Biology subjects

Petzold, C. J.

Publications and source records attributed to Petzold, C. J..

35 records · Page 2Linked to original sources

bioRxiv2023

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

Type I polyketide synthases (T1PKSs) hold an enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite the great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enables the production of an unnatural polyketide in each of the hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. Here, we not only highlight the significance of codon optimization but also establish the groundwork for high-throughput assembly and characterization of PKS pathways in alternative hosts.

synthetic biology↗

bioRxiv2023

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

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 optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized 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 revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. 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 via CRISPRi led to faster growth and uptake of 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 and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatics making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatics present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

microbiology↗

bioRxiv2023

Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane), and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

synthetic biology↗

bioRxiv2023

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para-coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para-coumarate minimal medium. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/532821v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f0754aorg.highwire.dtl.DTLVardef@a45d24org.highwire.dtl.DTLVardef@df0596org.highwire.dtl.DTLVardef@1e7e140_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

Revealing Pentose Catabolism in Pseudomonas putida

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 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. Originality-Significance StatementMembers of the Pseudomonas putida group are intensively studied for their role in plant growth promotion and biomass conversion. Despite this interest, the scope of pentose oxidation, key sugars in plant biomass, in this group is not known. Here, we report targeted isolation of members of the P. putida group that grow by xylose and arabinose oxidation. Using a combined genomic and proteomic approach, we identify gene products involved in pentose oxidation and identify conditionally essential genes for xylose oxidation using a CRISPRi gene repression approach. This work describes a targeted isolation and analysis strategy that may applied for many microbial groups of industrial and agricultural interest.

microbiology↗

bioRxiv2022

Mixed Heavy Metals Stress Induces Global Iron Starvation as Revealed by System Level Multi-Omic Analyses

Globally, multiple heavy metal contamination is an increasingly common problem. As heavy metals have the potential to disrupt microbially-mediated biogeochemical cycling, it is critical to understand their impact on microbial physiology. However, systems-level studies on the effects of a combination of heavy metals on bacteria are lacking. Here, we use a native Bacillus cereus isolate from the subsurface of the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) -- representing a highly abundant species at the site-- to assess the combined impact of eight metal contaminants. Using this metal mixture and individual metals, all at concentrations based on the ORR site geochemistry, we performed growth experiments and proteomic analyses of the B. cereus strain, in combination with targeted MS-based metabolomics and gene expression profiling. The combination of eight metals impacts cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Specifically, exposure to the metal mixture elicited global iron starvation responses not observed in any of the individual metal treatments. As nitrate is also a significant contaminant at the ORR site and nitrate and nitrite reductases are iron-containing enzymes, we also examined the effects of the metal mixture on reduction of nitrogen oxides. We found that the metal mixture inhibits the activity of these enzymes through a combination of direct enzymatic damage and post-transcriptional and post-translational regulation. Altogether, these data suggest that metal mixture studies are critical for understanding how multiple rather than individual metals influence microbial processes in the environment.

microbiology↗

bioRxiv2020

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr(R) and 2 L bioreactors.

bioengineering↗

bioRxiv2019

Succession of physiological stages hallmarks the transcriptomic response of fungus Aspergillus niger to lignocellulose

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergilllus niger over its life time to six substrates important for biofuel production.\n\nResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.\n\nConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

microbiology↗

bioRxiv2019

Allosteric priming of E. coli CheY by the flagellar motor protein FliM

Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliMN with and without D13K-Y106W have shown FliMN bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliMN interface; namely the closure of the 4-{beta}4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY.FliMN complex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated community sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far. Statement of SignificanceCheY affinity for FliMN, its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY.FliMN crystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliMN affinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliMN interface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliMN-tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

biophysics↗

bioRxiv2019

The response of Pseudomonas putida to a complex lignolysate

There is strong interest in the valorization of lignin derived from plant biomass to produce valuable products; however, the structural complexity of this biopolymer has been a major bottleneck to conversion. Chemical pretreatment liberates soluble fractions of lignin that may be upgraded by biological conversion. Here, ionic liquid pretreatment was employed to obtain soluble aromatic-rich fractions from sorghum, which were converted by Pseudomonas putida KT2440, a promising host for bioconversion of aromatics derived from lignin. Growth studies and mutational analysis demonstrated that P. putida growth on these soluble lignin-derived fractions, referred to as lignolysate, was dependent on aromatic monomers derived from lignin (p-coumarate and ferulate), but other, unknown factors in the lignolysate contributed to growth. Proteomic and metabolomic analyses provided evidence that these unknown factors were amino acids and residual ionic liquid. Proteomic measurements indicated a coordinated response in which these substrates were catabolized simultaneously. A cholinium catabolic pathway was identified and deletion of five genes in the pathway abrogated the ability of P. putida to grow on cholinium as a sole carbon source. This work demonstrates that lignolysates obtained through biomass pretreatment contain multiple substrates and conversion strategies for lignin-derived should take this complexity into account.\n\nImportanceLignin is one of the most abundant biopolymers on Earth and is generated as a co-product in the processing of lignocellulosic biomass. Valorization of these residual lignin streams is a promising method to enhance the economic viability of modern lignocellulosic biorefineries. In this study we developed a process to couple chemical depolymerization of lignin and biological conversion using Pseudomonas putida KT2440. Water-soluble and bioavailable lignolysate was obtained from sorghum and further characterized as a growth substrate for P. putida. Proteomic and metabolomic analyses demonstrated that P. putida metabolized other components of the lignolysate beyond monoaromatic compounds, which illuminates how microbes can process complex lignolysates obtained from plants. Understanding the underlying microbial responses in lignolysates will enable the design of rational strategies for lignin valorization.

