Genome-Scale Metabolic Model of Actinosynnema pretiosum ATCC 31280 and Its Application for Ansamitocin P-3 Production Improvement.

Jian Li,Renliang Sun,Xinjuan Ning,Xinran Wang,Zhuo Wang

doi:10.3390/genes9070364

Abstract

Actinosynnema pretiosum ATCC 31280 is the producer of antitumor agent ansamitocin P-3 (AP-3). Understanding of the AP-3 biosynthetic pathway and the whole metabolic network in A. pretiosum is important for the improvement of AP-3 titer. In this study, we reconstructed the first complete Genome-Scale Metabolic Model (GSMM) Aspm1282 for A. pretiosum ATCC 31280 based on the newly sequenced genome, with 87% reactions having definite functional annotation. The model has been validated by effectively predicting growth and the key genes for AP-3 biosynthesis. Then we built condition-specific models for an AP-3 high-yield mutant NXJ-24 by integrating Aspm1282 model with time-course transcriptome data. The changes of flux distribution reflect the metabolic shift from growth-related pathway to secondary metabolism pathway since the second day of cultivation. The AP-3 and methionine metabolisms were both enriched in active flux for the last two days, which uncovered the relationships among cell growth, activation of methionine metabolism, and the biosynthesis of AP-3. Furthermore, we identified four combinatorial gene modifications for overproducing AP-3 by in silico strain design, which improved the theoretical flux of AP-3 biosynthesis from 0.201 to 0.372 mmol/gDW/h. Upregulation of methionine metabolic pathway is a potential strategy to improve the production of AP-3.

Highlights

Actinosynnema pretiosum ATCC 31280 was isolated in 1977 [1] and is known as the producer of ansamitocins [2]
Ansamitocins are a series of complex polyketide compounds [3], among which ansamitocin P-3 (AP-3) was confirmed to be the most potent antitumor agent [4,5]
AP-3 has been used as the payload in many antibody-drug conjugants, such as trastuzumab emtansine, which was approved by the FDA for breast cancer treatment [6]

Summary

Introduction

Actinosynnema pretiosum ATCC 31280 was isolated in 1977 [1] and is known as the producer of ansamitocins [2]. The antitumor activity of AP-3 is highly effective, the commercial application of AP-3 is substantially limited by its low production titer [7]. In the past decades, many efforts have been made to improve the production of AP-3 [8,9,10]. These strategies includes mutant screening, medium optimization, and genetic engineering. The titer of AP-3 is still far from ideal. The reason for limited success in the improvement of AP-3 titer is probably due to a less understanding of the AP-3 biosynthetic

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Results

Conclusion