Accelerating evolution: engineering invertase enzymes to enhance fructan metabolism in Musa spp. (banana)

The equatorial countries of sub-saharan Africa have the highest worldwide mortality rates, primarily caused by infectious disease. Those who are immunosuppressed, including children, the elderly and the sick, are particularly at risk. Improving sanitation, and boosting the immune system with vaccination or improved diet, have been key for continuing to combat this issue.

Short-chain low-digestible carbohydrates (LDCs), act as human prebiotics, selectively stimulating the growth and activity of selected intestinal bacteria, creating a symbiotic relationship between the gut and its microflora. In this way, LDCs act as immunomodulators, and significantly ameliorate the onset of type II diabetes, coronary heart disease, GI diseases, cancers, dental cavities and obesity.

Of particular interest is fructan as a LDC. Fructans have roles in the plant stress response, as both ROS scavengers and cellular stabilizers. Abiotic stress is estimated to be the leading cause of crop loss, exceeding 50% worldwide. Genetic engineering has been suggested as a means of generating hardier plants - for example - heterologous over-expression of a fructosyltransferase in tobacco greatly improved the survival rate of plants submitted to abiotic stresses.

Musa spp. (bananas) are the staple food crop in Uganda, Tanzania, Burundi, Rwanda and other surrounding countries. Banana organs, especially the fruit, contain small inulin-type fructans (kestoses and small inulo-n-oses) as reserve polysaccharides. Preliminary findings suggest that banana lacks genuine fructosyltransferases for the production of longer fructans, but instead produces small fructans via promiscuous vacuolar invertase (VI) enzymes. This suggests that the “fructan syndrome” is already naturally evolving in banana, but residing at a premature stage. It is believed that all fructosyltransferases evolved from VIs within the family 32 of glycoside hydrolases.

The project aim is to identify and characterize the different banana vacuolar invertase enzymes, first by bioinformatic search, and then by heterologous expression in Pichia pastoris. The best fructan producing enzyme, as determined by in vitro assays, will be chosen to further enhance the fructosyltransferase properties of the enzyme through site directed mutagenesis, based on our extended previous expertise on structure-function relationships within family GH32. Mutations will then be introduced into this sequence to enhance the fructosyltransferase properties of the enzyme, for eventual re-introduction into banana, thereby creating a fruit with increased fructan content and hence a better prebiotic and immunomodulatory potential. Such fruits will have the greatest impact in the countries most susceptible to infectious disease where bananas are eaten as a staple crop. This project also provides the groundwork for engineering plants more resistant to abiotic stresses, which could be applied to other crops, as environmental extremes threaten increasing amounts of arable land.

The methods of this project encompass classic molecular biology and biochemistry, with future aims to employ newer techniques, such as CRISPR/Cas9. The research will be carried out in the Plant Molecular Biology lab at the University KU Leuven.