Genetic manipulation of Lactobacillus to produce optically pure lactic acid following fermentation using hydrolysed organic fraction of municipal solid waste as a feedstock

Lactic acid for polymer applications must be optically pure to allow the final plastic properties to be controlled. We have isolated a strain of Lactobacillus plantarum capable of utilising a range of carbohydrates and tolerant to high sugar and organic acid concentrations. However, like most Lactobacillus plantarum, this strain produces D- and L-lactic acid. To improve the commercial application of this strain gene editing has been used to ensure only one isoform is produced.

Using two-step homologous recombination, ldhL was replaced with a truncated version interrupted by a chloramphenicol acetyltransferase resistance marker, then with an unmarked truncated version.

The optical purity shift was quantified biochemically by enzymatic assay and HPLC and fermentation performance compared against the unmodified strain using enzymatically hydrolysed municipal solid waste pulp (MSW sugars) as a feedstock.

The ldhL gene was successfully deleted, leading to a dramatic shift in the optical purity of lactate produced in both synthetic media and MSW sugars. The unmodified strain produced D-lactic acid with an optical purity of 40%, while the mutant achieved 94%. In MSW sugars, the modified strain performed as well as the unmodified control, consuming all available glucose in less than 48 hours with a volumetric productivity of 1.02 g/L/h.

Deletion of ldhL from our strain of Lactobacillus plantarum allowed exclusive production of D-lactic acid from MSW sugars. This provides a laboratory scale demonstration of the production of optically pure lactic acid from renewable sugars for use in the production of biodegradable plastics.