Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

Abstract The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well‐characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth‐promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.


INTRODUCTION
Lignocellulosic biomass is an abundant and sustainable global source as feedstocks for the production of biofuels and bio-based products (Dahmen et al., 2019;Paul & Dutta, 2018). Biofuels and bio-based chemicals have been traditionally produced from lignocellulosic hydrolysates by microorganisms such as Saccharomyces cerevisiae and Escherichia coli (Liu et al., 2021). However, these microbial hosts are limited in substrate range and are sensitive to toxic inhibitors that are often present in hydrolysates (Piotrowski et al., 2014). Therefore, other potential hosts with broader substrate ranges and higher tolerance to inhibitors have been developed to complement S. cerevisiae and E. coli (Keasling et al., 2021). Among the most promising is Pseudomonas putida KT2440, which has been recently reclassified as Pseudomonas alloputida KT2440 (Bentley et al., 2020;Dong et al., 2019;Shi et al., 2017;Shields-Menard et al., 2018;Wang et al., 2018). Pseudomonas alloputida is a representative of the P. putida group in the Gammaproteobacteria, members of which have been intensively studied for their role in bioremediation and plant growth promotion. Pseudomonas alloputida KT2440 is of particular interest because of its ability to convert plant-derived aromatics and has been Mee-Rye Park and Rahul Gauttam contributed equally to this work. engineered to produce a variety of biofuels and biobased chemicals from both glucose and aromatics (Banerjee et al., 2020;Dong et al., 2019;Johnson et al., 2019).
Pentose sugars xylose and arabinose are the predominant constituents of hemicellulose; xylose makes up a substantial amount of the total plant sugars (10%-25% of dry biomass) followed by arabinose (usually 2%-3%, although some hydrolysates contain up to 20%) (Agrawal et al., 2015;Dehghanzad et al., 2020;Kumar, Binod, et al., 2018;Narisetty et al., 2022;Rocha et al., 2015;Zhang et al., 2014). However, P. alloputida KT2440 is not able to catabolize pentose sugars (Isikgor & Becer, 2015;Lim et al., 2021). Therefore, expanding the substrate range of P. alloptuida to include pentose sugars will improve the overall carbon conversion efficiency of lignocellulosic hydrolysates. Several approaches have been used to engineer P. alloputida to utilize pentose sugars. The xylose isomerase pathway from E. coli, which converts xylose to intermediates in the pentose phosphate pathway, has been expressed in P. alloputida and used to convert xylose to cis-cismuconic acid (Dvoř ak & de Lorenzo, 2018;Le Meur et al., 2012). Two oxidative pathways for xylose catabolism that proceed through xylonate as an intermediate, the Weimberg pathway, originally characterized in Caulobacter crescentus, and the Dahms pathway from E. coli, have been expressed in P. alloputida and have been used to produce rhamnolipids and indigoidine (Bator et al., 2020;Lim et al., 2021). Heterologous expression of isomerase (E. coli) and oxidative (Burkholderia ambifaria) pathways for arabinose catabolism has allowed it to grow on this pentose sugar . However, problems such as genetic instability, long lag-phases and low cell density have been encountered during these engineering efforts Jeffries, 2006;Kang et al., 2018;Meijnen et al., 2009).
A complementary approach to genetic engineering is to obtain isolates related to P. alloputida KT2440 that can natively grow on pentose sugars. These isolates may serve as alternative hosts for the production of biofuels and bio-based chemicals. One member of the P. putida group, Pseudomonas taiwanensis, grows on xylose (Köhler et al., 2015). Analysis of the P. tawianensis genome indicated that it possessed a variant of the oxidative xylose pathway found in C. crescentus, converting xylose through oxidative intermediates (xylonate and 2-keto-3-deoxyxylonate) to α-ketoglutarate. The P. taiwanensis pathway has been expressed in P. alloputida KT2440, conferring on it the ability to grow on xylose (Bator et al., 2020). Here, we describe a targeted isolation approach to obtaining Pseudomonas species related to P. alloputida KT2440 that grow on xylose and arabinose. These isolates illuminate the extent of pentose catabolism in the P. putida group and provide possible new hosts for metabolic engineering.

