Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

Lim, Hyun Gyu; Eng, Thomas; Banerjee, Deepanwita; Alarcon, Geovanni; Lau, Andrew K.; Park, Mee-Rye; Simmons, Blake A.; Palsson, Bernhard O.; Singer, Steven W.; Mukhopadhyay, Aindrila; Feist, Adam M.

doi:10.1021/acssuschemeng.1c03765

Title: Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

Journal Article · 18 August 2021 · ACS Sustainable Chemistry & Engineering

DOI: https://doi.org/10.1021/acssuschemeng.1c03765 · OSTI ID:1958747

^[1]; Eng, Thomas ^[2]; Banerjee, Deepanwita ^[2]; Alarcon, Geovanni ^[3]; Lau, Andrew K. ^[2]; Park, Mee-Rye ^[2];

^[2]; Palsson, Bernhard O. ^[4];

^[2];

^[4]

Univ. of California, La Jolla, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Univ. of California, La Jolla, CA (United States)
Univ. of California, La Jolla, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark)

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. Here, in this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (<0.1 h^–1 or none) was evolutionarily optimized to rates of up to 0.25 h^–1 on xylose and 0.52 h^–1 on galactose. Whole-genome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1958747

Journal Information:: ACS Sustainable Chemistry & Engineering, Journal Name: ACS Sustainable Chemistry & Engineering Journal Issue: 34 Vol. 9; ISSN 2168-0485

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Leloir pathway
Pseudomonas putida
Weimberg pathway
adaptive laboratory evolution
biomass
carbohydrates
galactose
genetics
metabolism
peptides and proteins
xylose

Title: Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

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