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Two-Step Bioprocess for Reducing Nucleus Degradation in Phytosterol Bioconversion by Mycobacterium neoaurum NwIB-R10hsd4A

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Abstract

In order to obtain high 4-androstene-3,17-dione (AD) yield, nucleus degradation needs to be avoided during phytosterol bioconversion process with Mycobacterium neoaurum NwIB-R10hsd4A. 3-Ketosteroid-Δ1-dehydrogenase (KstD) catalyzes 1,2-desaturation of steroids and is a key enzyme involved in steroid nucleus oxidation. Heterogeneous expression and characterization of two KstDs (KstD2, KstD3) from M. neoaurum NwIB-R10hsd4A showed that their activities were inhibited by shifting temperature from 30 to 37 °C. However, the total activities of KstD2 and KstD3 were replenished when M. neoaurum NwIB-R10hsd4A was cultured at 37 °C because the transcription levels of kstD2 and kstD3 were upregulated 1.61- and 1.43-fold respectively compared with the cultivation at 30 °C. As the optimal temperature for cell growth was 30 °C, we developed a two-step bioprocess, cell culture at 30 °C and bioconversion with resting cells at 37 °C avoiding higher transcriptional level of kstD2 and kstD3. This process repressed the activities of KstDs, resulted in the decrease of 1,2-desaturation products, and reduced the nucleus degradation (17.6%). AD production increased to 24.7 g l−1 at higher substrate concentration (50 g l−1). These results indicated that the two-step bioprocess was potential in phytosterol biotransformation industrially.

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Funding

This study was funded by the National Natural Science Foundation of China (No. 21276083, 31570079).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Xuedong Wang, Chaolong Hua, Xinwei Xu & Dongzhi Wei

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  1. Xuedong Wang
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  2. Chaolong Hua
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  3. Xinwei Xu
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  4. Dongzhi Wei
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Correspondence to Xuedong Wang.

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Fig. S1

A SDS-PAGE analysis of KstDs in induced E. coli BL21(DE3). Lane 1: Marker; Lane2: KstD2, supernatant of cell lysate; Lane 3: KstD2 sediment of cell lysate; Lane 4: KstD3, supernatant of cell lysate; Lane 5: KstD3, sediment of cell lysate (PNG 182 kb)

High Resolution (TIF 2693 kb)

Fig. S2

Determination of total KstDs apparent activities of Mycobacterium when cell culture was at 30 °C (■) and 37 °C (●). Consumption of AD (a) and production of ADD (b). The biotransformation were carried out in a reaction system containing 20 g l−1 cell pellets cultured at 30 °C or 37 °C, 2 g l−1 AD and 8 g l−1 HP-β-CD in 10 ml phosphate buffer (20 mmol l−1, pH 8.0) at 30 °C, with a rotary speed of 200 rpm, in a 250-ml flask. The apparent KstDs activities were determined by quantifying the amount of ADD formed in the reaction. The error bars represent mean ± SD (n = 3). (PNG 112 kb)

High Resolution (TIFF 20350 kb)

Fig. S3

(PNG 107 kb)

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Wang, X., Hua, C., Xu, X. et al. Two-Step Bioprocess for Reducing Nucleus Degradation in Phytosterol Bioconversion by Mycobacterium neoaurum NwIB-R10hsd4A. Appl Biochem Biotechnol 188, 138–146 (2019). https://doi.org/10.1007/s12010-018-2895-z

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