Cofactor engineering

Biocatalysts are required for the production of chiral building blocks needed in pharmaceuticals and other chemicals used by society. Many such biocatalysts require NADPH as a cofactor. NADPH, a cofactor quite similar to NADH, is both more expensive and less stable than its counterpart NADH. For these reasons, manufacturers would prefer that the biocatalysts they use in their production lines accept NADH over NADPH. Cofactor engineering has recently been successful in altering enzymes to prefer NADH as a cofactor instead of NADPH. In 2010, a group of scientists performed cofactor engineering on the enzyme Gre2p, an NADPH-preferring dehydrogenase found in Saccharomyces cerevisiae. Gre2p reduces the compound diketone 2,5-hexanedione into the chiral building blocks (5S)-hydroxy-2-hexanone and (2S,5S)-hexanediol. The scientists determined that Asn9 (Asparagine, position 9) was an important amino acid the active site of Gre2p. Specifically, Asn9 binds to the 3’-hydroxyl group and the 2’-oxygen atom of adenyl ribose moiety. Through direct mutagenesis, the scientists exchanged the Asn9 for both Asp (Aspartic Acid) and Glu (Glutamic Acid). This change caused Gre2p to have a decreased dependency on NADPH, and an increased affinity for NADH. This resulted in increased Gre2p activity when using NADH. It was observed that substituting Asn9 with Glu produced a greater effect than changing Asn9 to Asp. Asn contains a polar uncharged side chain, while both Asp and Glu contain a polar charged side chain. The increased effect of Glu is caused by the extra carbon in its side chain that brings it closer to adenyl ribose moiety. This allows for stronger hydrogen bonding between the 2’- and 3’- ribose hydroxyl groups and the side chain carboxyl group. The maximum velocity of the reaction doubled, while using NADH, when Asn9 was substituted with Glu. With these results, the scientists successfully engineered Gre2p to prefer NADH over NADPH and increased the speed of 2,5-hexanedione reduction. This will allow chemical companies to decrease their manufacturing costs by using NADH instead of NADPH at least for this particular reduction.[6]

An alternative example of changing an enzyme’s preference for cofactors is to change NADH dependent reaction to NADPH dependent reactions. In this example, the enzymes themselves are not changed, but instead different enzymes are selected that accomplish the same reaction with the use of a different cofactor. An engineered pathway was created to make 1-butanol from Acetyl-CoA by changing enzymes in the metabolic pathway of S. elongatus. The Clostridium genus is known to produce 1-butanol, providing a pathway that could be inserted in S. elongatus. This pathway synthesizes 1-butanol using the reverse β-oxidation pathway. The enzymes involved in this newly engineered pathway were NADH specific, which was problematic for replicating the pathway in S. elongatus as cyanobacteria produce much more NADPH than NADH.

The research group then identified enzymes that utilize NADPH or both NADPH and NADH by bioprospecting. Acetoacetyl-CoA reductase (PhaB) was found to be a suitable replacement for hydroxybutyric dehydrogenase (Hbd). To replace AdhE2, the researchers found that NADP-dependent alcohol dehydrogenase (YqhD) from E. coli to be effective for the pathway. Furthermore, the researchers needed a dehydrogenase to replace the aldehyde dehydrogenase capacity of AdhE2. CoA-acylating butyraldehyde dehydrogenase (Bldh) from C. saccharoperbutylacetonicum was found to be a good suit. Together, PhaB, Bldh, YqhD can replace Hbd and AdhE2, respectively, to change the cofactor preference of 3-ketobutyryl-CoA reduction from using NADH to using NADPH. The authors then constructed various combinations of the different enzymes (of those found in the reverse oxidation pathway and the NADPH utilizing enzymes) by overexpressing different genes in cultures of S. elongatus PCC 7942. In order to do so, they constructed plasmids containing the genes corresponding to the enzymes and combined them into the genome of S. elongates. After enzyme assays, the strain of cyanobacteria expressing the NADPH utilizing enzymes produced the greatest amount of 1-butanol (29.9 mg/L), exceeding that of strains that did not consist of the NADPH utilizing enzymes by four times. Overall, 1-butanol was produced in S. elongatus using a pathway from another organism. This pathway was modified in order to match the preferred reducing cofactor for the cyanobacteria.[7]

Example of how Cofactor Engineering can be used to engineer one pathway to influence another.

