L-lactic acid production by Aspergillus brasiliensis overexpressing the heterologous ldha gene from Rhizopus oryzae.
BACKGROUND
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. 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.


RESULTS
To 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.


CONCLUSIONS
We 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.
INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. nadege.liaud@univ-amu.fr.|Aix-Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. nadege.liaud@univ-amu.fr.|ARD Agro-Industrie Recherche et Développement, Route de Bazancourt, 51110, Pomacle, France. nadege.liaud@univ-amu.fr.
Nadège
N
Liaud
INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. marie-noelle.rosso@univ-amu.fr.
Marie-Noëlle
MN
Rosso
ARD Agro-Industrie Recherche et Développement, Route de Bazancourt, 51110, Pomacle, France. n.fabre@a-r-d.fr.
Nicolas
N
Fabre
ARD Agro-Industrie Recherche et Développement, Route de Bazancourt, 51110, Pomacle, France. s.crapart@a-r-d.fr.
Sylvaine
S
Crapart
INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. Isabelle.gimbert@univ-amu.fr.|Aix-Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. Isabelle.gimbert@univ-amu.fr.
Isabelle
I
Herpoël-Gimbert
INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. jean-claude.sigoillot@univ-amu.fr.|Aix-Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. jean-claude.sigoillot@univ-amu.fr.
Jean-Claude
JC
Sigoillot
INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. sana.raouche@univ-amu.fr.|Aix-Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, Polytech' Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille, Cedex 09, France. sana.raouche@univ-amu.fr.
Sana
S
Raouche
Aix-Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, INSERM U1095, IHU Méditerranée Infection, Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille, Faculté de Médecine, 27 Bd Jean Moulin, 13005, Marseille, France. anthony.levasseur@univ-amu.fr.
Anthony
A
Levasseur
D001230:Aspergillus / Q000378:metabolism; D015870:Gene Expression; D019344:Lactic Acid / Q000378:metabolism*; D011091:Polyesters; D011108:Polymers / Q000378:metabolism*; D012233:Rhizopus / Q000235:genetics*
D011091:Polyesters; D011108:Polymers; D019344:Lactic Acid; C033616:poly(lactide)