complex in a coordinated manner, ensuring that each subunit is produced at appropriate levels and in the correct stoichiometric ratios. This preliminary coordination of expression ensures proper assembly and functionality of the protein complex.
4Adaptability or versatility
If bicistronic mRNAs with non-structured junction sequence are used, then two proteins can be produced from one mRNA, but expression of downstream gene is lower than upstream gene
Solution Approach 1:
Instead of using a single bicistronic mRNA with a junction sequence, the patent segments the expression into multiple independent cassettes, each with its own promoter and regulatory elements. This ensures uniform expression levels for all genes without the downstream suppression effect of bicistronic constructs.
Data Source
AI summary
An expression vector for the co-expression of different polynucleotides in microalgal cells and to the expression cassettes having both polynucleotides is provided. Also provided are a host cell, a method for expressing the proteins of interest and a method for selecting a cell co-expressing two proteins of interest in a stoichiometric manner.