Process optimization with alternative carbon sources and modulation of secondary metabolism for enhanced ansamitocin P-3 production in Actinosynnema pretiosum

Fan, Yuxiang; Gao, Yang; Zhou, Jie; Wei, Liujing; Hua, Qiang

doi:10.1016/j.jbiotec.2014.10.020

Cited by 24 publications

(16 citation statements)

References 28 publications

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“…These results confirmed that overexpression of asmUdpg could pull more carbon flux from primary metabolism to UDP‐glucose pool and AHBA biosynthesis pathway in O asm13‐17 . 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.…”

Section: Discussionmentioning

confidence: 66%

“…As reported by Fan et al (), glucose negatively affected AP‐3 production when it was used as a sole carbon source. Different from glucose, fructose was transported by phosphotransferase system and may bypass the CCR effect of glucose; and it was reported to induce the expression of AP‐3 biosynthetic genes (Fan et al, ; Li et al, ). Those reports suggest that feeding fructose may be beneficial to precursor supply and stimulate the secondary metabolism in A. pretiosum .…”

Section: Introductionmentioning

confidence: 51%

“…Previous studies suggested that expression of asm14 and asm17 was coordinated with the AP‐3 biosynthesis (Fan et al, ; Lin et al, ). The glycolate unit supply was postulated to be the bottleneck in AP‐3 biosynthesis, but its single effect on AP‐3 production was unclear.…”

Section: Resultsmentioning

confidence: 94%

“…With pulse‐feeding of fructose, we finally obtained 757.7 mg/L of AP‐3, which was much higher than other reports in bioreactors (Table ). It was demonstrated that feeding fructose could result in flux rearrangement in primary metabolism for better providing biosynthetic precursors and stimulating the secondary metabolism in A. pretiosum (Fan et al, ). Similarly, compared to glucose and sorbitol, feeding fructose could promote the fengycin biosynthesis by improving the gene expression level, precursor supply and energy metabolism in Bacillus amyloliquefaciens fmb‐60 (Lu et al, ).…”

Section: Discussionmentioning

confidence: 99%

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

Zhong

2018

Biotech & Bioengineering

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Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is an important antitumor agent for cancer treatment, but its market supply suffers from a low production titer. The role of AP-3 unusual glycolate unit supply on its biosynthesis was investigated in this work by overexpressing the responsible gene cluster asm13-17 in A. pretiosum (WT). As a result, the accumulation of AP-3 and its intermediate glyceryl-S-ACP in the asm13-17-overexpressed strain (Oasm13-17) versus WT was enhanced by 1.94 and 1.49-fold, respectively. To provide a higher supply of another precursor 3-amino-5-hydroxybenzoic acid, asmUdpg was also overexpressed in Oasm13-17 (Oasm13-17:asmUdpg), and an improved AP-3 titer of 680.5 mg/L was achieved in shake flasks. To further enhance the AP-3 titer, a rational fed-batch strategy was developed in bioreactor fermentation of Oasm13-17:asmUdpg; and by pulse feeding 15 g/L fructose and 1.64 g/L isobutanol at 60, 96, and 120 hr, the AP-3 production level reached 757.7 mg/L, which is much higher than ever reported in bioreactors. This work demonstrated that a rational approach combining precursor pathway engineering with substrate feeding was very effective in enhancing the AP-3 titer, and this enabling methodology would be helpful to industrial production of this eye-catching drug.

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Section: Discussionmentioning

confidence: 66%

Section: Introductionmentioning

confidence: 51%

Section: Resultsmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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

Zhong

2018

Biotech & Bioengineering

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“…To facilitate further (pre)clinical study and wide commercial application of AP‐3, improvement of its production efficiency is still one of recent research focuses as the current fermentation titer is yet not so high. In the past decades, many strategies have been employed to enhance the biosynthesis of AP‐3, including mutant screening (Chung & Byng, ), medium optimization (Fan et al, ; Gao et al, ; Jia & Zhong, ; Li et al, ; Lin, Bai, Deng, & Zhong, ; Lin, Bai, Deng, & Zhong, ), and genetic engineering (Fan, Hu, Wei, Bai, & Hua, ; Fan, Zhao, et al, ; Zhao, Fan, Wei, Hu, & Hua, ). By applying traditional mutagenesis such as ultraviolet (UV) and N ‐methyl‐ N' ‐nitro‐ N ‐nitrosoguanidine (MNNG), a relatively high AP‐3 producing mutant was obtained (Chung & Byng, ), which kept its highest titer record of 401 mg/L in literature.…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Qian

et al. 2017

Biotech & Bioengineering

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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.

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Identification and Engineering of Post‐PKS Modification Bottlenecks for Ansamitocin P‐3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280

Ning

Wang

et al. 2017

Biotechnology Journal

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The type-I polyketide ansamitocin P-3 (AP-3) is a potent antitumor agent. Its production is most likely hampered by the required multiple substrate supplies and complicated post-PKS modifications in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. For titer improvement, gene ansa30, encoding for a glycosyltransferase competing for the N-demethyl-AP-3 (PND-3) intermediate for AP-3 biosynthesis, was initially inactivated. In the mutant NXJ-22, the AP-3 titer was increased by 66% along with an obvious accumulation of PND-3, indicating that the N-methylation is a rate-limiting step. Alternatively, when abundant upstream intermediate 19-chloroproansamitocin was fed into a PKS mutant, 3-O-acylation was further identified along with the N-methylation as the rate-limiting steps. Subsequent overexpression of N-methyltransferase gene asm10 in NXJ-22 resulted in a 93% increase of AP-3 and a corresponding 92% decrease of PND-3. Additional supplementation of L-methionine, the precursor for SAM biosynthesis, substantially decreased the accumulation of PND-3. In parallel, the 3-O-acylation bottleneck was relieved by feeding with L-valine to NXJ-22, resulting in a 126% increase of AP-3. Eventually, a combined asm10 overexpression and supplementation of L-methionine and L-valine resulted in a 5-fold increase of AP-3, from 42 ± 2 mg L to 246 ± 6 mg L , without any noticeable accumulation of PND-3.

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Process optimization with alternative carbon sources and modulation of secondary metabolism for enhanced ansamitocin P-3 production in Actinosynnema pretiosum

Cited by 24 publications

References 28 publications

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

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

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

Identification and Engineering of Post‐PKS Modification Bottlenecks for Ansamitocin P‐3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280

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