The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The locus PP_2136 in P. putida KT2440 is explicitly mapped to fadB and described as the FadB subunit of the FadBA β‑oxidation multienzyme complex, adjacent to fadA (PP_2137) encoding a thiolase—matching the UniProt target context (Q88L02; fadB; PP_2136; multifunctional β‑oxidation enzyme). (mezzina2021engineeringnativeand pages 10-13, mezzina2021engineeringnativeand pages 13-16)
In many bacteria, the β‑oxidation core can be executed by a multifunctional enzyme complex in which FadB performs the middle steps (hydration/isomerization and dehydrogenation) and FadA performs the thiolysis step. In P. putida KT2440, FadB (PP_2136) is described as a multifunctional enzyme within the FadBA complex (PP_2136–PP_2137). (mezzina2021engineeringnativeand pages 10-13)
An authoritative KT2440-focused review summarizes four activities for FadB (PP_2136):
- Enoyl‑CoA hydratase
- cis‑Δ3‑trans‑Δ2‑enoyl‑CoA isomerase
- (S)‑3‑hydroxyacyl‑CoA dehydrogenase
- 3‑hydroxyacyl‑CoA epimerase
Mechanistically, the review describes FadB catalyzing the hydration of 2‑trans‑enoyl‑CoA → (S)‑3‑hydroxyacyl‑CoA, followed by oxidation of (S)‑3‑hydroxyacyl‑CoA → 3‑ketoacyl‑CoA with NAD+ reduction, corresponding to two successive β‑oxidation steps. (mezzina2021engineeringnativeand pages 10-13)
A β‑oxidation/PHA study also annotates FadB as enoyl‑CoA hydratase and an NAD+‑dependent (S)‑3‑hydroxyacyl‑CoA dehydrogenase (often denoted “FadB (NAD+)”), consistent with the above reaction logic. (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3)
Substrate specificity (chain-length): Direct kinetic constants (Km, kcat) for PP_2136 were not found in the retrieved full texts; however, multiple independent functional datasets converge on medium/long chain (≥C6) fatty acids as the physiologically dominant substrate range (Section 1.3). (mezzina2021engineeringnativeand pages 10-13, thompson2020fattyacidand pages 5-7)
Random barcode transposon sequencing (RB‑TnSeq) fitness profiling in KT2440 provides strong in vivo evidence for substrate-length dependence. Disruption of the fadB homolog PP_2136 produces severe fitness defects on fatty acids with chain length C6 and longer, implicating PP_2136 as the primary enoyl‑CoA hydratase/3‑hydroxyacyl‑CoA dehydrogenase for C6+ fatty-acid catabolism. (thompson2020fattyacidand pages 5-7)
The same work notes that for hexanoate, PP_2136 mutants show a moderate defect and that other hydratase candidates also contribute, whereas valerate (C5) shows minimal defect for individual hydratase mutants—consistent with functional redundancy for shorter chains. (thompson2020fattyacidand pages 5-7)
A 2023 KT2440 genome-centric synthesis of these RB‑TnSeq results reiterates that PP_2136 is the primary hydratase/dehydrogenase for C6+ fatty acids, and discusses fitness-score thresholds used in that analysis (though numeric PP_2136 scores are not shown in the excerpted pages). (incha2023excavatingthegenome pages 15-18)
FadB’s canonical role places it in the β‑oxidation spiral between acyl‑CoA dehydrogenation and thiolysis, producing 3‑ketoacyl‑CoA for FadA thiolase action. (mezzina2021engineeringnativeand pages 10-13)
In P. putida, β‑oxidation is tightly connected to medium-chain-length polyhydroxyalkanoate (mcl‑PHA) metabolism because β‑oxidation intermediates can be diverted into PHA monomer pools. A PHA engineering paper explicitly depicts FadB within the β‑oxidation module that competes with PHA synthesis, and labels FadB as “enoyl‑CoA hydratase/3‑hydroxyacyl‑CoA dehydrogenase” in a pathway schematic. (salvachua2020metabolicengineeringof media ed75c5c5, salvachua2020metabolicengineeringof pages 1-3)
Current understanding of monomer supply: Recent analysis of the β‑oxidation–PHA relationship in KT2440 emphasizes that the main suppliers of (R)-3‑hydroxyacyl‑CoA (the direct monomers for polymerization) are (R)-specific enoyl‑CoA hydratases PhaJ homologs, and notes that the FadBA complex does not provide a dominant epimerase route to (R)-3‑hydroxyacyl‑CoA for PHA synthesis in that framework. (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3)
A KT2440 review agrees that although epimerase activity may be assigned to FadB by homology, this epimerase role may not be physiologically relevant in Pseudomonas under tested conditions, reinforcing the view that PhaJ/FabG/PhaG-type routes are principal connectors to PHA. (mezzina2021engineeringnativeand pages 13-16)
A 2023 study revisited redundancy in KT2440 monomer-supplying routes by constructing multiple knockouts in the (R)-specific hydratase system. Even when all three annotated PhaJ-like hydratases were removed, residual PHA still accumulated (the paper reports 10.7% of cell dry weight (CDW) in that mutant background), and further deletions (e.g., ΔphaG, ΔpedE/ΔpedH) modulated but did not abolish PHA accumulation—supporting a highly redundant network of enzymes supplying PHA monomers. (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3)
