Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

Abstract

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.

Fig 1. Metabolic fates of pyruvate in A. niger.

All enzymes catalyzing the reactions with pyruvate (as substrate or product) are depicted and the pathway shown with dotted arrow is from A. nidulans. ALT- alanine transaminase, LDH- lactate dehydrogenase [40], MAE- malic enzyme [41], PDC- pyruvate decarboxylase, PDH- pyruvate dehydrogenase complex, PKI- pyruvate kinase [42] and PYC- pyruvate carboxylase [43]. Respective K _m values for the enzymes are given in brackets. These are either taken from Aspergillus literature or for the corresponding yeast enzymes from BRENDA (www.brenda-enzymes.org).

Fig 2. Analysis of mldhA transformants by activity staining, genomic PCR and qPCR: a—Activity staining for LDH from A. niger and six of its transformants.

Desalted cell-free extract (5 μg protein each) was loaded on polyacrylamide gels. Purified rabbit skeletal muscle LDH served as positive control. b—PCR amplification of integrated PcitA- mldhA DNA from the transformants. Parent A. niger genomic DNA and pCBXCmldh served as negative and positive controls, respectively.c—PcitA-mldhA copy number determination in transformants by qPCR. The mldhA copy number was determined using the single copy A. niger actin gene as reference. The data are plotted as mean values obtained from different concentrations of genomic DNA. Error bars represent the standard deviation.

Fig 3. LDH specific activity and lactate and citrate levels in mldhA transformants: Extracellular (Panel a) and intracellular (Panel b) concentrations of L-lactate (filled circles) and citrate (open circles) are shown.

Data in panel b is an average of two measurements. Each LDH specific activity data point represents one individual transformant (see Table 3). Arrow indicates the data for C12 strain.

Fig 4. Effect of increasing medium glucose on the formation of lactate and citrate by A. niger C12 strain: Extracellular (Panel a) and intracellular (Panel b) concentrations of the two acids from C12 strain (circles) and the parent (squares) are shown.

Filled symbols represent L-lactate and open symbols represent citrate. Data are for 24 h growth.

Fig 5. Lactate formation from glucose by A. niger C12 strain: The shake flask growth over 7 days was performed and biomass (Panel a; wet weight), medium glucose (Panel b), medium pH (Panel c), LDH specific activity (Panel d) and extracellular (Panel e) and intracellular (Panel f) concentrations of the two acids are shown.

In panels a, b, c and d, filled triangles represent data for C12 strain while open triangles represent data for the parent strain. The symbols in panels e and f mean the same as in Fig 4.