Bacillus Subtilis-Assembled Plasmid for Multi-Host Gene Expression
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Solution Overview
Problem
Existing methods for producing polyketide compounds face challenges in compatibility between host cells and require frequent plasmid modifications for heterologous expression, necessitating a novel approach to construct compatible plasmids.
Innovation Solution
A method involving a DNA assembly using Bacillus subtilis to link a first gene cluster with replication origins of Bacillus subtilis and Escherichia coli, and a conjugation initiation sequence for actinomycetes, eliminating the need for plasmid modifications across different host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a plasmid is constructed for heterologous expression in different host cells, then the gene cluster can be expressed in multiple hosts, but the plasmid requires modification for each host cell due to compatibility issues
Solution Approach 1:
The plasmid is designed with multiple replication origins (Bacillus subtilis origin and Escherichia coli origin) and conjugation initiation sequences that enable it to function across different host cell types. This multi-functional design allows the single plasmid structure to be maintained without modification when transferred between Gram-positive and Gram-negative bacteria, resolving the contradiction between host adaptability and plasmid complexity
Solution Approach 2:
The plasmid structure is segmented into functional modules including gene clusters, replication origins, and conjugation sequences. This modular segmentation allows each component to be optimized for specific host compatibility while maintaining overall plasmid stability across different bacterial hosts, reducing the need for comprehensive plasmid re-engineering
2Productivity
If traditional chemical methods are used to produce polyketide compounds, then production can be achieved, but the production efficiency is low and the process is complex
Solution Approach 1:
The invention utilizes the host cell's own biosynthetic machinery to produce polyketide compounds by introducing heterologous gene clusters. The host cell automatically transcribes and translates the introduced genes using its endogenous RNA polymerase, ribosomes, and tRNA pools, eliminating the need for complex external production systems and significantly improving production efficiency while reducing process complexity
Solution Approach 2:
The plasmid serves as an intermediary carrier that introduces heterologous gene clusters into host cells. This intermediary mechanism enables the transfer and expression of biosynthetic pathways from one organism to another, facilitating efficient polyketide production through heterologous expression while avoiding the complexities of both traditional chemical synthesis and direct genetic manipulation of native strains
Data Source
AI summary
Provided are a method for producing a novel plasmid in heterologous expression of a gene and a plasmid. The method includes preparing a first gene cluster containing the plurality of genes by a DNA assembly method using a plasmid transformation system of Bacillus subtilis; and linking, to the first gene cluster, a second gene cluster containing a replication origin of Bacillus subtilis, a replication origin of Escherichia coli, and an initiation sequence for conjugation to an actinomycete.