Recombinant Microbial Cells for Melatonin Production
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Solution Overview
Problem
Current methods for producing melatonin and related compounds in recombinant microorganisms are inefficient, requiring exogenous nucleic acids and cofactors, and there is a need for improved recombinant microorganisms capable of efficient production.
Innovation Solution
Development of recombinant microbial cells with heterologous monooxygenases like TPH and PCD, optionally without exogenous nucleic acids encoding DHPR or DHMR, utilizing native E. coli compounds to support monooxygenase activity, and identification of variants of GCH1 and TPH with improved hydroxylation activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant microorganisms are used for melatonin production, then production capability is achieved, but efficiency is poor and exogenous nucleic acids and cofactors are required
Solution Approach 1:
The patent extracts and eliminates the requirement for exogenous nucleic acids and cofactors by utilizing the microorganism's own endogenous pathways. Specifically, it employs endogenous GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase to biosynthesize BH4 cofactor internally, removing the need for external supplementation and simplifying the production system.
Solution Approach 2:
The recombinant microorganism serves itself by utilizing its endogenous metabolic pathways to provide all necessary components for melatonin production. The cell's own GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase enzymes work together to generate BH4 cofactor autonomously, eliminating dependency on exogenous nucleic acids and cofactors.
2Productivity
If multiple enzymes and pathways are introduced for efficient production, then production yield improves, but the system becomes more complex
Solution Approach 1:
The patent employs a multi-functional approach where the introduced recombinant enzymes work synergistically with existing endogenous enzymes. The system utilizes both the introduced GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase alongside endogenous pathways, creating a universal biosynthetic network that efficiently produces BH4 and downstream melatonin products without requiring completely separate systems.
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
The recombinant microbial cells can produce oxidation products like 5HTP and melatonin efficiently without exogenous nucleic acids, achieving increased hydroxylation activity and production yields, and can include additional enzymes for producing serotonin and other compounds.
Implementation Method 1
The first step is this pathway, the conversion of L-tryptophan to 5HTP, is catalyzed by L-tryptophan hydroxylase (TPH). TPH and many other mammalian aromatic amino acid hydroxylases require oxygen and tetrahydropterin (BH4) as cofactors.
Implementation Method 2
a pathway comprising a GTP cyclohydrolase I (GCH1), a 6-pyruvoyl-tetrahydropterin synthase (PTS or PTPS) and a sepiapterin reductase (SRP)
Implementation Method 3
regenerated into BH4 via consecutive reactions by pterin-4a-carbolamine dehydratase (PCD) and dihydropterin reductase (DHPR)
Data Source
AI summary
Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.