In cofactor engineering, a metabolic pathway is altered by changing the concentrations of specific cofactors that are produced either in that particular pathway or in a separate pathway. For example, an hypothetical organism could have two arbitrary pathways called A and B where some enzymes in both A and B utilize the same cofactors. If scientists wanted to decrease the output of pathway A, they may first consider directly engineering the enzymes involved in A, perhaps to decrease a particular active site's affinity for its substrate. In some cases however, the enzymes in A may be difficult to engineer for various reasons, or it may be impossible to engineer them without dangerously affecting some third metabolic pathway C, which utilizes the same enzymes. As a separate option, scientists could increase the flux of B, which may be easier to engineer. This in turn could "tie up" the cofactors needed by A, which would slow enzymatic activity, decreasing output in A. This is one hypothetical example of how cofactor engineering can be used, but there are many other unique cases where scientists use cofactors as a way of altering metabolic pathways. A major advantage to cofactor engineering is that scientists can use it to successfully alter metabolic pathways that are difficult to engineer by means of ordinary metabolic engineering. This is achieved by targeting more easily engineered enzymes in separate pathways, which use the same cofactors. Since many cofactors are used by different enzymes in multiple pathways, cofactor engineering may be an efficient, cost effective alternative to current methods of metabolic engineering.[8]

Yeast are commonly used in the beer and wine industry because they are capable of efficiently producing ethanol through the metabolic pathway fermentation in the absence of oxygen. Fermentation requires the enzyme glycerol-3-phosphate dehydrogenase (GPDH) which depends on the cofactor NADH. This pathway involves the conversion of glucose to both ethanol and glycerol, both of which use NADH as a cofactor. Scientists engineered Saccharomyces cerevisiae to overproduce GPDH, which shifted the cells metabolic flux away from ethanol and toward glycerol, by limiting NADH availability in the ethanol production portion of the pathway. The opposite effect was achieved by influencing a separate pathway in the cell, the Glutamate Synthesis pathway. Inactivating the expression of the enzyme glutamate dehydrogenase, which is NADPH dependent, and over expressing the enzymes glutamine synthetase and glutamate synthetase, which rely on NADH as a cofactor shifted the cofactor balance in glutamate synthesis pathway. The pathway is now dependent on NADH rather than NADPH, which decreases NADH availability in the fermentation pathway. This in turn causes increased ethanol production and decreased glycerol production. This method of manipulating metabolic fluxes could be visualized much like global fuel markets, where the increased production of ethanol for use in the automotive industry would decrease its availability in the food industry. Essentially, producing more engines which run on ethanol could result in decreased consumption of processed sweets, which contain high fructose corn syrup. This engineering of cofactors is applicable to the beer and wine industry since it allows for the regulation of ethanol levels in alcoholic beverages. Advancements in the wine industry have caused a steady increase in ethanol content, so winemakers in particular would be interested in the possibility of reducing the ethanol levels of some of their wines.[3]

Coenzyme A (CoA) and acetyl-CoA are two intermediate metabolites, most notably found in the Citric Acid Cycle, which participate in over 100 different reactions in the metabolism of microorganisms. Recent experiments have shown that over expression of the enzyme pantothenate kinase and supplementation of pantothenic acid in the CoA biosynthesis pathway have allowed adjustments of both CoA and acetyl-CoA fluxes. This increased concentration of cofactors resulted in an increased carbon flux in the isoamyl acetate synthesis pathway, increase the production efficiency of isoamyl acetate. Isoamyl acetate is used industrially for artificial flavoring and for testing the effectiveness of respirators. In addition to the production of isoamyl acetate, the manipulation of CoA biosynthesis during the pyruvate hydrogenase reaction also causes an increase in the production of both succinate and lycopene, each of which have beneficial effects on the human body. An increase in succinate concentration, which is used as a catalyst, may lead to an increase in the speed of the Citric Acid Cycle, and consequently an individual's metabolism. Increasing lycopene concentrations, has been shown to decrease the risk of prostate cancer. The potential rewards of repeating such feat of cofactor engineering and successfully incorporating them into industry practices are innumerable.[4][9][10]

Many important industrial enzymes use cofactors to catalyze reactions. By using cofactors to manipulate metabolic pathways, it is possible to reduce material cost, eliminate steps in production, reduce production time, decrease pollution, and increase overall production efficiency. One case that demonstrates several of these manufacturing benefits involves the genetic engineering of aspen trees. In the paper production process, manufacturing plants must break down lignin, a biochemical compound that gives a tree trunk its stiffness, in order to form the pulp used throughout the rest of production. The chemical pulping process requires the manufacturing plant to use a significant amount of energy, as well as many expensive and toxic chemicals. A group of genetic engineers, through cofactor engineering, engineered a genetically superior aspen tree that produced less lignin. These genetically engineered trees have allowed for paper mills to reduce their costs, pollution, and manufacturing time.[1][11]