Here’s another highlight from the Cyanobacterial Workshop last week. It comes from the Bryant Lab at Penn State. The cyanobacterium Synechococcus sp. 7002* is a marine strain isolated from fish pens in Puerto Rico. It is a particularly attractive cyanobacterial strain to work with as far as developing engineered derivatives for various applications. It is a great genetic system, which means making modifications can be done quickly and easily. Also, it tolerates a range of growth conditions from brackish to marine water and is very tolerant of high light intensities. Under optimal conditions, 7002 has the fastest doubling time (the time it takes for the number of cells in a culture to double) of all cultured cyanobacteria. It clocks in at about 3.5 hours, while all other characterized strains have doubling times well into the double digits. However, 7002 has one unfortunate weakness; it requires the addition of cobalamin (aka vitamin B12) to its growth medium because the cells cannot manufacture it on their own. While this may not be a deal-breaker for the use of 7002 as a lab organism, when you start talking about using it for biotech purposes for which culture volumes are in hundreds of gallons or cubic meters of raceway ponds, this is a serious problem. Adding vitamin B12 on that scale becomes prohibitively expensive.
Adam Perez presented a poster (#62) and a short talk on his work tinkering with 7002’s cobalamin requirement. As it turns out, there is only one enzyme, called metH, that requires this cofactor. It is used for the synthesis of L-methionine, a molecule involved in a number of essential cell functions. Other cyanobacteria do not have this requirement because they have an additional enzyme, called metE, which does not use cobalamin. Perez showed that he could swap out 7002’s metH gene for another cyanobacterium’s metE gene and create a cobalamin-independent 7002 strain. This means that the metE-containing 7002 strain doesn’t need this additive in the media, which opens up new possibilities of 7002 on the application scale.
The feat described above is innovative on its own, but Perez didn’t stop there. Perez and co-workers used a regulatory strategy of metE to learn more about cobalamin transport in 7002. Bacteria must have specific transporters at their membranes to take up useful molecules like cobalamin, metals, and sugars. Because the native strain of 7002 requires cobalamin, it must have transporters to take it into the cell, but researchers never knew which genes were actually responsible for this activity. Here’s how they did it. Some cyanobacteria, like the metE donor strain used in this study, have both metE and metH. These genes are tightly controlled by the presence of cobalamin. When cobalamin is present, the metE gene is turned off so that the metH gene can be used. Perez et al. took advantage of this gene regulatory feature and used it as the basis for a screen. If a fluorescent protein is placed under the control of the metE regulatory sequence, then when cobalamin is present, fluorescence will not be detected. On the other hand, when cobalamin is absent, then fluorescence will be detected. So, if there is a mutation in the cobalamin transport machinery, then these cells would always have fluorescence whether cobalamin was added to the medium or not. That’s exactly what they looked for in a screen of random mutants with the fluorescent reporter system. They were able to find genes encoding a cobalamin transporter at the cell membrane.
So now we know how this cyanobacterial strain takes its vitamins and how to eliminate their requirement for this vitamin.
*Synechococcus (sin-eh-ko-cock-us) is too much to say over and over (not to mention typing), so let’s just abbreviate it 7002.