Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

Kang, Junhua; Gu, Pengfei; Wang, Yang; Li, Yikui; Yang, Fan; Wang, Qian; Qi, Qingsheng

doi:10.1016/j.ymben.2012.09.002

Cited by 41 publications

(40 citation statements)

References 38 publications

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“…Studies have further shown that E. coli O157 and other E. coli strains may compete for different nutrients in the intestine (9). In addition, it was shown that deletions in the catabolite pathways for N-acetylmuraminic acid and N-acetylglucosamine had the greatest impact (11). Fabich et al (12) demonstrated that gluconate, N-acetylglucosamine, N-acetylneuraminic acid (glucuronate), mannose, fucose, and ribose have an impact on E. coli nutrition in the mouse intestine.…”

mentioning

confidence: 99%

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

Polzin

Huber

Eylert

et al. 2013

Appl Environ Microbiol

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In this study, the intracellular proteome of Escherichia coli O157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-13 C 6 ]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 M 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.

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confidence: 99%

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

Polzin

Huber

Eylert

et al. 2013

Appl Environ Microbiol

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“…To this end, the operon responsible for Neu5Ac degradation ( nanATEK ) was deleted. Subsequently, since the wild‐type strain does not accumulate Neu5Ac (Kang et al, ; Vimr et al, ), a heterologous pathway was introduced enabling Neu5Ac production. This pathway comprises the neuB1 and neuC genes from Campylobacter jejuni , which encode a Neu5Ac synthase and a GlcNAc6P 2‐epimerase, respectively.…”

Section: Resultsmentioning

confidence: 99%

Development of N‐acetylneuraminic acid responsive biosensors based on the transcriptional regulator NanR

Peters

Paepe

Wannemaeker

et al. 2018

Biotech & Bioengineering

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Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high-throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences without the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental. In contrast, a bottom-up approach to design tailor-made biosensors was pursued here. Novel biosensors were created that respond to N-acetylneuraminic acid (Neu5Ac), an important sugar moiety with various biological functions, by employing native and engineered promoters that interact with the TF NanR. This bottom-up approach, whereby various tuned modules, e.g., the ribosome binding site (RBS) controlling NanR translation can be combined, enabled the reliable engineering of various response curve characteristics. The latter was validated by testing these biosensors in combination with various Neu5Ac-producing pathways, which allowed to produce up to 1.4 ± 0.4 g/L extracellular Neu5Ac. In this way, the repertoire of biosensors was expanded with seven novel functional Neu5Ac-responsive biosensors.

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“…nanT is the secondary transporter of NeuAc. The deletion of nanT avoids retro-transportation, and prevents consumption of NeuAc in E. coli [6]. Deletion of acetate production pathway can reduce the by-product, thus more pyruvate can be gotten, which is also a precursor of NeuAc production.…”

Section: Comparison With Optknock Gdls Gdbb and Optforce On Succinamentioning

confidence: 99%

OP‐Synthetic: identification of optimal genetic manipulations for the overproduction of native and non‐native metabolites

Liu

Wang

2015

Quant. Biol.

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Constraint-based flux analysis has been widely used in metabolic engineering to predict genetic optimization strategies. These methods seek to find genetic manipulations that maximally couple the desired metabolites with the cellular growth objective. However, such framework does not work well for overproducing chemicals that are not closely correlated with biomass, for example non-native biochemical production by introducing synthetic pathways into heterologous host cells. Here, we present a computational method called OP-Synthetic, which can identify effective manipulations (upregulation, downregulation and deletion of reactions) and produce a step-by-step optimization strategy for the overproduction of indigenous and non-native chemicals. We compared OP-Synthetic with several state-of-the-art computational approaches on the problems of succinate overproduction and Nacetylneuraminic acid synthetic pathway optimization in Escherichia coli. OP-Synthetic showed its advantage for efficiently handling multiple steps optimization problems on genome wide metabolic networks. And more importantly, the optimization strategies predicted by OP-Synthetic have a better match with existing engineered strains, especially for the engineering of synthetic metabolic pathways for non-native chemical production. OPSynthetic is freely available at:http://bioinfo.au.tsinghua.edu.cn/member/xwwang/OPSynthetic/.

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Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

Cited by 41 publications

References 38 publications

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

Development of N‐acetylneuraminic acid responsive biosensors based on the transcriptional regulator NanR

OP‐Synthetic: identification of optimal genetic manipulations for the overproduction of native and non‐native metabolites

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