Microalgae Chloroplast Transformation for Collagen Peptide Production
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Solution Overview
Problem
Current recombinant protein production systems face challenges in achieving high yields and native conformation of proteins with high purity, particularly due to limitations in post-translational modifications and genetic instability, which affects the solubility and activity of proteins like collagen and elastin derivatives in microalgae.
Innovation Solution
A method involving chloroplast genome transformation of microalgae with nucleic acid sequences encoding recombinant proteins or peptides, such as collagen and elastin derivatives, to enhance accumulation, stability, and solubility by utilizing homologous recombination and endogenous disulfide bond formation, allowing for efficient production and purification of these proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant proteins are produced in bacterial systems like E. coli, then production cost and speed are improved, but protein solubility and activity deteriorate due to inclusion body formation
Solution Approach 1:
The patent uses microalgae chloroplasts as an intermediary expression system between bacterial systems and eukaryotic systems. The chloroplast provides eukaryotic post-translational modification machinery (including disulfide bond formation) while maintaining bacterial-like genetic manipulation ease, thereby resolving the contradiction between production speed and protein solubility/activity
Solution Approach 2:
The patent changes the expression system parameter from prokaryotic (E. coli) to eukaryotic organelle (microalgae chloroplast), which fundamentally alters the cellular environment to enable proper protein folding and disulfide bond formation, thus improving protein solubility and activity while maintaining high productivity
2Reliability
If recombinant proteins are produced in eukaryotic systems with post-translational modification capability, then protein activity and conformation are improved, but production cost and complexity increase
Solution Approach 1:
The chloroplast serves as an intermediary that provides eukaryotic post-translational modification capabilities without requiring complex eukaryotic cellular machinery. The chloroplast genome can be directly transformed and manipulated like bacterial plasmids, simplifying the overall system while maintaining protein activity
Solution Approach 2:
The patent extracts the essential function of post-translational modification from the complex eukaryotic cytoplasm and concentrates it in the chloroplast organelle, which can be genetically manipulated with bacterial-like simplicity. This separation allows obtaining eukaryotic protein activity without eukaryotic system complexity
3Reliability
If DNA sequences encoding repeat units are used in microalgae, then genetic recombination occurs improving protein folding, but genetic instability arises leading to truncated proteins
Solution Approach 1:
The patent uses homologous recombination between identical chloroplast genomes to integrate the repeat unit sequences before expression. This preliminary integration ensures proper protein folding through disulfide bond formation while preventing subsequent genetic instability and truncation
Solution Approach 2:
The patent changes the integration mechanism parameter from simple transformation to homologous recombination-mediated integration. This ensures stable incorporation of repeat unit sequences into the chloroplast genome, preventing genetic instability while enabling proper protein folding
4Reliability
If chloroplast transformation is used in microalgae, then protein accumulation and stability are improved, but production yield remains low compared to microbial platforms
Solution Approach 1:
The patent optimizes multiple parameters including: using highly repetitive amino acid sequences that favor chloroplast expression, optimizing codon usage for chloroplast translation, using strong chloroplast promoters, and facilitating disulfide bond formation. These parameter changes collectively increase production yield while maintaining protein stability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly increases the accumulation and stability of recombinant peptides and proteins in microalgae, improving their solubility and activity, and facilitates their production with higher yields and purity, addressing the limitations of existing systems.
Implementation Method 1
utilizing homologous recombination and endogenous disulfide bond formation
Implementation Method 2
utilizing homologous recombination and endogenous disulfide bond formation
Data Source
AI summary
The present invention concerns a recombinant microalgae comprising a nucleic acid sequence encoding a recombinant protein, polypeptide or peptide comprising repeat units of amino acids, said recombinant protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives, and said nucleic acid sequence being located in the chloroplast of microalgae. It further relates to a method for producing a recombinant protein, polypeptide or peptide comprising repeat units of amino acids in the chloroplast of microalgae, said recombinant protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives wherein said method comprises transforming the chloroplast genome of a microalgae with a nucleic acid sequence encoding said recombinant protein, polypeptide or peptide.