Direct Fermentation of L-Lactic Acid from Starch By Genetically Engineered Aspergillus Oryzae

Lactic acid can be produced biologically, and it is a source of polylactate. Polylactate has been a focus of recent studies for its use as a biodegradable alternative to petroleum-based plastic products. Nowadays, bio-based lactic acid is produced from starchy biomass, while bioproduction by lactic acid bacteria require for utilization of expensive amylolytic enzymes. A filamentous fungus Aspergillus oryzae has been used in Japan for more than 1,000 years for the production of fermented foods such as sake, miso, and soy sauce, and it produces various amylolytic enzymes. Therefore, we attempted to examine bioproduction of lactic acid from starch by A. oryzae.

Lactic acid-producing A. oryzae strains were constructed by genetic engineering. The bovine (Bos taurus) L-LDH-A gene sequence was modified based on the codon usage of A. oryzae and synthesized. The synthesized artificial gene fragment was cloned into a plasmid vector pIS1, which contains the sodM promoter and glaB terminator from A. oryzae. Using the resulting plasmid, A. oryzae btLDH which expresses the bovine L-lactate dehydrogenase gene was constructed. Similarly, L-lactate dehydrogenase genes from other organisms, a lactate-producing filamentous fungous Rhizopus oryzae and lactic acid bacteria Lactobacillus casei, were synthesized and then transformants were constructed. Furthermore, an authentic lactate dehydrogenase gene (Ao-ldhA) and pyruvate decarboxylase gene (pdc) on A. oryzae chromosome were disrupted by replacing with pyrG fragment.

A. oryzae btLDH was selected from the transformant pool because it showed highest titer in the transformants constructed. It produced 38 g/L of lactate from 100 g/L of glucose. To improve the lactate titer, Ao-ldhA disruption mutant A. oryzae btLDHΔ871 was constructed and it produced approximately 50 g/L of lactate from 100 g/L of glucose. Furthermore, pdc disruption mutant A. oryzae btLDHΔPDC was also constructed, because ethanol was simultaneously produced during the cultivation. This strain produced approximately 50 g/L of lactate from 100 g/L of glucose.

Next, direct fermentation of lactate from starch using these strains was examined, because A. oryzae strains innately secrete amylases. A. oryzae btLDHΔ871 and A. oryzae btLDHΔPDC produced approximately 30 g/L and 40 g/L of lactate from 100 g/L of soluble starch, respectively. Although the both strains showed titers at the same in the fermentation from glucose, A. oryzae btLDHΔPDC showed higher titer compared with A. oryzae btLDHΔ871.

From these results, the disruption of pdc gene, that is disruption of ethanol production pathway, improved the lactate production in the genetically engineered A. oryzae. However, the titer and yield are lower than those of other lactic acid producers. Now we attempt to improve the titer and yield by constructing strains which a carbon flow into a TCA cycle is slowed down and the conversion of lactate from pyruvate is accelerated.