Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables a Significant Increase in Citric Acid Production

Abstract

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Keywords: Aspergillus niger; acetyl-CoA; citrate; phosphoketolase.

Figure 1

Roles of ACS in the synthesis of acetyl-CoA and citric acid in A. niger. (A) The metabolic pathway of citric acid in A. niger; the black arrows represent the canonical pathway of citric acid metabolism, the blue arrow means the putative cytosolic citrate synthesis pathway, the red arrows indicate the putative complementary pathway of cytosolic acetyl-CoA and the green arrow indicates that acetic acid addition can supplement the cytosolic acetyl-CoA in the acl-deletion strain [7]. (B) The concentrations of extracellular citric and acetic acid and intracellular acetyl-CoA of S469, S688 and S1422 during a 3-day citrate fermentation in a shake flask at 28 °C and 200 rpm. CT: complementation of ACK driven by the gpdA promoter at the amyA locus of S688; ACK: acetate kinase; ACS: acetyl-CoA synthetase; CitA: mitochondrial citrate synthase; CitB: cytosolic citrate synthase; PK: phosphoketolase.

Figure 2

Roles of ACK, PKA and PKB in the biosynthesis of acetyl-CoA and citric acid of A. niger. (A) the concentrations of extracellular citric and acetic acid and intracellular acetyl-CoA of the S469, S917 (Δack), S2738 (ΔpkA), S2908 (ΔpkB) and S3050 (ΔpkAΔpkB) during 3-day citrate fermentation in a shake flask at 28 °C and 200 rpm; (B) the concentrations of extracellular citric and acetic acid and intracellular acetyl-CoA of the S469, S1724 (Δack, CTack), S3098 (ΔpkAΔpkB, CTpkA) and S3104 (ΔpkAΔpkB,CTpkB) during 3-day citrate fermentation in a shake flask at 28 °C and 200 rpm. CT: complementation of genes driven by the gpdA promoter at the amyA locus.

Figure 3

Reconstruction of the heterologous PK-PTA pathway in A. niger S469. (A) The PK activities and citric acid production of S469, S3050 (ΔpkAΔpkB), S3098 (ΔpkAΔpkB, CTpkA), S3104 (ΔpkAΔpkB,CTpkB), S3081 (ΔpkAΔpkB, CTBl-pk), S3087 (ΔpkAΔpkB, CTBa-pk) and S3092 (ΔpkAΔpkB, CTCa-pk) for screening an efficient PK used for the construction of a heterologous PK-PTA pathway; (B) the reactions of the native ACK-ACS pathway of A. niger and heterologous PTA from bacteria; (C) the PTA activities and citric acid production of S469, S917 (Δack), S2909 (Δack, CTTs-pta) and S2914 (Δack, CTBs-pta) for screening an efficient PTA used for the construction of a heterologous PK-PTA pathway; (D) shake flask citrate fermentation of A. niger S469 and S3158 (OECa-pk, OETs-pta) at 28 °C for 3 d. Note: the activities of PK and PTA were assayed in shake flask citrate fermentation at 28 °C for 3 d. CT: complementation of genes driven by the gpdA promoter at the amyA locus. OE: overexpression of Ca-pk and Ts-pta in S469.

Figure 4

Citric acid fermentation of A. niger S469 and S3158 (OECa-pk, OETs-pta) in a 2 L bioreactor. (A) The citric acid yield, cell dry weight, residual sugar and pH in the citrate fermentation of the 2 L bioreactor at 28 °C for 120 h. (B) Mycelial morphology of S469 and S3158 in the citrate fermentation of the 2 L bioreactor at 28 °C for 120 h.