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Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N‑acetyl‑d‑neuraminic acid

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Abstract

N‑Acetyl‑d‑neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-d-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.

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Acknowledgements

We thank Dr. Thomas Kuhlman for providing pTKRed used in this research.

Supplementary Information

Supplementary file 1. Table S1: Bacterial strains and plasmids used in this research. Table S2: PCR primers and oligonucleotides used in the study.

Figure S1: Schematic presentation of the recombineering and I-SceI-mediated gene deletion. Figure S2: Confirmation of Δnan mutant. Figure S3: Confirmation of Δman mutant. Figure S4: Confirmation of Δnag mutant. Figure S5: Confirmation of ΔpoxB mutant. Figure S6: Outline of CRISPR/Cas9-assisted ssDNA recombineering for gene deletion. Figure S7: Confirmation of ΔldhA mutant. Figure S8: Confirmation of ΔackA mutant. Figure S9: HPLC analysis of Neu5Ac biotransformation.

Funding

This research was financially supported by the National Natural Science Foundation of China (No. 82073742).

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Authors and Affiliations

  1. Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, No. 1 Wenyuan Rd., Xixia District, Nanjing, 210023, Jiangsu Province, People’s Republic of China

    Qiong Zhang, Jiao Zhang, Yanhong Shao & Guangdong Shang

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  1. Qiong Zhang
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  2. Jiao Zhang
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  3. Yanhong Shao
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  4. Guangdong Shang
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Contributions

GS was responsible for the experimental design and drafted the manuscript. QZ, JZ and YS performed the experiments. All authors participated in data analysis, read and approved the final manuscript.

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Correspondence to Guangdong Shang.

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Zhang, Q., Zhang, J., Shao, Y. et al. Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N‑acetyl‑d‑neuraminic acid. Biotechnol Lett 45, 1521–1528 (2023). https://doi.org/10.1007/s10529-023-03426-3

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