Mutant strain lacking G6PD (glucose-6-phosphate dehydrogenase) showed no difference in rates of glucose
utilization, gellan production or CO2 production suggesting that this enzyme is not essential for glucose
metabolism in Sphingomonas. This indicates that either the main route of glucose utilization involves glucose dehydrogenase or gluconate kinase, or the absence of G6PD induces a compensatory increase in these enzymes. As yet, however, there is no clear indication of which mechanism occurs.
Martins and Sá-Correia (19) proposed a possible pathway for the synthesis of repeating tetrasaccharide
unit of gellan gum. They assumed that gellan synthesis requires activated precursors before the repeating unit is
assembled, similar to other exopolysaccharides synthesized in the cell wall of microorganisms. These gellan precursors were detected by enzyme assays, and were found to be nucleotide diphosphate sugars, viz. UDP-
-glucose, TDP-rhamnose and UDP-glucuronic acid. The proposed biosynthetic pathway is shown in Fig. 2.
Glucose-6-phosphate seems to occupy a key position from which two routes commence, one leading to
uridine-5-diphosphate glucose (UDPG) and the other leading to thymine-5-diphosphate glucose (TDPG). In
turn, UDPG induces D-glucose and D-glucuronic acid synthesis and TDPG leads to the synthesis of rhamnose.
The combination of these three compounds presumably results in the synthesis of gellan (22). However, the reactions leading to binding of these three monomers have not been clearly elucidated.
Specific activities of gellan synthetic enzymes
Conditions that favour gellan gum formation might be expected to increase the levels of the enzyme responsible for the formation of precursors. Phosphoglucose isomerase (PGI) and phosphoglucose mutase (PGM)
possess the highest activities in cell-free extracts (in vitro) as they play multiple roles in the cell metabolism.
The enzymes UDPG phosphorylase (UGP) and TDPG phosphorylase (TGP) appeared to have values of specific activities lower than PGI and PGM. TDPR synthetase (TRS) and UGD are the least active and the most thermosensitive enzymes above 30 °C. They are essential for synthesis of rhamnose (TRS) and glucuronic acid (UGD).The activity of these enzymes presumably limits gellan synthesis, especially at temperatures higher than 30 °C
Additionally, it has been found that isocitrate isomerase, which is involved in CO2 production through the carboxylic acid cycle, has very high specific activity in vitro. This might represent an unfavourable reaction
for industrial purposes, because glucose is not channelled towards gellan gum production. Apart from the enzymes mentioned, there must be other enzymes that influence gellan synthesis after the formation of gellan
precursors.
Genetic engineering of the gellan pathway
The most exciting prospects for gellan modification and increasing production yield are found in genetic engineering. Some attempts have been made to increase the relatively low conversion efficiency of gellan from
glucose in S. paucimobilis ATCC 31461.
By site-specific mutagenesis, the G6PD gene encoding glucose-6-phosphate dehydrogenase was inactivated, envisaging diversion of the carbon flow toward gellan synthesis, apparently without the expected results.
Identification of a few genes and elucidation of crucial steps of the gellan biosynthesis pathway indicated
some possibilities of exerting control over gellan production at any of the three levels of its biosynthesis:
(i)at the level of synthesis of sugar-activated precursors,
(ii) at the level of the repeat unit assembly and of gellan,
(iii) at polymerization and export. By modifying expres- sion of any of the individual, or of a group of these
genes the conversion efficiency and gellan gum yield can be increased.
In spite of recent advances in the elucidation of the gellan biosynthetic pathway, a better knowledge of the
poorly understood steps and of the regulation and bottlenecks of the pathway is crucial for the eventual success of the metabolic engineering of gellan production.
