Microbial NADPH Engineering for Higher Biosynthesis Yield
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Solution Overview
Problem
The production of chemical compounds like 1,3-butanediol, methyl methacrylate, (3R)-hydroxybutyl (3R)-hydroxybutyrate, and amino acids is limited by the availability of co-factors such as NADPH, leading to imbalanced redox levels and reduced yields.
Innovation Solution
Engineering non-naturally occurring microbial organisms with exogenous nucleic acids encoding enzymes that increase NADPH availability, such as ATP-NADH kinase and gene attenuations, to enhance the production of desired products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional microbial production methods are used, then the production process is simple, but the titer, rate, and yield of chemical compounds are limited due to co-factor availability
Solution Approach 1:
The patent changes the biochemical parameters of the microbial system by introducing exogenous nucleic acids that encode enzymes (ATP-NADH kinase, NAD(P) transhydrogenase) to alter the NADPH/NADH ratio and increase NADPH availability, thereby improving productivity without fundamentally changing the production system architecture
Solution Approach 2:
The patent uses exogenous nucleic acids and engineered enzymes as intermediaries to transfer energy and reducing equivalents from available co-factors to the NADPH pool, enabling increased product synthesis without directly modifying the product pathway enzymes
2Productivity
If co-factor availability is increased through microbial engineering, then the yield of desired products increases, but the complexity of the production system increases
Solution Approach 1:
The engineered enzymes (ATP-NADH kinase, NAD(P) transhydrogenase) serve multiple functions: they regenerate NADPH for product synthesis, maintain redox balance, and can operate with various substrates, making the system more versatile and reducing the need for separate systems for each function
Solution Approach 2:
The microbial system is engineered to self-regulate its co-factor pool by incorporating feedback mechanisms where the engineered enzymes respond to cellular redox state and automatically adjust NADPH regeneration rates, reducing the need for external control 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 increased NADPH availability leads to higher titers, rates, and yields of 1,3-butanediol, methyl methacrylate, (3R)-hydroxybutyl (3R)-hydroxybutyrate, and amino acids, optimizing industrial production processes.
Implementation Method 1
The non-naturally occurring microbial organism includes one or more exogenous nucleic acids encoding an enzyme expressed in an amount sufficient to increase the availability of reduced nicotinamide adenine dinucleotide phosphate (NADPH)
Implementation Method 2
NADPH provides the reducing equivalents for biosynthetic reactions, such as lipid and nucleic acid synthesis, and the oxidation-reduction involved in protecting against the toxicity of reactive oxygen species (ROS)
Data Source
AI summary
The present disclosure provides microbial organisms having increased availability of co-factors, such as NADPH, for increasing production of various products, including 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, amino acids, 3HB-CoA, adipate, caprolactam, 6-ACA, HMD A, or MAA, and products made from any of these. Also provided are one or more exogenous nucleic acids encoding an enzyme expressed in a sufficient amount to increase availability of NADPH, where the exogenous nucleic acid includes one or more of ATP-NADH kinase, pntAB, nadK, and gapN. Also provided are one or more gene attenuations occurring in genes, such as NDH-2, that result in an increased ratio of NADPH to NADH. Various combinations of the exogenous nucleic acids and gene deletions are also provided in the present disclosure. The present disclosure also provides methods of making and using the same, including methods for culturing cells, and for the production of the various products.