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

Abstract

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. 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. Wholegenome 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.

Machine-learning fromPseudomonas putidaTranscriptomes Reveals Its Transcriptional Regulatory Network

“…putida PRECISE321 includes seventeen gene expression profiles with 11 different carbon source conditions: glucose (as a reference), gluconate, fructose, galactose, xylose, citrate, coumarate, ferulate, serine, acetate, and myristic acid. In addition, it should be noted that gene expression profiles with xylose and galactose utilization were obtained from engineered P. putida strains with heterologous gene expression 26 .…”

“…We named the compendium putidaPRECISE321, for putida Precision RNA-seq Expression Compendium for Independent Signal Exploration. It consists of 321 samples from 118 unique experimental conditions across 21 projects [22][23][24][25][26][27][28][29][30][31] . We centered each project to a baseline condition in order to remove batch effects (Supplementary Fig.…”

Engineering Pseudomonas putida for improved utilization of syringyl aromatics

“…Li et al, 2020; W. J. Li et al, 2019), and xylose (Lim et al, 2021). Our ALE process spanned over 3 months to improve an average growth rate of the ALE culture from 0.4 to 2.8 day −1 , which is a combined result of shortened lag phase and increased growth rate.…”

Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms

“…The soil bacterium Pseudomonas putida maintains significant advantages over common production chassis due to its natural biodegradation pathways and oxidative stress tolerance, which has contributed to its broad proliferation in many environmental niches. Several studies have explored substrate tolerance through toxicity adaptive laboratory evolution (TALE) of P. putida ( Mohamed et al, 2020 ; Lim et al, 2021 ). A recent work integrated three different xylose pathways (Dahms, Isomerase, and Weimberg) on plasmids to enable growth on xylose, a prominent component of degraded hemicellulose ( Bator et al, 2019 ).…”