During the course of this PhD new methods have been developed for the high throughput screening to determine the lignin degrading ability of micro- organisms. Application of these assays has led to the identification of R. jostii
RHA1, Acetinobacter PC/4 and a number of S. coelicolor strainsas being lignin degraders. In addition, the assays allowed the identification of the peroxidase DypB, the first recombinant bacterial enzyme shown to degrade lignin. The nitrated lignin and fluorescence assay represent a step forwards in lignin degradation assays, as they are fast and more specific than conventional general peroxidase assays.
In the future there are further opportunities for the assays developed here. Currently in other projects within the Bugg group the nitrated lignin assay is being employed to help purify more lignin degrading proteins, with ongoing studies focusing on R. erythropolis and R. jostii RHA1, as well as new lignin- degrading bacteria. The solid agar plate assay is currently being employed to screen for new degrading strains from geothermal soils and composted straw (with aim of finding thermophilic strains).
The studies of the genetic knockouts of R. jostii RHA1 showed a potentially interesting laccase encoded for by the gene ro2377. This enzyme is currently undergoing further studies in our group. As postulated in Chapter V its activity
towards lignin seems low, but it is possible that this could be enhanced by the selective mutations to increase its redox potential. Alternatively, it could be that the use of mediators or other supporting enzymes may increase its activity.
It was shown that growing R. jostii RHA1 and P.putida on lignocellulose gave rise to a number of small organic compounds. These included oxalic acid and ferulic acid for which there are already established commercial markets. This highlights that there is potential in the use of bacteria to valorise lignin. However, these studies were hampered by the formation of a wide mixture of products similar to the results of chemical degradation.23 In the studies on DypB it was found that product formation could be controlled by addition of the enzyme diaphorase. It is particularly interesting that vanillin could be formed. This is a potential tool to access higher yields of products of interest. In addition, current studies in our group are also looking at using enzyme inhibitors to interfere with the degradation of aromatic compounds by bacteria and then examining changes in the products formed using the methods developed in Chapter IV.
Figure 7.1 Metabolism of vanillin could possibly be stopped by the general aldehyde dehydrogenase inhibitor disulfiram leading to an increase in the amount of vanillin in the culture
Evidence has been gathered to suggest that bacteria have a specificity for particular types of lignin. This is a new observation with little prior discussion in the literature.81 At a molecular level it was shown that DypB was specific for - aryl ether 27, such enzymes specificities may be responsible for the specificity of the parent organism. The preference of different micro-organisms for different feedstocks should be considered carefully when designing potential processes for a biorefinery. There is need for ongoing work to look at variation in product formation with different lignin types as this too will have a significant effect on what material and micro-organism will be best for industrial application. The nitrated lignin assay is well suited to screening a set of lignin degraders against a range of plant feedstocks (e.g. varieties of wheat).
The cellular location of DypB must logically be extracellular, since activity is seen with the R. jostii RHA1 supernatant but not with the supernatant from the DypB knockouts. The protein sequence for Rhodococcus jostii RHA1 DypB contains neither a Sec signal sequence nor a Tat signal sequence, though DypB homologues in some other bacteria do contain possible Tat signal sequences.151 As discussed in chapter V the gene encoding for DypB is immediately adjacent to the gene encoding encapsulin in R. jostii RHA1. It is probable that the transportation of DypB to the outside of the cell is related to encapsulin but the mechanism for this is unknown at this time.
In nature DypB is probably one of a suite of enzymes, as proposed in chapter IV, hence its modest activity towards lignin in vivo (Figure 6.6). This is analogous to the fungal system and without all of the pieces lignin degrading
activity can be expected to be low.78 Despite this DypB represents the first recombinant bacterial lignin peroxidase to be kinetically characterised.
The observation of oxalic acid in the culture media of bacteria grown on lignocellulose offers the first explanation of how the hydrogen peroxide for lignin degradation is generated in bacteria. Previous work on the fungal system has shown that oxalic acid is the final product of a series of oxidations each of which reduces oxygen to hydrogen peroxide.78
This PhD has produced a number of tools for investigating lignin degradation, it has also identified a number of new lignin degrading bacterial strains. It has identified a number of products formed by R. jostii RHA1 and P. putida from lignin. It has also provided the first detailed characterisation of a recombinant bacterial protein. It is hoped that the tools developed here will be of further help to develop methods of using bacteria to form renewable chemicals.