Lactococcus Promoters for High-Level Protein Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is a need for strong promoters derived from Lactococcus that can achieve high levels of protein expression in Lactic acid bacteria for industrial and therapeutic applications, while maintaining the 'generally regarded as safe' (GRAS) status, as exogenous promoters can compromise this safety.
Innovation Solution
Identification and utilization of native nucleic acid sequences from Lactococcus as strong promoters, such as non-heme iron-binding ferritin (PdpsA), dipeptidase PepV, and superoxide dismutase (PsodA), which are operably linked to open reading frames, exceeding the strength of the currently known strongest Lactococcus-derived promoter, thyA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If exogenous promoters are introduced to achieve strong protein expression in Lactic acid bacteria, then protein expression level is improved, but the GRAS (generally regarded as safe) status is compromised
Solution Approach 1:
The patent uses copying by identifying and utilizing native promoter sequences from Lactococcus that replicate the safety profile of the host organism while providing strong expression capability. The promoters are derived from the same species (Lactococcus) as the host, ensuring they are GRAS-compliant while achieving high protein expression levels through careful selection of native promoters with high transcriptional activity.
Solution Approach 2:
The patent applies parameter changes by modifying or optimizing native promoter sequences through mutagenesis and computational analysis to enhance their strength and expression capability. By altering specific nucleotide sequences while maintaining their native origin, the promoters achieve higher protein expression levels without compromising their GRAS status, as they remain derived from Lactococcus.
2Reliability
If native Lactococcus promoters are used to maintain GRAS status, then safety is preserved, but protein expression level may be insufficient for industrial applications
Solution Approach 1:
The patent employs parameter changes by optimizing native promoter sequences through directed mutagenesis and computational algorithms to enhance their transcriptional strength. By carefully modifying specific parameters of the promoter sequences (nucleotide composition, spacing, etc.) while maintaining their Lactococcus origin, the invention achieves high protein expression levels that satisfy industrial requirements without compromising GRAS status.
Solution Approach 2:
The patent creates composite promoter elements by combining different functional sequences and motifs from native Lactococcus promoters to generate chimeric or hybrid promoters with enhanced strength. These composite promoters integrate the best features of multiple native promoters, achieving both high expression levels and GRAS compliance through their derived-from-Lactococcus nature.
3Productivity
If existing strong promoters like thyA are used, then high protein expression is achieved, but the promoter strength can be further improved for better protein yield
Solution Approach 1:
The patent applies preliminary action by using computational algorithms and in silico analysis to predict and identify the strongest potential promoters from Lactococcus genomic data before experimental validation. This preliminary computational screening allows systematic evaluation of multiple promoter candidates and their potential expression levels, enabling selection of the optimal promoters for high protein yield without requiring exhaustive experimental testing of all possible promoters.
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
The invention is in the field of molecular biology, and relates to recombinant engineering and protein expression. More in particular, the invention relates to nucleic acids for recombinant expression of proteins comprising sequences derived from Lactococcus and useful as promoters. The invention further relates to vectors comprising the said nucleic acids and host cells transformed therewith. The invention also covers the use of host cells comprising the said nucleic acids or vectors for expressing heterologous or homologous proteins; and also for delivery, especially therapeutic delivery, of the said proteins to subjects.