1). The author believes that microbiology will lead the way in meeting this challenge by identifying and isolating stable enzymes from extremophiles, resulting in a much more efficient metabolic pathway that operates at higher temperatures. Such a pathway would use extremophile enzymes as a starting template and culminate with an elegantly engineered pathway, one in which every nuance could be tweaked for maximum efficiency and proven by mathematical analysis of weight-based turnover in terms of product yield and catalyst consumption.
As the author delineates the process of SyPaB, he simultaneously pinpoints its inherent advantages. The absence of a living cell reduces all metabolic processes other than those that yield the desired product. Such a system design, independent of living cells, not only performs more efficiently, but also is simpler to modify and manipulate than living organisms, given its freedom from physiology, cell maintenance, replication, reaction regulation, membranes, cell walls, etc., etc. In addition, carbon emissions could very nearly be lowered to zero, while yielding 12 hydrogen molecules per glucose molecule (compared to the 4 free hydrogen molecules theoretically produced by natural biological pathways). A hypothetical reaction sequence explains the means for such enhanced hydrogen and limited carbon yield. .
SyPaB involves a five-step development process "(i) pathway reconstruction, (ii) enzyme selection, (iii) enzyme engineering, (iv) enzyme production and purification, and (v) process engineering" (p. 2). The author states that enzyme selection is of particular importance and that the issue of stability necessitates a reliance on extremophiles. Two specific enzymes that have been identified as thermostable building blocks of a potential SyPaB are named in the article, both derivatives of thermophiles, clostridium thermocellum and Thermotoga martima. These enzymes can functionally withstand temperatures of 60 degrees Celsius, which makes them extremely stable and valuable catalysts.