EXPERIMENTAL PROCEDURES
Soil collection, microbial isolation, and screening A total of 40 environmental samples were collected from the diverse habitats in Emeryville, CA, USA to isolate microbes. The samples were collected in sterile zip-lock plastic maintaining aseptic conditions and brought to the laboratory, then stored at 4 C. Culturable bacteria were isolated from liquid suspensions prepared at approximately 2.5% (w/v) in autoclaved minimal medium. Serial dilution of the sample suspensions was plated on Pseudomonas isolation agar (PIA) plates (Sigma-Aldrich, St. Louis, MO, USA) first and incubated for 1-2 days at 30 C. Visible bacterial colonies were selected and were sub-cultured by streaking on M9 agar plates containing 0.5% (w/v) xylose and p-coumarate, respectively, in a sequence. For purification, a single bacterial colony was re-streaked on the same medium several times. The growth of each colony was subsequently screened in M9 minimal media supplemented with 0.5% (w/v) glucose, xylose and pcoumarate as a sole carbon source at 30 C. Following the screening of isolation to obtain pure cultures, selected strains were routinely cultured in LB at 30 C overnight. The pure cultures of the isolates were preserved in 20% glycerol stock at À80 C. The soil isolates were grown in 5 ml LB broth overnight at 30 C for high-molecular-weight genomic DNA sequencing as previously described (Park et al., 2022). The genome sequences for the soil isolates have been deposited in GenBank (Park et al., 2022).

Cultivation conditions
All the Pseudomonas strains were routinely propagated in Luria Bertani (LB) media (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 2.5 g/L) at 30 C and 200 rpm. For the growth experiments and sample preparation for omics analysis, the cells were grown in minimal medium containing 6 g/L (Na 2 HPO 4 ), 3 g/L KH 2 PO 4 , 1.4 g/L (NH 4 ) 2 SO 4 , 0.5 g/L NaCl, 0.2 g/L MgSO 4 .7H 2 O, 0.015 g/L CaCl 2 .H 2 O, and 1 ml/L trace element solution purchased from Teknova (Hollister, CA, USA). Depending on the substrate of interest (glucose, xylose, arabinose, and p-coumarate), the minimal medium was supplemented with a carbon source (5 g/L). The seed cultures were prepared by inoculating a single colony from a freshly prepared LB agar plate into the minimal medium supplemented with a carbon source (5 g/L). The next day, seed cultures were used as an inoculum to prepare the first pre-culture in minimal medium with a corresponding carbon source. Following day, the strains were inoculated from pre-culture to the main culture (minimal medium with the corresponding carbon source) to start the growth kinetics experiment in 48 well plates with 250 μl of cell culture in each well and for the sample preparation of multi-omics analysis in 50 ml glass tubes. All cultivations were performed in triplicate. The growth was monitored by measuring optical density at 600 nm (OD 600nm ) using a Synergy plate reader (Biotek Instruments, Inc, Winooski, VT, USA). Maximum specific growth rates (h À1 ) were estimated by calculating the slope of the semi-log plot of OD 600nm versus time in the exponential growth phase in 15 ml culture tubes.

Phylogenetic analysis
Phylogenetic analysis and tree reconstruction were performed for each genome of isolates obtained in this study, including representative type strains in Pseudomonas strains (Keshavarz-Tohid et al., 2017 with complete or draft genome sequences retrieved from GenBank. Cellvibrio japonicus Ueda107T was included as outgroup. Nucleotide sequences were aligned using MUSCLE v3.8.425 (Edgar, 2004). Alignments were used to compute Maximum Likelihood analysis using KBase FastTree2 (Price et al., 2010) with built-in branch support values. The resulting tree with maximum likelihood analysis was visualized with iTOL software (Heidelberg, Germany) (Letunic & Bork, 2021). Taxonomic species assignment to all newly assembled and downloaded isolates was performed with tool FastANI (Jain et al., 2018) to compute whole-genome similarity metrics such as average nucleotide identity (ANI) values. Each assembly was mapped to each reference strain genome to find orthologous regions using the Mashmap method (reference), for which the average nucleotide index was then calculated and used for comparison. A 95% ANI cut-off is the most frequently used standard for species demarcation (Jain et al., 2018).

