Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation

Abstract

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

Synergistic effects of components in deep eutectic solvents relieve toxicity and improve the performance of steroid biotransformation catalyzed by Arthrobacter simplex

“…Prednisone acetate (PA) is an important anti‐inflammatory and anti‐allergic glucocorticoid agent . It can be produced by the Δ1, 2‐dehydrogeneration of cortisone acetate (CA) using Arthrobacter simplex , and many attempts have been made to improve the efficiency of this biotransformation . As a promising and attractive alternative to organic solvents and ILs, three ChCl‐based DESs were used as co‐solvents to enhance the efficiency of C1,2‐dehydrogeneration by improving the solubility of the highly hydrophobic substrate .…”

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

“…The ksdD4 gene (GenBank ID: CP009896.1; Protein ID: WP_038682825.1) was ligated into the vector pET28a(+) (Novagen, Madison, WI, USA) with a His tag (HHHHHH) at the N-terminus from the genome DNA Arthrobacter simplex (also called Pimelobacter simplex). 15,43 The PCR product of KsdD was obtained with the KsdD forward primer F (GGAATTCCATATG TCG-GACACCACCGTGGACC) and the reverse primer R (CCGGAATTCCAT GGCGGTGGCGGCGTG), and PrimeSTAR MAX as DNA polymerase. The following PCR program was used: 98 ∘ C for 3 min followed by 30 cycles of 10 s at 98 ∘ C, 15 s at 54 ∘ C, 9 s at 72 ∘ C, and then 72 ∘ C for 10 min.…”

“…Various microorganisms can be exploited for biotransformation and the great diversity of microbial metabolic reactions represents a rich repository of desirable chemical modifications of steroids . Δ 1 ‐dehydrogenation in steroids is one of the crucial modifications owing to the increasing biological activity and economic value of the resulting steroid derivatives, in which a C1‐C2 double bond is introduced into the ring A of 3‐ketosteroid substrates by 3‐ketosteroid‐Δ 1 ‐dehydrogenase …”

“…a C1-C2 double bond is introduced into the ring A of 3-ketosteroid substrates by 3-ketosteroid-Δ 1 -dehydrogenase. 15, 16 3-ketosteroid-Δ 1 -dehydrogenase (KsdD, EC 1.3.99.4) was found in several steroid-degrading bacteria, including bacteria from Arthrobacter, 17 Mycobacterium, 18,19 Rhodococcus, 7,[20][21][22][23][24] Comamonas 25 and Nocardia. 26,27 The KsdD enzyme is a flavoprotein with the ability to dehydrogenate a wide variety of 3-ketosteroids, with a preference for substrates unsaturated at the C4-C5 position, such as AD, 9 -OH-AD, or progesterone.…”

“…37,38 However, no efficient genetic tools are available for A. simplex. 15,39 Besides, recombinant ksdDs produced in E. coli was found mainly in the insoluble inclusion bodies fraction, and only a small proportion was soluble and active, 1,25 leading to the overexpression of nonfunctional form in the E. coli expression system, 40 and limiting its practical application in steroid conversion. 41,42 Here, we reported a 3-ketosteroid-Δ 1 -dehydrogenase (KsdD4) from A. simplex expressed in E. coli BL21(DE3).…”