The Role of Enoyl Reductase in the Monacolin K Biosynthesis Pathway in Monascus spp

Abstract

Monacolin K (MK), a secondary metabolite produced by Monascus spp. with the ability to inhibit cholesterol production, is structurally identical to lovastatin produced by Aspergillus terreus. In the lovastatin biosynthetic pathway, the polyketide synthase (PKS) encoded by lovB must work together with the enoyl reductase encoded by lovC to ensure lovastatin production. However, it is unclear whether mokA and mokE in the MK gene cluster of Monascus spp., both of which are highly homologous to lovB and lovC, respectively, also have the same functions for MK biosynthesis. In the current study, the high-yielding MK M. pilosus MS-1 was used as the research object, and it was found that the enoyl reductase domain of MokA may be non-functional due to the lack of amino acids at active sites, a function that may be compensated for by MokE in the MK synthesis pathway. Then, the mokE-deleted (ΔmokE), -complemented (ΔmokE::mokE), and -overexpressed (PgpdA-mokE) strains were constructed, and the results showed that ΔmokE did not produce MK, and ΔmokE::mokE restored MK synthesis, while the ability of PgpdA-mokE to produce MK was increased by 32.1% compared with the original strain MS-1. These results suggest that the MokA synthesized by Monascus spp. must be assisted by MokE to produce MK, just as lovastatin produced by A. terreus, which provides clues for further genetic engineering to improve the yield of MK in Monascus spp.

Keywords: Monascus pilosus; biosynthetic pathway; gene cluster; monacolin K.

Figure 1

Comparison of monacolin K gene clusters in A. terreus and M. pilosus.

Figure 2

The biosynthetic pathway of monacolin K. MokA: polyketide synthase; MokB: polyketide synthase; MokC: P450 monooxygenase; MokD: oxidoreductase; MokE: enoyl reductase; MokF: transesterase.

Figure 3

Domains of MokA from M. pilosus MS-1 and LovB of A. terreus. KS: ketoacyl synthase; AT: acyltransferase; DH: dehydratase; C-MeT: carbon methyl transferase; ER: enoyl reductase (ER* means inactive); KR: ketoreductase; ACP: acyl carrier protein; CON: condensation (participate in the Diels–Alder reaction during the synthesis of nonaketide from MokA and MokE).

Figure 4

Alignments of ER domains of MokA and LovB to those of MokE and LovC. α: α helix; β: β fold; xL1 (151–162) and xL2 (290–301): the extension loop; nucleotide-binding region (166–215): nicotinamide adenine dinucleotide phosphate (NADP)-binding region; GXXTXXA (172–178): the conserved motif. The color of the background is intended to correspond to the color of the comment.

Figure 5

Three-dimensional structure diagrams of LovC and MokE. (a) Three-dimensional structure diagram of LovC; (b) three-dimensional structure diagram of MokE; (c) comparison of the overlay of the 3D structure of LovC and MokE.

Figure 6

Schematic diagram of the mokE deletion strain and PCR validation diagram. (A) Schematic diagram of the homologous recombination strategy of the mokE deletion strain. (B) PCR validation of mokE deletion vector constructed by seamless cloning. Lane 1: 5’ flanking region of mokE, Lane 2: hph gene fragment, Lane 3: 3’ flanking region of mokE. (C) PCR validation image of mokE deletion strains. Using 3 pairs of primers, PCR amplification revealed different fragments in different strains. Lane 1: MS-1 strain; Lane 2: ΔmokE strain.

Figure 7

Schematic diagram of the mokE complementation strain and PCR validation diagram. (A) Schematic diagram of the homologous recombination strategy of an mokE complementation strain. (B) PCR validation of mokE complementation vector constructed by seamless cloning. Lane 1: 5’ flanking region of mokE (containing mokE open reading frame region), Lane 2: TtrpC fragment, Lane 3: neo gene fragment, Lane 4: 3’ flanking region of mokE. (C) PCR validation image of mokE complementation strains. Using 5 pairs of primers, PCR amplification revealed different bands in different strains. Lane 1: ΔmokE strain; Lane 2: ΔmokE::mokE strain.

Figure 8

Schematic diagram of the mokE overexpression strain and PCR validation diagram. (A) Schematic diagram of the homologous recombination strategy of an mokE overexpression strain. (B) PCR validation of mokE overexpression vector constructed by seamless cloning. Lane 1: 5’ flanking region of mokE, Lane 2: neo gene fragment, Lane 3: PgpdA gene fragment, Lane 4: 3’ flanking region of mokE (containing mokE open reading frame region). (C) PCR validation image of mokE overexpression strains. Using 4 pairs of primers, PCR amplification revealed different bands in different strains. Lane 1: MS-1 strain; Lane 2: the putative PgpdA-mokE strain.

Figure 9

Transcriptional level analysis of key genes in MK gene clusters of ΔmokE, ΔmokE::mokE, and PgpdA-mokE. The expression level of the corresponding gene in MS-1 is 1, which is represented as y = 1.

Figure 10

Transcriptional level analysis of key genes in MPs gene clusters of ΔmokE, ΔmokE::mokE, and PgpdA-mokE. The expression level of the corresponding gene in MS-1 is 1, which is represented as y = 1.

Figure 11

MK produced by M. pilosus MS-1, ΔmokE, ΔmokE::mokE, and PgpdA-mokE. Error bars represent the standard deviation between the three replicates. * means p < 0.05; ** means p < 0.01.

Figure 12

MPs produced by M. pilosus MS-1, ΔmokE, ΔmokE::mokE, and PgpdA-mokE. The yields of yellow MPs (a), orange MPs (b), and red MPs (c) of the strains were detected at 380, 470, and 520 nm. Error bars represent the standard deviation between the three replicates. * means p < 0.05; ** means p < 0.01.