Proteomics and data analysis
Three biological replicates of each type of substrategrown cells were harvested in mid-log phase. Cell pellets of the isolates (M2, BP6, and BP8) were prepared for shotgun proteomic analysis according to a previously described chloroform/methanol precipitation method (https://doi.org/10.17504/protocols.io.bfx6jpre). Briefly, cells were resuspended in a solution with 80 μl of methanol and 20 μl of chloroform and thoroughly mixed by pipetting. Sixty microliters of water were subsequently added to the samples and mixed. Phase separation was induced with 1 min of centrifugation at 3220 Â g. The methanol and water layers were removed, and then methanol (100 μl) was added to each well. The plate was centrifuged for 2 min at 3220 Â g and then the supernatant layers were decanted. The protein pellets were resuspended in the 100 mM ammonium bicarbonate buffer supplemented with 20% methanol, and the protein concentration was determined by the DC assay (Bio-Rad). proteins from each sample were reduced by the addition of tris 2-(carboxyethyl) phosphine to 5 mM for 30 min at room temperature and followed by alkylation with iodoacetamide at 10 mM for 30 min at room temperature in the dark. Protein digestion with trypsin at 1 μg/μl concentration was accomplished with a 1:50 (w/w) trypsin/total protein ratio overnight. The resulting peptide samples were subjected to standard shotgun proteomic analysis protocol (dx.doi.org/10.17504/protocols.io.buthnwj6). Briefly, 20 μg of peptides were separated on a Sigma-Aldrich Ascentis Peptides 588 ES-C18 column (2.1 mm Â 100 mm, 2.7 μm particle size, operated at 60 C) at a 0.400 ml/min flow rate and eluted with the following gradient: initial condition was 98% solvent A (0.1% formic acid) and 2% solvent B (99.9% acetonitrile, 0.1% formic acid). Solvent B was increased to 35% over 11.5 minand then increased to 80% over 0.5 min, and held for 1.5 min, followed by a ramp back down to 2% B over 0.5 min where it was held for 1 min to re-equilibrate the column to original conditions. The eluted peptides were ionized via OptaMaxTM NG electrospray ion source operating in positive ion mode with source and acquisition parameters detailed in the protocol. The MS raw data were acquired using Thermo Scientific Xcalibur version 4.3.73, and the acquired raw data were converted to .mgf files using RawConverter tool and searched against the three annotated P. putida strains protein databases with Mascot search engine version 2.3.02 (Matrix Science). Mascot search results are refined by using Scaffold 5.0. Identified peptides are filtered by a 1% peptide-level false discovery rate. In addition, the false discovery rate at the protein level is calculated, and only the proteins with false discovery rate ≤1% are reported. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2022) with the dataset identifier PXD031549 (10.6019/PXD031549).

Constructs and strains for CRISPR interference-based gene repression in M2
For adaptation of CRISPR interference (CRISPRi) in M2 for gene repression studies, Streptococcus pasteurianus dCas9-based pRGPdCas9bad and Streptococcus pyogenes spdCas9-based pRGPspdCas9badwere used, which were previously adapted for gene repression in KT2440 (Gauttam et al., 2021). The sgRNAs and phenotypic growth measurements were designed following a previously described strategy (Gauttam et al., 2021). The cloning strategy for generating gene-specific CRISPRi vectors, the complete list for primers containing the sequence for 20-bp homology sequence for gene targeting (Table S8), and the target sequence (Table S9) for a corresponding gene can be found in the Supplementary Information S1. The test strains were generated in Pseudomonas sp. M2 and deposited in the public instance of the JBEI registry (http://public-registry.jbei.org/). The strain PP2M491 carrying pRGPspdCas9-bad (with no targeting sgRNA sequence) was used as control. The recombinant strains were grown in minimal medium supplemented with an appropriate carbon source (0.5% w/v), namely, glucose, xylose and arabinose. The oligonucleotides used in this study were ordered from Integrated DNA Technologies (IDT, San Diego, CA, USA) (Table S8).

Isolation, screening, and growth characteristics
A screen was designed to isolate Pseudomonas species that grew on xylose and plant-derived aromatics (e.g. p-coumarate) (Park et al., 2022). Sequential agar plate-based screens yielded 40 colonies, of which 5 colonies grew in liquid culture on xylose and p-coumarate. The growth of these isolates was characterized in minimal medium supplemented with 0.5% (w/v) glucose, xylose, and p-coumarate, respectively, as a sole carbon source and compared with P. alloputida KT2440 and P. taiwanensis VLB120 as controls. Five isolates were able to grow on all these substrates ( Figure 1). P. alloputida KT2440 showed slightly better growth compared with five isolates and P. taiwanensis VLB120 on glucose ( Figure 1A and Table 1). In the presence of xylose, the five isolates showed higher growth rates when compared to P. taiwanensis VLB120 and BP8 showed the highest optical density among our five isolates ( Figure 1B and Table 1); as expected, P. alloputida KT2440 was unable to grow on xylose. The growth rates of the isolates on p-coumarate were comparable to P. alloputida KT2440 and P. taiwanensis VLB120 showed no growth ( Figure 1C and Table 1). Two isolates (M2 and M5) grew on arabinose, whereas the other isolates (BP6-BP8) were not able to grow on arabinose ( Figure 1D and Table 1).

Whole genome-derived phylogenetic classification of the isolates
The phylogenetic affiliation of the five pentose-utilizing isolates was determined by whole-genome comparisons to a representative set of Pseudomonas isolates. The five isolates were affiliated with the P. putida group and are clearly distinguished from other Pseudomonas groups (Figure 2 Figure 2). Further, a FastANI analysis with an estimation of ANI represented that BP8 could be considered a new species since the ANI values between BP8 and the closest taxa were much lower than 95% (Table S1), which is the cut-off value for recognizing a new species (Jain et al., 2018). As these isolates grew on glucose and p-coumarate, the metabolic pathways for these substrates were interrogated in each genome. Strains M2, BP6, and BP8 were chosen as representatives for each phylogenetically distinct pentose-utilizing clade. Each of the isolates contained genes for a complete periplasmic oxidation pathway (gcd, gnuD [PP3382-3384], gnuK, kguK, and kguD), the Entner-Doudoroff pathway (ED; edd and eda), a phosphorylation protein (glk), and the pentose phosphate pathway (PP; zwfA and pgl); all these enzymes are involved in glucose catabolism ( Figure S1A,B). The isolates possess complete pathways for p-coumarate catabolism including side-chain oxidation (fcs, ech, vdh, and pobA), ring cleavage (pcaGH, pcaB, pcaC, and pcaD) and the β-ketoadipate conversion (pcaIJ and pcaF) ( Figure S1A,C). The predicted proteins in the glucose catabolism and aromatic degradation pathways are closely related to genes present in P. alloputida KT2440 (Nikel et al., 2015;Park et al., 2020).

