Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Abstract

a b s t r a c tBackground: 3-Ketosteroid-Δ 1 -dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd II ) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The K m and V max values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

“…Zhang et al . reported that the highest transformation rate of AD to ADD by M. neoaurum KsdD in Corynebacterium crenatum was about 83.87% after 10 h . Shao et al .…”

“…However, maximum ADD concentrations (2.66 g L −1 ) were obtained at 21 h during the third fed-batch fermentation process (bioconversion from 18 h to 27 h). Zhang et al reported that the highest transformation rate of AD to ADD by M. neoaurum KsdD in Corynebacterium crenatum was about 83.87% after 10 h. 50 Shao et al reported that ADD conversion rate of M. neoaurum KsdD in E. coli achieved 95% in 36 h using a fed-batch strategy. 37 However, the bioconversion of AD to ADD with A. simplex KsdD-expressing B. subtilis was only 45.3%.…”

Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

“…P. pastoris expresssion KstD F from Aspergillus fumigatus CICC 40167 is able to transform 1 g L −1 AD to ADD after 4 h of fermentation, however, the cell culture needs a further 6 days to establish [ 20 ]. In addition, KstD1 from M. neoaurum JC-12 has been expressed in C. crenatum , E. coli and B. subtili s to produce ADD from AD [ 22 – 24 ]. The highest yield (8.76 g L −1 ) was obtained in B. subtili s, with a fed-batch strategy in 50 h, after codon optimization and co-expression with catalase to remove H 2 O 2 toxicity [ 24 ].…”

“…A B. subtilis, expressing codon-optimized kstD gene from M. neoaurum JC-12, produced 8.76 g L −1 ADD by whole-cell biocatalysis [ 24 ]. Prominently, a KstD overexpressing C. crenatum can transform 83.87% of the AD to ADD [ 22 ].…”

Production of 4-Ene-3-ketosteroids in Corynebacterium glutamicum

“…The only reports related to this issue are the work of Charney et al [6] concerning biotransformations of steroid substrates using Corynebacterium simplex (previously classified as Arthrobacter simplex and Pimelobacter simplex, and currently known as Nocardioides simplex) and the recent work of Zhang et al [7] who used Corynebacterium crenatum as host to express the 3-ketosteroid-∆ 1 -dehydrogenase from Mycobacterium neoaurum to catalyze the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD).…”