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Modification of an engineered Escherichia coli by a combinatorial strategy to improve 3,4-dihydroxybutyric acid production

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Abstract

Objectives

3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy.

Results

Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, d-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from d-xylose (0.190 g 3,4-DHBA/g d-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g d-xylose).

Conclusions

These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli.

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Acknowledgements

This work was supported by the Natural Science Foundation of Shanghai (No. 19ZR1412700), the Fundamental Research Funds for the Central Universities (No. 22221818014), and partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Supplementary Information

Supplementary Table 1—List of plasmids this study.

Supplementary Table 2—List of primer sequences this study.

Supplementary Figure 1—Schematic illustration of this fusion construct.

Supplementary Figure 2—HPLC-MS in negative ion mode for 3,4-DHBA verification by strain E0-E01-A01. 3,4-DHBA (C4H8O4) was corresponded to the retention time of 1.0 min and the peak of 119.03 Da.

Supplementary Figure 3—SDS-PAGE analysis of target proteins of the engineered strains. Lane 1: whole cell of E0-E01-A02; Lane 2: whole cell of E1-E01-A02; Lane 3: whole cell of E2-E01-A02; Lane 4: whole cell of E3-E01-A02; Lane 5: whole cell of E4-E01-A02; Lane 6: whole cell of E5-E01-A02. M: protein molecular weight marker.

Supplementary Figure 4—Cell growth of engineered strains.

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Yidi Liu, Xinlei Mao, Baoqi Zhang, Jinping Lin & Dongzhi Wei

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  1. Yidi Liu
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  2. Xinlei Mao
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  3. Baoqi Zhang
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  4. Jinping Lin
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  5. Dongzhi Wei
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Correspondence to Jinping Lin.

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Liu, Y., Mao, X., Zhang, B. et al. Modification of an engineered Escherichia coli by a combinatorial strategy to improve 3,4-dihydroxybutyric acid production. Biotechnol Lett 43, 2035–2043 (2021). https://doi.org/10.1007/s10529-021-03169-z

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