E. coli 7α-HSDH Knockout for Pure UDCS Synthesis
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Solution Overview
Problem
Current methods for producing ursodeoxycholic acid (UDCS) are contaminated with impurities like lithocholic acid (LCS) due to enzymatic contamination from 7α-HSDH activity during the enzymatic/chemical production process, which cannot be effectively separated from the desired product.
Innovation Solution
The production of UDCS is achieved by knocking out the 7α-HSDH gene in E. coli BL21(DE3) host cells, preventing 7α-HSDH activity and thus eliminating the formation of undesirable by-products, using a recombinant strain E. coli BL21(Δ7α-HSDH) to produce 12α-HSDH or 7β-HSDH enzymes that are free from 7α-HSDH contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If 12α-HSDH is produced using E. coli BL21(DE3) host cells, then the enzyme can be efficiently produced, but the product is contaminated with 7α-HSDH activity that causes formation of unwanted by-products
Solution Approach 1:
The invention removes the harmful 7α-HSDH gene from the E. coli host genome through targeted gene knockout. By eliminating the endogenous 7α-HSDH gene, the host cell can no longer produce the contaminating enzyme, thus removing the source of harmful by-products while preserving the ability to produce desired HSDH enzymes.
Solution Approach 2:
The invention exploits the fact that E. coli BL21(DE3) naturally possesses 7α-HSDH activity by knocking out this specific gene. The knockout mutant strain is then used as the host for recombinant HSDH production, converting a previously problematic feature into a controlled advantage where only the desired enzyme activity remains without contaminating 7α-HSDH activity.
2Productivity
If 7β-HSDH is used for reductive production of UDCS, then UDCS can be produced by reductive route, but 3,12-diketo-CDCS by-product accumulates due to 7α-HSDH activity
Solution Approach 1:
The invention removes the harmful 7α-HSDH gene from the E. coli host genome through targeted gene knockout. By eliminating the endogenous 7α-HSDH gene, the host cell can no longer produce the contaminating enzyme, thus removing the source of harmful by-products while preserving the ability to produce desired HSDH enzymes.
Solution Approach 2:
The gene knockout is performed in advance before the recombinant HSDH production process. This preliminary genetic modification ensures that the host strain is incapable of producing 7α-HSDH contamination, preventing the formation of 3,12-diketo-CDCS by-product before the actual UDCS synthesis begins.
3Productivity
If chemical oxidation is used to produce UDCS from cholic acid, then UDCS can be synthesized, but multiple protection steps are required due to lack of selectivity
Solution Approach 1:
The invention replaces non-selective chemical oxidation with selective enzymatic oxidation using purified HSDH enzymes. The enzymatic catalyst provides inherent stereoselectivity and regioselectivity, eliminating the need for multiple chemical protection and deprotection steps that are required when using conventional chemical oxidation methods.
Solution Approach 2:
The invention changes the catalytic mechanism from chemical to enzymatic, utilizing the specific catalytic properties of HSDH enzymes. This parameter change introduces high selectivity into the oxidation process, allowing direct conversion of cholic acid to UDCS without requiring protective group chemistry.
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 allows for the synthesis of LCS-free UDCS by ensuring that the enzymatic conversion steps are free from 7α-HSDH activity, resulting in a purer product without the formation of unwanted by-products like 3,12-diketo-CDCS.
Implementation Method 1
The 12α-HSDH selectively oxidizes CS to 12-keto-CDCS
Implementation Method 2
The 12α-HSDH selectively oxidizes CS to 12-keto-CDCS
Implementation Method 3
the 12α-HSDH enzyme catalyzing the enzymatic conversion step has an enzymatic contamination due to 7α-HSDH activity. The result of this is the oxidation of 12-keto-CDCS to 7,12-diketo-LCS
Implementation Method 4
the oxidation of 12-keto-CDCS to 7,12-diketo-LCS
Implementation Method 5
The recombinant E. coli hosts also produce endogenous 7α-HSDH
Implementation Method 6
The enzyme is preferably produced by recombinant E. coli hosts
Data Source
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Figure 2A~2C
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AI summary
The invention relates to novel microbial 7a-hydroxysteroid dehydrogenase (7a-HSDH) knockout mutants and to the use thereof for producing other HSDHs having various functionalities, such as 3a-, 7ß- or 12a-HSDH, and to the use of thus-produced HSDH enzymes in enzymatic reactions of cholic acid compounds, and in particular for producing ursodeoxycholic acid (UDCS). The invention relates in particular to novel methods for synthesizing UDCS.