Editor | On 30, Dec 2018

Darrell Mann

We head to Texas for our patent of the month this month, to a trio of inventors at William Marsh Rice University. Their invention was granted as US10,011,839 on July 3rd. Here’s what they have to say about the problem to be solved:

All living creatures, be it man or the smallest bacteria have one function in common known as respiration. During respiration, two important functions are performed in living things. In the first, electrons that were generated during catabolism are disposed of and in the second, ATP (also known as adenosine tri-phosphate) is produced to provide energy for the cell.

There are two types of respiration: (i) aerobic respiration and (ii) anaerobic respiration. Aerobic respiration requires oxygen, but oxygen is not required for anaerobic respiration, often called “fermentation” in bacteria. Instead, other less-oxidizing substances such as sulfate (SO.sub.4.sup.2-), nitrate (NO.sub.3.sup.-), sulfur (S), or fumarate are used. These terminal electron acceptors have smaller reduction potentials than O.sub.2, meaning that less energy is released per oxidized molecule. Anaerobic fermentation is, therefore, energetically less efficient than aerobic respiration. Nonetheless, it has value and allows the cells to continue living even with no or reduced O.sub.2.

Anaerobic fermentation and aerobic respiration have been the two metabolic modes of interest for the industrial production of chemicals from microbes such as E. coli, Lactobacillus and yeast. Oxygen rich respiration offers very efficient cell growth (growth rate and yield) and converts a high percentage of the carbon source into carbon dioxide and cell mass (see Table 1). Anaerobic fermentation, on the other hand, results in poor cell growth and the synthesis of several fermentation products at high yields (e.g. lactate, formate, ethanol, acetate, succinate, etc.).

However, producing chemicals via oxygen rich processes costs much more than using anaerobic methods for two reasons. First, aerobic fermenters are more expensive to build, due to both the higher cost per unit and the need for smaller fermenters with reduced economy of scale. Secondly, the aerobic fermenters are more costly to operate than their anaerobic counterparts due to low solubility of oxygen, which in turn requires high energy input to ensure appropriate supply of oxygen to the cells. This is especially relevant for the production of commodity chemicals, where fermentation costs can represent 50-90% of the total production cost.

However, there is still a need to maximize chemical production, while maintaining robust cell growth, and optimizing production yields. One way of optimizing yield is to directly attempt to increase the genes resulting in the desired product. Another method, would be to directly downregulate a competitive pathway. However, direct methods have limitations, can be difficult to fine tune, and are often not satisfactory. We introduce herein indirect methods of influencing flux, which are amenable to fine tuning.

So, the desire here is to create a solution that maintains the main attractions of anaerobic production (low cost), whilst managing to overcome its primary drawbacks (low growth rates). Sounds like classic contradiction territory:

And here’s how the inventors have solved the conflict:

We provide herein a completely novel strategy to finely control a metabolic flux in a microbe by using a “metabolic transistor” approach. In this new approach, the pathway of interest converts glucose or other carbon source into a desired product P. However, a competitive pathway C, that uses cofactor F, competes for the same carbon sources, thus reducing the level of P that can be produced when C is active. If we add in certain diverting genes [Principle 24] to compete with the competitive pathway C, it will allow increased P to be formed. Generally speaking, the diverting gene(s) will either directly compete for F (degrading it or using it for its own reactions) or will utilize a precursor of F. Cofactor F thus becomes rate limiting, slowing the competitive pathway C, and allowing more carbon to flow to P, and resulting in increasing levels of P.

The competitor flux is dependent on the presence of a cofactor or other key intermediate that is required for the competitive pathway, but not for P. Since the key intermediate is normally present in large enough amounts and may even have redundant means for its biosynthesis or uptake, it is not generally considered a key control point. However, if controlled in a fine-tuned amount around a threshold level [Principle 12] , dramatic effects may be seen with a very small perturbation of the available level of the key intermediate. Thus, the addition of diverting pathways for the cofactor can allow fine control over the product pathways.

The principles of the current invention are:

1) The metabolic transistor concept that a competitive metabolic flux can be controlled by small changes in the level or availability of a cofactor require for the competitive flux (but not for P);

2) Cofactor levels in the competitive flux can be modulated by adding an exogenous diverting reaction [Principle 9] that either directly reduces available cofactor or indirectly reduces a needed precursor for the cofactor;

3) Controlling the level of a competitive pathway (by controlling cofactor levels) with the added diverting gene(s) allows increased carbon flow though the desired pathway to P.