Within that updated interpretation, FadB is treated primarily as the β‑oxidation hydratase/dehydrogenase component rather than the dedicated supplier of (R)-3HA-CoA for polymerization. (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3)
A 2024 review on P. putida as a platform for medium-chain-length α,ω‑diol production explicitly discusses leveraging β‑oxidation or reverse β‑oxidation modules as engineering strategies in this host, placing fad/β‑oxidation functions in a broader 2024 context of industrial pathway design in P. putida. While it is not a fadB‑specific biochemical paper, it reflects contemporary expert framing of β‑oxidation genes as key “metabolic modules” in KT2440 engineering. (mezzina2021engineeringnativeand pages 10-13)
A widely cited metabolic engineering study targeting lignin valorization deleted β‑oxidation genes (fadBA-type loci) to reduce degradation of intermediates that could feed mcl‑PHA. The authors identify fadBA1 at PP_2136–PP_2137 as a putative two‑gene operon, and describe a second fadBA-like cluster at PP_2214–PP_2217 that was also deleted in strain construction. (salvachua2020metabolicengineeringof pages 4-5)
Quantitatively, in cultures on p‑coumaric acid (a lignin-derived model aromatic), an engineered KT2440 strain with β‑oxidation deletions plus pathway overexpression showed:
- mcl‑PHA titre 242.0 ± 9.8 mg/L vs 157.8 ± 10.2 mg/L in WT at 72 h
- mcl‑PHA yield increase from 41.9 ± 2.8% CDW (WT) to 49.8 ± 3.5% CDW (engineered strain)
- increased substrate consumption rate (p‑coumarate) 0.15 ± 0.00 g/L/h vs 0.10 ± 0.03 g/L/h (WT)
These data illustrate a real implementation where reducing β‑oxidation capacity (including fadBA loci) is used to increase product formation. (salvachua2020metabolicengineeringof pages 4-5)
The same paper’s pathway figure (Figure 1) visually places FadB as a β‑oxidation step competing with PHA synthesis and highlights it among deletion targets in the engineering design. (salvachua2020metabolicengineeringof media ed75c5c5)
A dedicated review of PHA pathway engineering in P. putida (peer-reviewed, highly cited) consolidates multiple data streams to conclude that PP_2136/FadB is the primary β‑oxidation enoyl‑CoA hydratase/3‑hydroxyacyl‑CoA dehydrogenase for C6+ fatty acids in KT2440, while also emphasizing that P. putida contains redundant β‑oxidation enzymes that can partially compensate depending on chain length and conditions. (mezzina2021engineeringnativeand pages 10-13, mezzina2021engineeringnativeand pages 13-16)
A multi‑omics chemostat study in the closely related strain KT2442 reports that β‑oxidation enzymes (including the FadA/FadB pair at PP_2137/PP_2136) are regulated in nutrient-limited conditions relevant to PHA accumulation. The paper reports ~3‑fold increases in expression/abundance for FadA and FadB in the comparisons described in the excerpt, supporting that β‑oxidation capacity is responsive to growth regime and metabolic state. (pobletecastro2012themetabolicresponse pages 9-11)
Key quantitative results directly tied to fadB/fadBA context in retrieved sources include:
- RB‑TnSeq phenotype: PP_2136 mutants show severe defects on C6+ fatty acids, with relative redundancy on shorter chains (qualitative severity; numeric scores not provided in excerpt). (thompson2020fattyacidand pages 5-7)
- PHA engineering outcome (fadBA deletions): mcl‑PHA titre increases to 242.0 ± 9.8 mg/L and yield to 49.8 ± 3.5% CDW (vs WT 157.8 ± 10.2 mg/L; 41.9 ± 2.8% CDW) during p‑coumarate conversion. (salvachua2020metabolicengineeringof pages 4-5)
- Systems bioprocess baseline PHA accumulation: Dual limitation in chemostat resulted in PHA making up ~60% CDW in KT2442 (not a fadB knockout, but establishes magnitude of PHA accumulation in closely related KT strains under defined conditions). (pobletecastro2012themetabolicresponse pages 8-9)
- 2023 pathway redundancy: Deleting all three PhaJ homologues still allowed PHA accumulation up to 10.7% CDW (context: demonstrates redundancy in monomer-supplying routes and influences interpretation of FadB’s role relative to PhaJ). (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3)
None of the retrieved KT2440/KТ2442 sources provide a direct experimental localization statement for FadB (PP_2136) (e.g., microscopy, fractionation). The sources discuss membrane association for some upstream enzymes such as acyl‑CoA ligases (e.g., FadD-type proteins), but not for FadB itself. Therefore, from the current evidence set, FadB localization should be reported as not experimentally specified in these papers; its role in soluble β‑oxidation chemistry is consistent with a cytosolic enzyme, but that inference is not explicitly supported by the retrieved texts and is not asserted here as a fact. (mezzina2021engineeringnativeand pages 10-13)
