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Simultaneous Optimization of Multi-lignocellulosic-sugar Catabolism via Systematic Laboratory Evolution in Dynamic Conditions

ABSTRACT: The development of microbial bioproduction is challenged by the complexity and variability of industrial feedstocks. This study aimed to systematically and simultaneously optimize catabolism of multiple sugars present in common renewable lignocellulose by adaptive laboratory evolution (ALE) using dynamic selection regimes. The engineered strain, JE3692, was subjected to ALE using three dynamic regimes— a high sugar mixture (HSM), low sugar mixture (LSM), and glucose-xylose-arabinose (GXA) series—and was compared with static ALE protocols using individual sugars. Physiological characterization of evolved strains revealed that the LSM strategy yielded the most significant improvements in catabolic efficiency and growth rates under industrially relevant mixed sugar conditions. Genomic analysis identified key mutations in heterologous pathways that contributed to the enhanced catabolism of xylose and arabinose, while proteomic analysis linked these mutations to changes in protein expression profiles in the LSM1 strain. The LSM1 strain demonstrated superior performance in producing the value-added product indigoidine, highlighting the potential of dynamic ALE for developing robust microbial strains for industrial bioproduction using lignocellulosic biomass

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ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD055215 | Pride | 2026-06-29

REPOSITORIES: Pride

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Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

			Action	DRS
	JE3692_Glc-R1.raw	Raw
	JE3692_Glc-R2.raw	Raw
	JE3692_Glc-R3.raw	Raw
	JE3692_Glc-R4.raw	Raw
	JE3692_HSM-R1.raw	Raw

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Simultaneous Optimization of Multi-lignocellulosic-sugar Catabolism via Systematic Laboratory Evolution in Dynamic Conditions

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