A procedure for removal of cyanuric acid in swimming pools using a cell-free thermostable cyanuric acid hydrolase.

Cyanuric acid (CYA) is used commercially for maintaining active chlorine to inactivate microbial and viral pathogens in swimming pools and hot tubs. Repeated CYA addition can cause a lack of available chlorine and adequate disinfection. Acceptable CYA levels can potentially be restored via cyanuric acid hydrolases (CAH), enzymes that hydrolyze CYA to biuret under mild conditions. Here we describe a previously unknown CAH enzyme from Pseudolabrys sp. Root1462 (CAH-PR), mined from public databases by bioinformatic analysis of potential CAH genes, which we show to be suitable in a cell-free form for industrial applications based upon favorable enzymatic and physical properties, combined with high-yield expression in aerobic cell culture. The kinetic parameters and modeled structure were similar to known CAH enzymes, but the new enzyme displayed a surprising thermal and storage stability. The new CAH enzyme was applied, following addition of inexpensive sodium sulfite, to hydrolyze CYA to biuret. At the desired endpoint, hypochlorite addition inactivated remaining enzyme and oxidized biuret to primarily dinitrogen and carbon dioxide gases. The mechanism of biuret oxidation with hypochlorite under conditions relevant to recreational pools is described.

Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene.

There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C5H8), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C10 and C15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii, used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation.IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii, an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway.

Metabolic engineering for the microbial production of 1,3-propanediol.

Improvements in the biological production of 1,3-propanediol, a key component of an emerging polymer business, have been realized. Utilizing genes from natural strains that produce 1,3-propanediol from glycerol, metabolic engineering has enabled the development of a recombinant strain that utilizes the lower cost feedstock D-glucose. This accomplishment bodes well for future metabolic engineering efforts and, ultimately, for increased societal benefit obtained through the production of chemicals from renewable resources.