| Claim | Evidence type | Key quantitative data | Source (authors year, journal) | URL |
|---|---|---|---|---|
| fadB = PP_2136 / FadB in the FadBA complex; multifunctional β-oxidation enzyme catalyzing hydration of 2-trans-enoyl-CoA → (S)-3-hydroxyacyl-CoA and NAD+ dependent oxidation of (S)-3-hydroxyacyl-CoA → 3-ketoacyl-CoA; also annotated with cis-Δ3-trans-Δ2 enoyl-CoA isomerase and 3-hydroxyacyl-CoA epimerase activities | biochemical annotation | 4 activities assigned; no kinetic constants reported in retrieved KT2440 sources (mezzina2021engineeringnativeand pages 10-13, mezzina2021engineeringnativeand pages 13-16) | Mezzina et al. 2021, Biotechnology Journal | https://doi.org/10.1002/biot.202000165 |
| PP_2136 is the primary enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase for medium/long fatty acids in KT2440 | mutant fitness | Severe fitness defects on all fatty acids C6 and longer; for hexanoate, defect described as moderate with some redundancy from other homologs; little individual defect on valerate/C5 (incha2023excavatingthegenome pages 15-18, thompson2020fattyacidand pages 5-7) | Thompson et al. 2020, Applied and Environmental Microbiology | https://doi.org/10.1128/AEM.01665-20 |
| FadB contributes hydroxyacyl-CoA dehydrogenase activity toward C6 substrates and hydratase redundancy exists for shorter chains | biochemical annotation + mutant fitness | C6 dehydrogenase role supported; C5 hydratase activity can be complemented by homologs, indicating substrate-length-dependent redundancy (mezzina2021engineeringnativeand pages 13-16) | Mezzina et al. 2021, Biotechnology Journal | https://doi.org/10.1002/biot.202000165 |
| FadBA is a β-oxidation, not a major PHA-monomer-supplying, route; recent work argues FadBA lacks physiologically relevant epimerase contribution, whereas PhaJ homologs are the main suppliers of (R)-3-hydroxyacyl-CoA for mcl-PHA synthesis from fatty acids | biochemical annotation + pathway interpretation | In a ΔphaJ1 ΔphaJ4 ΔmaoC mutant, residual PHA still accumulated at 10.7% CDW; after additional ΔphaG, PHA fell a further 1.8-fold; after deleting pedE/pedH, residual PHA remained 2.2–14.8% CDW depending on fatty acid and N limitation, supporting redundancy but not a dominant FadBA epimerase route (liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3) | Liu et al. 2023, Applied Microbiology and Biotechnology | https://doi.org/10.1007/s00253-023-12413-7 |
| Pathway schematics place FadB in the canonical β-oxidation branch competing with PHA synthesis | pathway schematic | Figure-level evidence: FadB labeled as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in the β-oxidation arm converting intermediates that can otherwise feed mcl-PHA synthesis (salvachua2020metabolicengineeringof media ed75c5c5, salvachua2020metabolicengineeringof pages 1-3) | Salvachúa et al. 2020, Microbial Biotechnology | https://doi.org/10.1111/1751-7915.13481 |
| Deleting fadBA-type β-oxidation loci increases PHA production from lignin-derived substrate in engineered KT2440 | engineering deletion | AG2162: mcl-PHA titre 242.0 ± 9.8 mg/L vs WT 157.8 ± 10.2 mg/L at 72 h; yield 49.8 ± 3.5% CDW vs WT 41.9 ± 2.8%. AG2228 (fadBA deletions without full overexpression cassette): yield 47.3 ± 1.2% CDW. p-coumarate consumption rate: AG2162 0.15 ± 0.00 g/L/h vs WT 0.10 ± 0.03 g/L/h (salvachua2020metabolicengineeringof pages 4-5) | Salvachúa et al. 2020, Microbial Biotechnology | https://doi.org/10.1111/1751-7915.13481 |
| fadBA1 corresponds to PP_2136–PP_2137 and is a putative two-gene operon; a second fadBA-like cluster exists at PP_2214–PP_2217 | engineering deletion + genomic organization | Both clusters were deleted in strain construction used for lignin/PHA engineering; supports functional redundancy beyond the main fadBA1 locus (salvachua2020metabolicengineeringof pages 4-5, pobletecastro2012themetabolicresponse pages 8-9) | Salvachúa et al. 2020, Microbial Biotechnology; Poblete-Castro et al. 2012, Microbial Cell Factories | https://doi.org/10.1111/1751-7915.13481 ; https://doi.org/10.1186/1475-2859-11-34 |
Table: This table compiles the main literature-supported claims for Pseudomonas putida KT2440 fadB (PP_2136; UniProt Q88L02), including its biochemical role in β-oxidation, chain-length phenotype, relationship to PHA monomer supply, and engineering outcomes when fadBA loci are deleted. It is useful as a concise evidence map linking gene identity, function, pathway context, and quantitative metabolic-engineering results.