microbiology↗

bioRxiv2019

Structural control of regioselectivity in an unusual bacterial acyl-CoA dehydrogenase

Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an {gamma},{delta}-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical ,{beta}-ACADs, it acts regioselectively at the {gamma},{delta}-position and only on ,{beta}-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the ,{beta} position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative {gamma},{delta}-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homolog of TcsD also regioselectively oxidizes ,{beta}-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.

biochemistry↗

bioRxiv2019

Host engineering for improved valerolactam production in Pseudomonas putida

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

synthetic biology↗

bioRxiv2019

The metabolic response of Pseudomonas taiwanensis to NADH dehydrogenase deficiency

Obligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions.\n\nWe here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed none or only a marginal phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics and enzyme activity assays revealed distinct responses to type II and type I dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes by rerouting of metabolic fluxes.\n\nThis metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate metabolic engineering efforts.\n\nImportanceWhile Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and consequently competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

physiology↗

bioRxiv2019

Versatile polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

synthetic biology↗

bioRxiv2019

Large-scale chemical-genetics of the human gut bacterium Bacteroides thetaiotaomicron

The genomic catalogue of the human microbiota has expanded dramatically in recent years, and insights derived from human microbiota genomics has vast potential to generate treatments for human diseases. However, predictably harnessing the microbiota for beneficial outcomes is currently limited by our lack of understanding of the physiology of the constituent bacteria. For instance, the functions of most of their genes are not known. Here, we systematically measure mutant phenotypes for genes from the gut commensal Bacteroides thetaiotaomicron. Using a barcoded transposon mutant library, we measured the fitness of 4,055 B. thetaiotaomicron genes across 492 experiments, including growth on 45 carbon substrates and in the presence of 57 stress-inducing compounds. Our data is in strong agreement with previous studies, and more importantly also uncovers the biological roles of poorly annotated genes. We identified 497 genes with a specific phenotype in only one or a handful of conditions, thus enabling informed predictions of gene function for a subset of these genes. For example, we identified a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a DUF1080 protein for disaccharide utilization, and a tripartite multidrug resistance system specifically important for bile salt tolerance. Our approach can be applied to other members of the human microbiota to experimentally characterize their genes.

microbiology↗

bioRxiv2018

Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism

The lysine metabolism of Pseudomonas putida can produce multiple important commodity chemicals and is implicated in rhizosphere colonization. However, despite intensive study, the biochemical and genetic links between lysine metabolism and central metabolism remain unresolved in P. putida. Here, we leverage Random Barcode Transposon Sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L- and D-lysine metabolism. We first describe three pathway enzymes that catabolize 2-aminoadipate (2AA) to 2-ketoglutarate (2KG) connecting D-lysine to the TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family without a previously described biological function. We demonstrate PP_5260 converts 2-oxoadipate (2OA) to 2-hydroxyglutarate (2HG), a novel biochemical reaction. We expand on recent work showing that the glutarate hydroxylase, CsiD, can co-utilize both 2OA and 2KG as a co-substrate in the hydroxylation of glutarate. Finally we demonstrate that the cellular abundance of D- and L-lysine pathway proteins are highly sensitive to pathway specific substrates. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria.\n\nImportanceO_LIP. putida is an attractive host for metabolic engineering as its lysine metabolism can be utilized for the production of multiple important commodity chemicals.\nC_LIO_LIWe demonstrate the first biochemical evidence of a bacterial 2OA catabolic pathway to central metabolites.\nC_LIO_LIDUF1338 proteins are widely dispersed across many kingdoms of life. Here we demonstrate the first biochemical evidence of function for a member of this protein family.\nC_LI

microbiology↗

bioRxiv2018

Sulfated RaxX, which represents an unclassified group of ribosomally synthesized post-translationally modified peptides, binds a host immune receptor

The rice immune receptor XA21 is activated by the sulfated microbial peptide RaxX (required for activation of XA21-mediated immunity X) produced by Xanthomonas oryzae pv. oryzae (Xoo). Mutational studies and targeted proteomics revealed that RaxX is processed and secreted by the protease/transporter RaxB, whose function can be partially fulfilled by a noncognate peptidase-containing transporter B (PctB). RaxX is cleaved at a Gly-Gly motif, yielding a mature peptide that retains the necessary elements for RaxX function as an immunogen and host peptide hormone mimic. These results indicate that RaxX is a founding member of a previously unclassified and understudied group of tyrosine sulfated RiPPs (ribosomally synthesized, post-translationally modified peptides). We further demonstrate that sulfated RaxX directly binds XA21 with high affinity. This work reveals a complete, previously uncharacterized biological process: bacterial RiPP biosynthesis, secretion, binding to a eukaryotic receptor and triggering of a robust host immune response.

microbiology↗