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  • br Disclosure and conflicts of interest

    2019-11-02


    Disclosure and conflicts of interest
    Acknowledgements This research was supported by grant (BT/PR/11293/BRB/10/849/2008) from Department of Biotechnology (DBT), Government of India and a DST YOS Chair Professorship to PB. The mass spectrometry facility is supported by an institutional program grant from the DBT.
    -4-Hydroxy--proline (-4Hyp) is a valuable chiral building block for the synthesis of pharmaceuticals such as carbapenem antibiotics, which are regarded as the last resort against serious gram-negative bacterial infections , , , . Conventional industrial production of -4Hyp employs 740 Y-P hydrolysis of mammalian collagen, which suffered from the serious drawbacks of a low recovery rate (4–7%) and heavy environmental pollution , . Due to the discovery of microbial -P4Hs , , which can hydroxylate free -proline to free -4Hyp utilizing dioxygen and α-oxoglutarate as co-substrates, the microbial production of -4Hyp has attracted more attention because of its economic advantages and environmental benefits. Engineered strains of or expressing diverse -P4Hs have been used for -4Hyp production (), wherein the most commonly used -P4H is from sp. RH1 (DaP4H) and codon optimization was generally required to adjust the codon usage and high G + C content (74%). Based on the substrates added in the production process, it can be divided into three categories: (i) Both of the hydroxylation substrates, -proline and α-oxoglutarate, are simultaneously loaded in the -4Hyp production via a whole cell biocatalytic process. Chen et al. constructed a recombinant BL21 CodonPlus(DE3) strain expressing a codon-optimized and truncated variant of gene, and 25.44 g/L of -4Hyp with a high productivity of 0.53 g/L/h was achieved after 48 h catalysis. (ii) Only -proline is added additionally but α-oxoglutarate is supplied by cell metabolism of other carbon source. Shibasaki et al. introduced a codon-optimized gene into W1485 Δ mutant, which resulted in 41 g/L of -4Hyp at 100 h, the highest titer reported so far, with a relatively high productivity of 0.41 g/L/h by using -proline and glucose as carbon source. Wang et al. introduced a codon-optimized gene into BL21 and engineered the strain to utilize glycerol. The resulting strain yielded 25.4 g/L of -4Hyp at 48 h by feeding -proline and supplying α-oxoglutarate from glycerol . (iii) Both -proline and α-oxoglutarate are supplied from cellular metabolism of carbon source such as glucose or glycerol. Shibasaki et al. introduced 740 Y-P DaP4H into an -proline-producing and 25 g/L of -4Hyp was synthesized after 96 h fermentation in a 5 L reactor by using glucose as the sole carbon source, the productivity was 0.26 g/L/h. Falcioni et al. introduced a codon-optimized gene into an -proline-producing strain, and 7.1 g/L of -4Hyp was obtained at 23 h by using a minimal medium containing glucose. Considering high expense of -proline (about 16 US dollar/kg) and available technology for efficiently producing -proline from glucose , , , , the strategy of producing -4Hyp directly from glucose with an integrated proline hydroxylation process into -proline-producing strains, seems to be a more economically attractive approach for industrialization. Therefore, -P4H, the crucial enzyme in converting -proline to -4Hyp, becomes the major focus on improving hydroxylation efficiency towards higher -4Hyp titer and productivity. However, few -P4Hs were found in microorganisms, including 3720 , P8648 , SANK 14591 , sp. JCM6894 , sp. RH1 , , , RB50 , ATCC 74030 and . Only five -P4H genes from sp. RH1 , ATCC 74030 , , RB50 and have been identified. Obviously, more -P4H should be explored. Over the past decades, genome mining has been proven as an effective approach to discover new enzymes , , . In this study, ten putative -P4Hs from NCBI database were selected using sequence-based homology search and phylogenetic tree analysis. Three out of ten enzymes were identified with the activity of -P4H, wherein the -P4H from (AlP4H) exhibited the highest ability on -4Hyp production in an -proline-producing chassis cell. This study provides a promising hydroxylase candidate for the efficient industrial production of -4Hyp.