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Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

ABSTRACT: Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Pseudomonas Putida

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD041554 | Pride | 2023-09-07

REPOSITORIES: Pride

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			Action	DRS
	KT2440_Fer-R1.mzid.gz	Mzid
	KT2440_Fer-R1.mzid_KT2440_Fer-R1.MGF	Mzid
	KT2440_Fer-R1.raw	Raw
	KT2440_Fer-R2.mzid.gz	Mzid
	KT2440_Fer-R2.mzid_KT2440_Fer-R2.MGF	Mzid

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Project description:Pseudomonas alloputida KT2440 (previously misclassified as P. putida KT2440 based on 16S rRNA gene homology) has emerged as an ideal host strain for plan t biomass valorization. However, P. alloputida KT2440 is unable to natively utilize abundant pentose sugars (e.g., xylose and arabinose) in hydrolysate streams, which may account for up to 25% of lignocellulosic biomass. In the last decades, microbes have been engineered to utilize the pentose sugars. However, most of the engineered strains were either slow-growing or displayed phenotypes that could not be replicated. In this work, we successfully isolated five Pseudomonas species with the native capability to utilize glucose, xylose and p-coumarate as a sole carbon source. These isolates were in two clusters; one set of isolates (M2 and M5) and the second set of isolates (BP6 and BP7) showed 85.6% and 96.2% ANI, respectively, to P. alloputida KT24440. BP8 showed 84.6% ANI to P. putida KT2440 and does not belong to any neighboring type strains indicating a new species. Notably, the isolates showed robust growth solely on xylose and higher growth rates (m, 0.36-0.49 h-1) when compared to only known xylose-utilizing Pseudomonas taiwanenesis VLB120 (m, 0.28 h-1) as a control. Unexpectedly, among five isolates, M2 and M5 grew solely on arabinose as well. Comprehensive analysis of genomics, transcriptomics and proteomics revealed the isolates utilize xylose and arabinose via Weimberg pathway (xylD-xylX-xylA) and oxidative pathway (araD-araX-araA), respectively. Furthermore, a preliminary result demonstrated the production of flaviolin solely on xylose and arabinose in the isolate, showing noteworthy potential to be an alternative host for lignocellulosic feedstocks into valuable products. This is the first report on isolating Pseudomonas strains natively capable of utilizing all of the major carbon sources in lignocellulosic biomass, and leading to higher consumption of available substrates and therefore maximizing the product yield.

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Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

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