Currently we are dependent on petroleum for the production of a wide range of bulk chemicals and structural materials like polyesters. Considering the depleting petroleum sources, instable political situations in petroleum exporting countries and the increasing concerns on environmental impact, the 21st century can no longer be dependent on petroleum as the only raw material for production of bulk chemicals and structural materials.

The major interest of the present study is a new bio-originated route for the production of polyesters to replace existing petrochemical routes. So far, the best known biological polyesters are the poly-hydroxyalkanoates (PHAs) which can contain  3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate  etc., and which can have application ranges from hard crystalline plastics to very elastic rubbers by varying the monomer composition. Although microorganisms can produce these polymers at high yields, the extraction of the polymer requires cell disruption. Therefore, in current study the focus is on production of the excreted monomers. The selected model compound is 4-HB and the selected final product is GBL (the lactone of 4-HB).  During the study the 4-HB production pathways are constructed.

Three possible metabolic pathways from glucose to 4-HB have been considered. The last step, in which succinic semialdehyde is reduced to 4-HB, is common in all three pathways. Pathway (1) is via 2-oxoglutarate. Glutamate dehydrogenase converts 2-oxoglutarate into L-glutamate, which is consecutively converted into 4-aminobutyrate and CO₂ by glutamate decarboxylase. In the next step, 4-aminobutyrate is converted to succinic semialdehyde (SSA). Pathway (2) uses a direct conversion of 2-oxoglutarate into SSA and CO₂ by 2-oxoglutarate decarboxylase. In pathway (3) succinic semialdehyde dehydrogenase converts succinate to SSA.

Equilibrium calculations revealed that for the physiological operation conditions (pH 6.5-7 and NAD(P)⁺/NAD(P)H=10-25) the yield of 4-HB will be  low (5-10%) and succinic semialdehyde (precursor of 4-HB) accumulates in the cell. However, equilibrium values for related reactions indicate that such accumulation of aldehyde is improbable. When the cofactor couple is NADP⁺/NADPH, it is seen that for the same pH the yields of 4-HB on converted citrate increases significantly for physiological cofactor ratios (0.1-10) when compared to NAD⁺/NADH couple.  Conversions up to 100% can be obtained by NADP⁺/NADPH couple.

Lactonization experiments revealed that Candida antactica Lipase B has significant lactonization activity for 4-HB at pH 4 and 60 ºC. However the conditions point out in-vitro rather than in-vivo lactonization.

The results of the theoretical study indicates that more detailed experimental studies are required to determine the feasibility of biochemical production of 4-HB from renewable resources.

This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability).