Combination of traditional mutation and metabolic engineering to enhance ansamitocin P‐3 production in Actinosynnema pretiosum

Abstract

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.

Rational approach to improve ansamitocin P‐3 production by integrating pathway engineering and substrate feeding in Actinosynnema pretiosum

“…In the O asm13‐17 : asmUdpg strain, the concentration of AHBA was higher than WT and O asmUdpg at Day 1 and 2 (Figure d), and its concentration of glyceryl‐ S ‐ACP was higher than WT and O asm13‐17 at Day 1 (Figure e). The results reflected that overexpression of asmUdpg in O asm13‐17 improved the AHBA biosynthesis, thus further enhanced the AP‐3 production, which is similar to the improvement of AP‐3 production after coexpression of asm13‐17 : asmUdpg in a mutant strain (Du et al, ). It is clear that the AP‐3 production was greatly improved by co‐enforcing the supply of UDP‐glucose (AHBA pathway) and glycolate unit in WT.…”

“…As a result, the AP‐3 production titer reached 680.5 mg/L in O asm13‐17 : asmUdpg , which is much higher than previous reports, including those by random mutagenesis (Chung & Byng, ; Kuo, Byng, & Widdison, ), medium optimization (Fan et al, ; Gao et al, ; Jia & Zhong, ; Li et al, ; Lin et al, ), modification of regulator genes (Bandi et al, ; Ng, Chin, & Wong, ; Pan, Kang, Wang, Bai, & Deng, ), and pathway engineering (Du et al, ; Fan, Hu, Wei, Bai, & Hua, ; Fan, Zhao et al, ; Ning, Wang, Wu, Kang, & Bai, ; M. Zhao et al, ). As summarized in Table , the recent record of AP‐3 titer in M‐ asmUdpg:asm13‐17 , which coexpressed asm13‐17 and asmUdpg in a high‐producing mutant M (Du et al, ), was lower than O asm13‐17 : asmUdpg here. In reality, the concentration of AHBA in O asm13‐17 : asmUdpg (Figure d) was two‐fold at the 1st and 2nd day compared to M‐ asmUdpg:asm13‐17 (Du et al, ).…”

“…When necessary, ampicillin (100 μg/ml), apramycin (30 μg/ml), and chloramphenicol (25 μg/ml) were added into the culture medium of Escherichia coli and A. pretiosum . The culture medium, construction of plasmids and recombinant strains were the same as reported (Du et al, ). The recombinant strains (O asm13‐17 , O asmUdpg and O asmUdpg:asm13‐17 ) were selected based on apramycin resistance and verified by colony polymerase chain reaction (PCR) using primer pair erm E‐F/15R (1.84 kb), erm E‐F/Udpg‐R (1.38 kb), and 17F/Udpg‐R (1.88 kb), respectively.…”

“…The AP‐3 structure was confirmed by extensive NMR analyses, including COSY, HSQC, and HMBC (Supporting Information Table SI, Figures S5–S11). The AP‐3 production titer was determined using ODS‐BP C18 column (Agilent) eluting with 60% acetonitrile supplemented with 0.1% trifluoroacetic acid (TFA) with a flow rate of 1 ml/min at 28°C as previously described (Supporting Information Figure S3; Du et al, ).…”

“…The ADCs of AP‐3‐Ado‐trastuzumab emtansine (Kadcyla ® ), approved by Food and Drug Administration of USA, is already used for targeted therapy of metastatic breast cancer (Chari, Miller, & Widdison, ). To meet the need of commercial application with cheap supply to patients as well as further (pre)clinical studies, without doubt a higher AP‐3 production titer is urgently required (Du, Zhang, Qian, Xiao, & Zhong, ; Wang et al, ).…”