Alg3 Inactivation in Aspergillus niger for Citric Acid Yield
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Solution Overview
Problem
Current methods for citric acid production in Aspergillus niger do not fully optimize carbohydrate conversion to citric acid, as they do not effectively alter post-translation modifications like N-glycosylation, which can impact metabolic pathways and product output.
Innovation Solution
Genetic inactivation of the dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene and/or overexpression of the LaeA gene to enhance citric acid production, altering protein glycosylation and metabolic flux.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional citric acid production methods are used in Aspergillus niger, then standard metabolic pathways are maintained, but carbohydrate conversion efficiency to citric acid is not fully optimized
Solution Approach 1:
The patent extracts and removes the Alg3 gene responsible for N-glycosylation from the fungal metabolic pathway. By deleting this gene, the pathway that consumes carbohydrates for protein glycosylation is eliminated, redirecting carbohydrate flux toward citric acid production and improving overall productivity
Solution Approach 2:
The patent changes the metabolic parameter by overexpressing the LaeA gene, which is a global regulator of secondary metabolism. This parameter change alters the expression of multiple downstream genes involved in metabolic pathways, thereby optimizing carbohydrate conversion efficiency to citric acid
2Quantity of substance
If protein glycosylation pathways are active, then proper protein folding and secretion occur, but carbohydrates are consumed reducing citric acid yield
Solution Approach 1:
The Alg3 gene encoding the mannosyltransferase enzyme is extracted from the genome through targeted deletion. This removes the capability to perform N-linked glycosylation at the dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase step, preventing carbohydrate consumption in this pathway and increasing substrate availability for citric acid synthesis
Solution Approach 2:
The patent discards the N-glycosylation pathway by deleting Alg3, and recovers the consumed carbohydrates by redirecting them into the citric acid production pathway. The metabolic flux that previously went to glycosylation is now recovered and channeled toward the desired product
3Productivity
If Alg3 gene is inactivated to reduce carbohydrate consumption, then citric acid production increases, but protein glycosylation is affected
Solution Approach 1:
The LaeA gene acts as an intermediary regulator that compensates for the Alg3 deletion. By overexpressing LaeA, the patent modulates the expression of multiple downstream genes involved in protein secretion, cell wall integrity, and alternative metabolic pathways, thereby maintaining cellular reliability despite the loss of N-glycosylation
Solution Approach 2:
Overexpression of LaeA changes the regulatory parameter of secondary metabolism and protein secretion pathways. This parameter change compensates for the Alg3 deletion by upregulating alternative pathways that maintain protein secretion and cellular function while allowing increased citric acid production
Data Source
AI summary
Provided herein are fungi, such as Aspergillus niger, having a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene genetic inactivation, increased expression of a loss of aflR expression A (Lae), or both. In some examples, such mutants have several phenotypes, including an increased production of citric acid relative to the parental strain. Methods of using the disclosed fungi to make citric acid are also provided, as are compositions and kits including the disclosed fungi.