Histidine Production via Feedback-Resistant Enzymes and Folate Mediation
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Solution Overview
Problem
The biosynthesis of histidine in organisms is an energy-intensive process with strict transcriptional and translational regulation, making it challenging to engineer host cells for high-level production due to its high ATP demand and complex regulatory mechanisms.
Innovation Solution
Engineered host cells expressing non-naturally occurring nucleic acids with specific promoter and ribosome binding site combinations, including genes like hisG, hisD, hisC, hisB, hisH, hisA, hisF, and hisI, along with ribose phosphate pyrophosphokinase (RPPK) and 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methylene-tetrahydrofolate cyclohydrolase (MTHFDC) enzymes, under synthetic promoters to enhance histidine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If histidine biosynthesis pathway is engineered in host cells, then histidine production is improved, but energy consumption increases due to high ATP demand
Solution Approach 1:
The patent modifies key parameters of the histidine biosynthesis pathway by introducing feedback-resistant mutant enzymes (HisG with E271K mutation, HisD with feedback resistance, HisC with feedback resistance) that maintain catalytic activity while being insensitive to product inhibition. This allows the pathway to operate at high flux without requiring excessive ATP consumption for overcoming regulatory barriers, thus improving histidine production while managing energy expenditure.
Solution Approach 2:
The patent introduces an intermediary mechanism by expressing multiple copies of the folD gene encoding 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase. This enzyme acts as a mediator that converts 5,10-CH2-THF to 10-CHO-THF, providing formyl groups for purine biosynthesis and indirectly supporting histidine production. This intermediary pathway helps balance the energy metabolism by utilizing folate derivatives to generate necessary intermediates without directly competing for ATP.
2Productivity
If histidine biosynthesis pathway is engineered in host cells, then histidine production is improved, but cellular stability deteriorates due to toxicity and instability issues
Solution Approach 1:
The patent employs feedback-resistant mutant enzymes that have been engineered to withstand high concentrations of histidine and pathway intermediates without losing activity or causing cellular stress. The HisG(E271K) mutant and other feedback-resistant variants maintain stable cellular function even when producing high levels of histidine, thereby improving productivity while preserving cellular stability.
Solution Approach 2:
The patent implements prior cushioning by introducing the folD gene multiple copies before the stress of high-level histidine production occurs. This preemptive measure ensures that the cell has adequate pools of 10-CHO-THF and formyl-THF intermediates available to support purine biosynthesis and maintain nucleotide balance, preventing metabolic stress and instability that would otherwise occur during high-flux histidine production.
3Reliability
If strict transcriptional and translational regulation is maintained, then cellular homeostasis is preserved, but histidine production is limited
Solution Approach 1:
The patent extracts the regulatory constraints from the histidine biosynthesis pathway by using feedback-resistant mutant enzymes that are decoupled from the normal feedback inhibition mechanisms. The HisG(E271K) mutant and other feedback-resistant variants operate independently of histidine-mediated repression, allowing the pathway to function at high flux without being constrained by cellular homeostatic regulation, thus enabling high-level histidine production.
Solution Approach 2:
The patent segments the regulatory control from the catalytic function by introducing separately engineered feedback-resistant enzyme variants. Each mutant enzyme (HisG, HisD, HisC) maintains its catalytic role while having its regulatory properties modified independently. This segmentation allows the pathway to achieve high productivity while the cell can still maintain homeostasis through other regulatory mechanisms unaffected by the mutated enzymes.
Data Source
AI summary
Aspects of the disclosure relate to biosynthesis of histidine in host cells. For example, host cells may comprise: a promoter; a ribosome binding site (RBS); and a nucleic acid comprising: hisG; hisD; hisC hisB; hisH; hisA; hisF; and/or hisI. Host cells may further comprise a nucleic acid encoding a ribose phosphate pyrophosphokinase (RPPK), optionally comprising one or more amino acid substitutions relative to the sequence of wildtype E. coli RPPK. Host cells of the disclosure may comprise a nucleic acid encoding a 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methylene-tetrahydrofolate cyclohydrolase (MTHFDC) enzyme. Further aspects of the disclosure relate to production of purine pathway metabolites and/or plasmid DNA in host cells.