Genes for pentose catabolism in P. putida isolates
The putative genes for xylose catabolism in the P. putida isolates were identified by comparison to the characterized pathway for xylose oxidation in P. taiwanensis VLB120. Homologs of xylonate dehydratase (xylD), 2-keto-3-deoxyxylonate dehydratase (xylX) and α-ketoglutarate dehydrogenase (xylA) were present in the M2, BP6 and BP8 genomes, suggesting that the isolates utilize xylose via Weimberg pathway ( Figure 3A-D). Comparing their sequences with the corresponding genes in P. taiwanensis VLB120, XylD, XlyX and XylA of three isolates showed 93.8%-97.0%, 82.2%-90.3%, and 80.3%-88.2% identity, respectively (Table S2). In addition, the three genomes and P. taiwianensis shared homologs for a LysR-type transcriptional regulator and a permease with three genomes. All the genomes had a second annotated transporter gene (annotated as MHS family metabolite: H+ symporter) between xylX and xylD and a dehydrogenase related to F I G U R E 3 Xylose and arabinose catabolism in three isolates and P. taiwanensis VLB120. Schematic overview of xylose and/or arabinose catabolic pathways of (A) M2, (B) BP6, and (C) BP8. Homologous gene clusters are involved in (D) xylose and (E) arabinose catabolic pathways. Red, orange, and grey arrows indicate cytoplasmic, cytoplasmic membrane, and unknown genes, respectively. Arrow sizes do not represent gene lengths. CIT, citrate; FUM, fumarate; ICT, isocitrate; 2-KGT, 2-ketoglutarate; MAL, malate; OAA, oxaloacetate; SucCoA, succinyl-CoA; SUC, succinate hydroxypyruvate reductase that were absent in the P. taiwanenesis genome.
The BP8 genome had an additional annotated dehydrogenase (renamed as dehydrogenase 1 in this study) and lyase adjacent to the putative hydropyruvate reductase (renamed as dehydrogenase 2 in this study), as shown in Figure 3D. Recent studies reported a nonphosphorylative pathway for xylose catabolism in Herbaspirillum seropedicae Z69 (Mal an et al., 2021) (identical to H. seropedicae SmR1) and Herbaspirillum huttiense (Watanabe et al., 2019), respectively, which transform the intermediate of 2-keto-3-deoxy-pentonate (KDP) to 5-hydroxy-2,4-dioxo-pentanonate (HDOP) by a dehydrogenase, and then a HDOP hydrolase is involved in the synthesis of pyruvate and glycolate. In this study, the dehydrogenase 1 and lyase of BP8 are closely related to dehydrogenases (62% identity from both strains) and hydrolases (72% identity from both strains) from Z69 (Mal an et al., 2021) and H. huttiense (Watanabe et al., 2019). The genomic analysis suggested that BP8 metabolizes xylose using the Weimberg pathway as well as the non-phosphorylative pathway via the KDP intermediate ( Figure 3C).
The sequences of the xylD and araD genes were used to determine the prevalence of xylose and arabinose oxidation in the P. putida group (Figure 4 and Tables S3 and S4). Interestingly, only a few strains affiliated with the P. putida group include the homologues of xylD in M2/M5 (Figure 4). For example, the xylD sequence of M2/M5 were >95% similar to the genes of BP6/BP7, P. putida W619, P. alloputida LF54 and P. taiwanensis VLB120 and showed 93% identity with that of BP8 (Table S3). Likewise, when araD of M2/M5 was compared against the Pseudomonas database, the predicted protein homologues were found in the following three species: P. putida W619 (100.0% identity), P. monteilii NBRC 103158 (98.8% identity) and P. plecoglossicida NBRC 103162 (96.2% identity) (Table S4).
Comparative proteomics analysis revealed that proteins encoded in the putative operon for xylose oxidation identified by genomic analysis were present at significantly higher abundance during growth on xylose as compared to growth on glucose in the isolates (Table 2). For example, XylD, which converts xylonate to α-keto-3-deoxy-xylonate was present at significantly higher abundance in all three strains (3.7 log 2 FC in M2; 2.9 log 2 FC in BP6; 4.6 log 2 FC in BP8). XylX, which dehydrates α-keto-3-deoxyxylonate to α-ketoglutarate semialdehyde was also present at significantly higher abundance (5.4 log 2 FC in M2; 4.5 log 2 FC in BP6; 5.3 log 2 FC in BP8), as well as XylA, which reduces α-ketoglutarate semialdehyde dehydrogenase to α-ketoglutarate (4.7 log 2 FC in M2; 4.3 log 2 FC in BP6; 5.3 log 2 FC in BP8). The proteins encoding for the predicted transporter (2.4 log 2 FC in M2; 0.6 log 2 FC in BP6; 2.5 log 2 FC in BP8) and permease (2.1 log 2 FC in M2; 1.0 log 2 FC in BP6; 2.0 log 2 FC in BP8) in putative xylose oxidation operon were also at higher abundance, suggesting that they also played a role in xylose catabolism. The annotated hydroxypyruvate reductase did not show any evidence of differential abundance. In addition, for strain BP8, the dehydrogenase 1 (4.4 log 2 FC), the lyase (4.2 log 2 FC) and the hydroxypyurvate dehydrogenase (dehydrogenase 2) (4.9 log 2 FC) were present at higher abundance in xylose-grown cells.
For P. putida M2, the arabinose-grown cells had the predicted arabinose oxidation proteins, AraDXA, at higher relative abundance compared to glucose-grown cells (3.4-4.9 log 2 FC) ( Table S5). The permease located between araX and araD, was also upregulated, F I G U R E 4 Maximum likelihood trees of xylonate dehydratase gene (xylD) from the isolates and closely related proteins (>95% sequence identity threshold) in the P. putida group using JTT matrix-based model in MEGA X(MEGA, 2018). The xylD of Caulobacter vibrioides CB15 was used as the outgroup. Numbers at each node are bootstrap probabilities by 1000 replications. The isolates in this study are in bold.
suggesting it may be involved in transport of intermediates into the cytosol.

