An NADH/NAD+-favored aldo-keto reductase facilitates avilamycin A biosynthesis by primarily catalyzing oxidation of avilamycin C

Abstract

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD⁺ or NADPH/NADP⁺. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD⁺-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD⁺ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD⁺ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD⁺ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.

Keywords: aldo-keto reductase; avilamycins; biosynthesis; branched-chain sugar; rational strain design.

Fig 1

Structure and biosynthetic gene cluster of avilamycins. (A) Avilamycins A and C show their only structural difference in the two-carbon branched-chain of the terminal octose moiety (5). (B) Organization of the clustered genes encoding avilamycin biosynthesis. Gene functions were assigned by bioinformatic analysis and previous genetic studies (4, 7–13).

Fig 2

In vivo examination of the fermentation products of the S. viridochromogenes strains. (A) HPLC analysis of the fermentation products of LK171, AVI101, and AVI102. (B) ESI-HR-MS analysis of avilamycins A and C. The protonated molecular ion [M+H]⁺ and ammonium adduct ion [M+NH₄]⁺ signals characteristic of avilamycin A were observed at m/z 1403.4738 (calculated 1403.4709) and m/z 1420.5001 (calculated 1420.4974), respectively. The [M+H]⁺ and [M+NH₄]⁺ signals characteristic of avilamycin C were observed at m/z 1405.4899 (calculated 1405.4865) and m/z 1422.5164 (calculated 1422.5131), respectively.

Fig 3

In vitro enzymatic activity assays to determine the redox activity of AviZ1. HPLC analysis of the AviZ1-catalyzed redox reactions. AviZ1 catalyzed the conversion of avilamycin A to avilamycin C in the presence of excessive NADPH (i) or NADH (ii). In the absence of NADPH and NADH, the reduction reaction did not happen (iii). AviZ1 catalyzed the conversion of avilamycin C to avilamycin A in the presence of excessive NADP⁺ (iv) or NAD⁺ (v). In the absence of NADP⁺ and NAD⁺, the oxidation reaction did not happen (vi).

Fig 4

Effects of opposing cofactors on the NAD⁺-dependent oxidation of avilamycin C and the NADPH-dependent reduction of avilamycin A catalyzed by AviZ1. (A) The NAD⁺-dependent oxidation of avilamycin C catalyzed by AviZ1 in the presence of increasing concentrations of NADPH as inhibitor. (B) The NADPH-dependent reduction of avilamycin A catalyzed by AviZ1 in the presence of increasing concentrations of NAD⁺ as inhibitor. The scatter symbols represent independent data points from four parallel tests. The polylines are plotted from the average values of these data points, and the error bars represent the standard deviations.

Fig 5

Sequence analysis, structural prediction, and site-directed mutagenesis proposing the basis for the dual cofactor specificity of AviZ1. (A) Multiple sequence alignment with members of families 1 and 2 that exhibit dual cofactor specificity, along with members of family 11. Amino acid sequences within β/α repeat 8 were represented. Amino acids important for binding both the 2’-phosphate of NADP⁺ and the 2’-hydroxyl of NAD⁺ are highlighted in blue, while amino acids specifically binding to the 2'-phosphate of NADP⁺ are highlighted in red. (B) Schematic diagrams of binding of NAD⁺ and NADP⁺ to AviZ1. Residue numbers for amino acids involved in binding both the 2’-phosphate of NADP⁺ and the 2’-hydroxyl of NAD⁺ are shown in blue, while those specifically binding to the 2’- phosphate of NADP⁺ in AviZ1-G279K/A280S are shown in red. (C) Examination of enzymatic NADPH/NADP⁺-dependent redox reactions by HPLC. Reactions were conducted within 30 min for the NADPH-dependent reduction of avilamycin A (i, enzyme-free control) in the presence of AviZ1 (ii), AviZ1-G279K/A280S (iii), AviZ1-G279K (iv), or AviZ1-A280S (v), and for the NADP⁺-dependent oxidation of avilamycin C (vi, enzyme-free control) in the presence of AviZ1 (vii), AviZ1-G279K/A280S (viii), AviZ1-G279K (ix), or AviZ1-A280S (x). (D) Examination of enzymatic NADH/NAD⁺-dependent redox reactions by HPLC. Reactions were conducted within 30 min for the NADH-dependent reduction of avilamycin A (i, enzyme-free control) in the presence of AviZ1 (ii), AviZ1-G279K/A280S (iii), AviZ1-G279K (iv), or AviZ1-A280S (v), and for the NAD⁺-dependent oxidation of avilamycin C (vi, enzyme-free control) in the presence of AviZ1 (vii), AviZ1-G279K/A280S (viii), AviZ1-G279K (ix), or AviZ1-A280S (x).

Fig 6

Content and proportion of avilamycin A from day 6 shake flask fermentation cultures of the S. viridochromogenes strains. The scatter dots represent independent data points from three parallel tests. The histograms are plotted from the average values of these data points, and the error bars represent the standard deviations. Asterisks indicate significant difference between fermentation yields of avilamycins A and C of the engineered strains and those of LK171 (t-test, P < 0.05).