Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

Cheng, Jie; Luo, Qing; Duan, Huaichuan; Peng, Hao; Zhen, Yin; Hu, Jianping; Lü, Yao

doi:10.1038/s41598-020-57752-x

Cited by 17 publications

(14 citation statements)

References 37 publications

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“…Compared with another new synthesis pathway for the fermentative production of 5AVA, in which the titer was only 5.1 g/L (seen in Table 1 ; Jorge et al, 2017 ), and the titer was greatly increased in this study. Compared with another whole-cell catalysis work, this synthetic pathway increased the titer of 5AVA by about 3.20% from 50.62 to 52.24 g/L ( Cheng et al, 2020 ). Importantly, the industrial production of 5AVA without the addition of ethanol and H 2 O 2 was more safe and economical in this study.…”

Section: Resultsmentioning

confidence: 89%

“…There are four strategies used in this study to increase the production of 5AVA. Firstly, lysine decarboxylase gene cadA was knocked out and L -lys HCl was selected as the industrial substrate for enhancing the utilization of L -lys ( Cheng et al, 2018a , b , 2020 ). Thirdly, H 2 O 2 could inhibit cell growth, thus affecting the production of goal production ( Niu et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…On the contrary, it decreased the OD 600 , possibly because the increase in gene expression caused an increase in cell burden ( Camara et al, 2017 ). However, in the fermentation tank, H 2 O 2 could significantly inhibit cell growth, resulting in limited production of 5AVA ( Cheng et al, 2018b , 2020 ). In addition, a lysine transporter gene lysP was overexpressed and inserted into the plasmid pZAkatE to form a new plasmid pZAKL.…”

Section: Resultsmentioning

confidence: 99%

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A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli

Cheng

Luo

et al. 2021

Front. Bioeng. Biotechnol.

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Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56. In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established. Combination of L-Lysine α-oxidase from Scomber japonicus, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could achieve the biosynthesis of 5AVA from biobased L-Lysine in E. coli. The H2O2 produced by L-Lysine α-oxidase was decomposed by the expression of catalase KatE. Finally, 52.24 g/L of 5AVA were obtained through fed-batch biotransformation. Moreover, homology modeling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed might be beneficial for the substrates stretch. In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli

Cheng

Luo

et al. 2021

Front. Bioeng. Biotechnol.

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“…Glutarate can be derived from l -lysine by four pathways that converge to 5AVA, which is converted to glutarate by GABA/5AVA aminotransferase (GabT) and the succinate/glutarate semialdehyde dehydrogenase (GabD). The first pathway from l -lysine to 5AVA employs l -lysine-α-oxidase (RaiP) from Scomber japonicus that catalyzes oxidative desamination of l -lysine using molecular oxygen followed by spontaneous decarboxylation (Cheng et al, 2020 ). The second pathway to 5AVA combines oxidative decarboxylation by l -lysine monooxygenase using molecular oxygen followed by desamidation by γ-aminovaleramidase from P. putida (Adkins et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis

Prell

Burgardt

Meyer

et al. 2021

Front. Bioeng. Biotechnol.

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l-2-hydroxyglutarate (l-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural l-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of l-2HG. Since l-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined l-lysine overproducing strain was required to enable the conversion of l-lysine to l-2HG in a six-step synthetic pathway. To this end, l-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported l-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of l-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved l-2HG production. The availability of Fe2+ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to l-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a l-2HG titer of 3.5 g L−1 was obtained at the higher of two tested aeration levels. Production of l-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of l-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability.

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“…All four pathways converge to 5-aminovalerate (5AVA), which then is converted to glutarate in two enzymatic steps catalyzed by GABA/5AVA aminotransferase (GabT) and succinate/glutarate semialdehyde dehydrogenase (GabD). The first pathway from l -lysine to 5AVA employs l -lysine-α-oxidase (RaiP) from Scomber japonicus that catalyzes oxidative deamination of l -lysine using molecular oxygen followed by spontaneous decarboxylation [ 21 ]. The second pathway to 5AVA combines oxidative decarboxylation by l -lysine monooxygenase (DavA) using molecular oxygen followed by desamidation by γ-aminovaleramidase (DavB) from Pseudomonas putida [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

et al. 2021

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Background The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. Results In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the l-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h−1 by the parental strain to 0.17 h−1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L−1 h−1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of l-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L−1, a yield of 0.23 g g−1 and a volumetric productivity of 0.35 g L−1 h−1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results. Conclusion Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

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Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

Cited by 17 publications

References 37 publications

A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli

A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli

Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

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