Identifying conditionally essential genes in Strain M2 using CRISPRi
Previously, Duet vectors were adapted to express a CRISPRi/dCas9-based gene repression system in P. alloputida KT2440 (Gauttam et al., 2021). The study demonstrated the use of two dCas9 homologs from S. pasteurianus (dCas9) and S. pyogenes (spdCas9) for screening of conditionally essential genes in minimal media. To translate this system to P. putida, both dCas9 proteins were used to target six endogenous genes, (acetylglutamate kinase [argB], argininosuccinate lyase [argH], 2-keto-3-deoxy-6-phosphogluconate aldolase [eda] phosphogluconate dehydratase [edd], chorismate mutase [pheA], and orotidine-5 0 -phosphate decarboxylase [pyrF]), which were shown to be essential for strain KT2440 growth in minimal medium with glucose as sole carbon source. Minimal repression of these conditionally essential genes was not observed using the S. pasteurianus dCas9-mediated CRISPRi system ( Figure S2). In contrast, growth inhibition was observed for all six genes targeted using the S. pyogenes spdCas9-mediated CRISPRi system ( Figure S3).
To demonstrate the utility of the spdCas9-based CRISPRi system in M2, genes shown to be involved in xylose catabolism by proteomics were targeted for gene repression by CRISPRi. (Tables S6-S9). Repression of these genes did not affect growth on glucose ( Figure S4); however, repression of xylD and the predicted transporter located between xylD and xylX repressed growth on xylose ( Figure 5).

Glucose dehydrogenase is responsible for xylose and arabinose oxidation
In comparison to the well-characterized oxidative pentose pathway in Caulobacter crescentus, the oxidative pathway in the Pseudomonas isolates lacked an evident xylose or arabinose dehydrogenase. The annotated hydroxypyruvate reductase (xylose) and phosphogluconate-2-dehydrogenase (arabinose) from P. putida M2 were expressed in E. coli but showed minimal activity on xylose and arabinose, suggesting that they were not involved in C5 catabolism (data not T A B L E 2 Differential abundance profiles of proteins predicted to be involved in xylose metabolism in M2, BP6 and BP8 grown in 0.5% (w/v) xylose as a sole carbon source compared to glucose in proteomics analysis The logarithms fold changes (FC) of protein regulation in three biological replicates with their associated p value: *p < 0.01; **p < 1.0.
shown). Since these dehydrogenases characterized from M2 were not pentose dehydrogenases, the enzymes responsible for xylose and arabinose oxidation remained unknown. A gene coding for glucose dehydrogenase ( gcd) was targeted for gene repression, based on previous work in P. taiwanensis (Köhler et al., 2015) and P. alloputida KT2440 (Dvoř ak & de Lorenzo, 2018, which demonstrated that xylose oxidation was dependent on Gcd. The recombinant CRISPRi strains were cultured in minimal media supplemented with glucose, xylose or arabinose (0.5% w/v). Repression of gcd inhibited strain M2 growth in minimal media with xylose and arabinose as a sole carbon source ( Figure 6B,C). However, the growth was not affected on glucose, suggesting gcd is not a conditionally essential gene for M2 growth on glucose ( Figure 6).

