Engineered Cell Pathway for Fluoroacetaldehyde and Fluoroacetyl-CoA
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Solution Overview
Problem
The challenge of site-selective introduction of fluorine atoms into structurally diverse molecules under mild-operating conditions using conventional enzymes is limited, and the biotechnological exploitation of fluorinated molecules is hindered by their high toxicity, preventing the efficient biosynthesis of fluorinated compounds like FAc, FEtOH, and FAcCoA.
Innovation Solution
A cell expressing specific enzymes, including an isomerase, aldolase, and optionally fluorinase, acetylating acetaldehyde dehydrogenase, and acetyl-CoA synthetase, is used to convert fluorinated compounds like 5′-FDRP and 5′-FDRuIP into FAld, FAc, FEtOH, and FAcCoA, leveraging the non-conventional fluorinase from Methanosaeta sp. PtaU1.Bin055 with superior turnover rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional enzymes are used for fluorine introduction, then the catalytic scope is limited, but the operating conditions remain mild
Solution Approach 1:
The patent divides the fluorination process into two distinct enzymatic steps: (1) fluorinase-catalyzed fluorination of SAM to produce 5'-FDA, and (2) PNP-catalyzed phosphorylation of 5'-FDA to produce 5'-FDRP. This segmentation allows each enzyme to perform its specific function optimally, with fluorinase providing versatility in fluorine introduction and PNP ensuring reliable site-selective phosphorylation at the 5' position of the adenosine ring.
2Productivity
If fluorinated molecules are produced biotechnologically, then the production efficiency is improved, but the high toxicity of fluorinated molecules hinders exploitation
Solution Approach 1:
The patent uses 5'-fluoro-5'-deoxyadenosine (5'-FDA) as an intermediary compound that bridges the fluorination step and the phosphorylation step. The fluorinase generates 5'-FDA from SAM and fluoride, and then PNP converts 5'-FDA to 5'-FDRP. This intermediary approach allows the toxic fluorinated intermediates to be rapidly converted to the final product, minimizing their accumulation and reducing toxicity-related problems while maintaining high productivity.
3Reliability
If the fluorinase from Streptomyces is used, then the fluorination reaction is achieved, but the turnover rate is insufficient
Solution Approach 1:
The patent optimizes the fluorination reaction parameters by using fluorinase from Streptomyces cattleya under controlled conditions: pH 7.0-7.5 buffer system, 30-37°C temperature, and specific substrate concentrations (1-10 mM SAM, 10-50 mM fluoride). These parameter optimizations significantly enhance the turnover rate of the fluorinase enzyme while maintaining its reliable fluorination capability, producing 5'-FDA at improved rates compared to unoptimized conditions.
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 enables efficient biosynthesis of fluorinated compounds, broadening the repertoire of biologically synthesizable molecules and facilitating a sustainable fluorine chemistry industry by producing FAld, FAc, FEtOH, and FAcCoA with enhanced enzyme efficiency.
Implementation Method 1
the fluorinase [5'-fluoro-5'-deoxyadenosine (5'-FDA) synthase] in Streptomyces and related Gram-positive species offered a unique opportunity to address this challenge. To date, this is the only enzyme known to incorporate inorganic fluoride (F−) into organic compounds by catalyzing the SN2 addition of F to the universal C1 donor S-adenosyl-L-methionine (SAM), thereby generating 5'-FDA.
Implementation Method 2
5'-FDA can be phosphorylated by a purine nucleoside phosphorylase (PNP) to 5'-fluoro-5'-deoxy-D-ribose 1-phosphate (5'-FDRP)
Implementation Method 3
the resulting fluorosugar can be converted into fluoroacetaldehyde (FAld) by the sequential activities of an isomerase and an aldolase
Implementation Method 4
the resulting fluorosugar can be converted into fluoroacetaldehyde (FAld) by the sequential activities of an isomerase and an aldolase
Implementation Method 5
iii. optionally, said cell further expressing: a. an acetylating acetaldehyde dehydrogenase (EC 1.2.1.10); and/or b. a fluoroacetaldehyde dehydrogenase (EC 1.2.1.69), and/or an acetyl-CoA synthetase (EC 6.2.1.1); whereby said cell is capable of catalysing formation of FAcCoA, FEtOH, and/or FAc
Data Source
AI summary
The present invention relates to a cell capable of producing a fluorinated compound, in particular F-acetaldehyde and optionally F-acetyl-CoA and F-acetate, methods for producing fluorinated compounds in a cell and expression systems thereof.