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Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli.

ABSTRACT: Dehydrogenase pathway, one of diaminopimelate pathway, is important to the biosynthesis of L-lysine and peptidoglycan via one single reaction catalyzed by meso-diaminopimelate dehydrogenase (DapDH). In this study, the thermostable DapDH was introduced into diaminopimelate pathway that increased the final titer (from 71.8 to 119.5?g/L), carbon yield (from 35.3% to 49.1%) and productivity (from 1.80 to 2.99?g/(L?h)) of L-lysine by LATR12-2?rpiB::ddh_St in fed-batch fermentation. To do this, the kinetic properties and the effects of different DapDHs on L-lysine production were investigated, and the results indicated that overexpression of StDapDH in LATR12-2 was beneficial to construct an L-lysine producer with good productive performance because it exhibited the best of kinetic characteristics and optimal temperature as well as thermostability in reductive amination. Furthermore, ammonium availability was optimized, and found that 20?g/L of (NH₄)₂SO₄ was the optimal ammonium concentration for improving the efficiency of L-lysine production by LATR12-2?rpiB::ddh_St. Metabolomics analysis showed that introducing the StDapDH significantly enhanced carbon flux into pentose phosphate pathway and L-lysine biosynthetic pathway, thus increasing the levels of NADPH and precursors for L-lysine biosynthesis. This is the first report of a rational modification of diaminopimelate pathway that improves the efficiency of L-lysine production through overexpression of thermostable DapDH in E. coli.

SUBMITTER: Xu JZ

PROVIDER: S-EPMC6382763 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli.

Xu Jian-Zhong JZ Ruan Hao-Zhe HZ Liu Li-Ming LM Wang Lu-Ping LP Zhang Wei-Guo WG

Scientific reports 20190220 1

Dehydrogenase pathway, one of diaminopimelate pathway, is important to the biosynthesis of L-lysine and peptidoglycan via one single reaction catalyzed by meso-diaminopimelate dehydrogenase (DapDH). In this study, the thermostable DapDH was introduced into diaminopimelate pathway that increased the final titer (from 71.8 to 119.5 g/L), carbon yield (from 35.3% to 49.1%) and productivity (from 1.80 to 2.99 g/(L∙h)) of L-lysine by LATR12-2∆rpiB::ddh<sub>St</sub> in fed-batch fermentation. To do th ...[more]

PMID: 30787467

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Project description:meso-Diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) is the first member of the meso-DAPDH family known to catalyze the asymmetric reductive amination of 2-keto acids to produce d-amino acids. It is important to understand the catalytic mechanisms of StDAPDH and other enzymes in this family. In this study, based on an evolutionary analysis and examination of catalytic activity, the meso-DAPDH enzymes can be divided into two types. Type I showed highly preferable activity toward meso-diaminopimelate (meso-DAP), and type II exhibited obviously reversible amination activity with a broad substrate spectrum. StDAPDH belongs to type II. A quaternary structure analysis revealed that insertions/deletions (indels) and a loss of quaternary structure resulted in divergence among members of the meso-DAPDH family. A structure alignment of StDAPDH with a representative of type I, the meso-DAPDH from Corynebacterium glutamicum (CgDAPDH), indicated that they had the same folding. Based on sequence and conservation analyses, two amino acid residues of StDAPDH, R35 and R71, were found to be highly conserved within type II while also distinct from each other between the subtypes. Site mutagenesis studies identified R71 as a substrate preference-related residue of StDAPDH, which may serve as an indicator of the amination preference of type II. These results deepen the present understanding of the meso-DAPDH family and provide a solid foundation for the discovery and engineering of meso-DAPDH for d-amino acid biosynthesis.IMPORTANCE The l-form of amino acids is typically more abundant than the d-form. However, the d-form has many important pharmaceutical applications. meso-Diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) was the first member of meso-DAPDH known to catalyze the amination of 2-keto acids to produce d-amino acids. Accordingly, we analyzed the evolution of meso-DAPDH proteins and found that they form two groups, i.e., type I proteins, which show high preference toward meso-diaminopimelate (meso-DAP), and type II proteins, which show a broad substrate spectrum. We examined the differences in sequence, ternary structure, and quaternary structure to determine the mechanisms underlying the functional differences between the type I and type II lineages. These results will facilitate the identification of additional meso-DAPDHs and may provide guidance to protein engineering studies for d-amino acid biosynthesis.

Dataset Information

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli.

Publications

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli.

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