DISCUSSION
This study has provided a broad perspective on the ability of members of the P. putida group to metabolize pentose sugars relevant to lignocellulose bioconversion. Targeted isolation studies carried out with xylose and p-coumarate as the targeted substrates provided isolates that expressed an oxidative pathway for xylose catabolism similar to the Weimberg pathway in C. crescentus (Shen et al., 2020). Comparison of the isolates to the P. putida group genomes indicates that xylose oxidation is relatively restricted in this group. Strain BP6 and BP7 cluster with P. alloputida, the species which is the most thoroughly characterized of the P. putida strains, including strain KT2440 (Keshavarz-Tohid et al., 2019). The BP6 and BP7 strains are most closely related to P. alloputida LF54, which is a representative of the most divergent clade of P. alloputida (Passarelli-Araujo et al., 2021). The M2 and M5 strains are affiliated with P. putida W619, which has been characterized for its ability to promote the growth of plants (Taghavi et al., 2005). P putida W619 has been shown to grow on both xylose and arabinose (Davis et al., 2013). The survey also revealed other members of the P. putida group (Pseudomonas sp. BP8 and P. vranovensis) that grew on xylose along with the F I G U R E 5 Characterization of putative conditionally essential genes for xylose catabolism in strain M2 using S. pyogenes spdCas9-based CRISPRi. The sgRNAs were designed to downregulate the expression of target genes (Gauttam et al., 2021) and sequences are listed in Table S9. For strain descriptions, refer to Table S6. The growth phenotype was assessed in minimal medium with different C-sources namely, glucose (A) and xylose (B) for the recombinant strains PP2M512 (pRGPspdCas9bad-xyl transporter) and PP2M516 (pRGPspdCas9bad-xylD).
The growth was compared to control strain PP2M491 (pRGPspdCas9bad) carrying a vector with no targeting sgRNA sequence. Data represent mean value of three biological replicates and error bars represent standard deviations.
F I G U R E 6 Phenotypic characterization of Gcd using S. pyogenes spdCas9-based CRISPR interference in minimal medium using different carbon sources: (A) glucose, (B) xylose and (C) arabinose for recombinant M2 strain JPUB_020123 (pRGPspdCas9bad-gcd). The growth was compared to control strain PP2M491 (pRGPspdCas9bad) carrying a vector with no targeting sgRNA sequence. Data represent mean value of three biological replicates and error bars represent standard deviations.
previously characterized P. taiwanensis. Interestingly, the ability to grow on arabinose is even more constrained and is only present in P. monteilii and P. plecoglossicida along with the clade containing P. putida M2/M5 and W619. These results are consistent with pentose oxidation being a niche activity in the P. putida group. Strain BP8 had genes for additional pathway for xylose oxidation that may divert intermediates in the oxidative pathway to pyruvate and glycolate. Proteins for both pathways were present in the xylose-grown cells.
Integrated genomic and proteomic analyses demonstrated that homologues of the Weimberg pathway (XylDXA and AraDXA) were responsible for xylose and arabinose oxidation in the P. putida group strains. These proteins are likely responsible for converting the oxidized sugar (xylonate and arabinoate) through multiple dehydrations and an oxidation to produce α-ketoglutarate. Repression of the genes encoding these proteins using CRISPRi in P. putida M2 demonstrated that only xylD interference repressed xylose growth. This result is consistent with studies in P. alloputida KT2440 that expression of only xylD is required to confer growth on xylose by these isolates, and that the activities of XylX and XylA can be recruited using other proteins (Lim et al., 2021). In the putative operon for xylonate oxidation, there is a transcriptional regulator and permease found in common in all the strains. However, the newly isolated strains have an annotated transporter (MHS family metabolite: H+ symporter) that is not present in P. tawianensis. The presence of the transporter, which was identified in the proteomes of Strains M2, BP6 and BP8, may improve growth on xylose relative to P. taiwanensis VLB120 (Table 1). The importance of this transporter was reinforced in the P. putida M2 CRISPRi experiments, as repression of this transporter inhibited the xylose growth of Strain M2. The possible substrate for the transporter is xylonate, as both CRISPRi-based repression of gcd in Strain M2 and deletion of gcd in Strain BP6 affected the ability of the strains to grow on xylose, indicating that Gcd oxidizes xylose to xylonate in the periplasm and then it is transported into the cytosol by the transporter present in the isolates described here. The xylose dehydrogenase activity of Gcd is consistent with previous studies on P. alloputida KT2440 (Dvoř ak & de Lorenzo, 2018), P. putida NCTC 10936 (Hardy et al., 1993) and P. taiwanensis VLB120 (Köhler et al., 2015), which demonstrated that Gcd was required for xylose oxidation. Interestingly, CRISPRi experiments also demonstrated that Gcd was required for arabinose growth, suggesting that arabinose is also oxidized to arabinoate in the periplasm and then the arabinoate is transported into the cytosol. The requirement of Gcd for pentose-based growth in multiple P. putida group strains and for multiple substrates indicates there is a link in the metabolism of all three major lignocellulose-derived sugars in the members of the P. putida group. The catalytic promiscuity of Gcd for lignocellulose-derived hexose and pentose sugars has also been demonstrated in sugar catabolism in Sulfolobus solfataricus and Sulfolobus acidocaldarius (Nunn et al., 2010).
The growth rates of the native xylose-oxidizing strains in the P. putida group are comparable or better than strains of P. alloputida KT2440 that have been engineered to grow on xylose (Elmore et al., 2022). Therefore, these isolates may be suitable complements to strain KT2440 for metabolic engineering focused on lignocellulose bioconversion.