Abstract
BackgroundLactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production.ResultsTo investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 × 10−2 U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan.ConclusionWe obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed enabled a 1.8-fold increase in conversion yields. The strain produced lactic acid from plant biomass. Our findings make A. brasiliensis a strong contender microorganism for low-pH acid production from various complex substrates, especially hemicellulose.
Highlights
Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics
Selection of transformants in ammonium medium without pH regulation After co-transformation of A. brasiliensis BRFM103 with the lactate dehydrogenase A gene and the hygromycin resistance gene, isolated transformants were screened for L-lactic acid (L-LA) production in MM1 liquid medium without pH regulation. pH of the medium dropped from 5 to 2.5 in 40 h slowly decreased to a final pH of 1.6 at 138 h culture
For the transformants exhibiting L-LA conversion yields higher than 23% (w/w), ethanol production was nearly abolished with final conversion yields below 1% (w/w), suggesting that the heterologous lactate dehydrogenase A gene (ldhA) efficiently compete with native pyruvate decarboxylase (PDC) for pyruvate utilization in high L-LAproducing strains
Summary
Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. They have never been used for lactic acid production. Lactic acid (LA) is a colorless, odorless monocarboxylic acid naturally produced by many organisms This weak acid has low buffering power and is tasteless, and has found broad use as an excipient in the food, cosmetics, pharmaceutical and chemical industries [1,2]. Low-pH production processes that do not require neutralizing chemicals allow undissociated (free) LA to be recovered directly from the broth without any acidification step in the purification process [1]
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