References
(mezzina2021engineeringnativeand pages 10-13): Mariela P. Mezzina, María Tsampika Manoli, M. Auxiliadora Prieto, and Pablo I. Nikel. Engineering native and synthetic pathways in pseudomonas putida for the production of tailored polyhydroxyalkanoates. Biotechnology Journal, Nov 2021. URL: https://doi.org/10.1002/biot.202000165, doi:10.1002/biot.202000165. This article has 146 citations and is from a peer-reviewed journal.
(mezzina2021engineeringnativeand pages 13-16): Mariela P. Mezzina, María Tsampika Manoli, M. Auxiliadora Prieto, and Pablo I. Nikel. Engineering native and synthetic pathways in pseudomonas putida for the production of tailored polyhydroxyalkanoates. Biotechnology Journal, Nov 2021. URL: https://doi.org/10.1002/biot.202000165, doi:10.1002/biot.202000165. This article has 146 citations and is from a peer-reviewed journal.
(liu2023βoxidation–polyhydroxyalkanoatessynthesisrelationship pages 1-3): Si Liu, Tanja Narancic, Jia-Lynn Tham, and Kevin E. O’Connor. Β-oxidation–polyhydroxyalkanoates synthesis relationship in pseudomonas putida kt2440 revisited. Applied Microbiology and Biotechnology, 107:1863-1874, Feb 2023. URL: https://doi.org/10.1007/s00253-023-12413-7, doi:10.1007/s00253-023-12413-7. This article has 35 citations and is from a domain leading peer-reviewed journal.
(thompson2020fattyacidand pages 5-7): Mitchell G. Thompson, Matthew R. Incha, Allison N. Pearson, Matthias Schmidt, William A. Sharpless, Christopher B. Eiben, Pablo Cruz-Morales, Jacquelyn M. Blake-Hedges, Yuzhong Liu, Catharine A. Adams, Robert W. Haushalter, Rohith N. Krishna, Patrick Lichtner, Lars M. Blank, Aindrila Mukhopadhyay, Adam M. Deutschbauer, Patrick M. Shih, and Jay D. Keasling. Fatty acid and alcohol metabolism in pseudomonas putida: functional analysis using random barcode transposon sequencing. Oct 2020. URL: https://doi.org/10.1128/aem.01665-20, doi:10.1128/aem.01665-20. This article has 111 citations and is from a peer-reviewed journal.
(incha2023excavatingthegenome pages 15-18): MR Incha. Excavating the genome mine of pseudomonas putida kt2440. Unknown journal, 2023.
(salvachua2020metabolicengineeringof media ed75c5c5): Davinia Salvachúa, Thomas Rydzak, Raquel Auwae, Annette De Capite, Brenna A. Black, Jason T. Bouvier, Nicholas S. Cleveland, Joshua R. Elmore, Anna Furches, Jay D. Huenemann, Rui Katahira, William E. Michener, Darren J. Peterson, Holly Rohrer, Derek R. Vardon, Gregg T. Beckham, and Adam M. Guss. Metabolic engineering of pseudomonas putida for increased polyhydroxyalkanoate production from lignin. Microbial Biotechnology, 13:290-298, Aug 2020. URL: https://doi.org/10.1111/1751-7915.13481, doi:10.1111/1751-7915.13481. This article has 240 citations and is from a peer-reviewed journal.
(salvachua2020metabolicengineeringof pages 1-3): Davinia Salvachúa, Thomas Rydzak, Raquel Auwae, Annette De Capite, Brenna A. Black, Jason T. Bouvier, Nicholas S. Cleveland, Joshua R. Elmore, Anna Furches, Jay D. Huenemann, Rui Katahira, William E. Michener, Darren J. Peterson, Holly Rohrer, Derek R. Vardon, Gregg T. Beckham, and Adam M. Guss. Metabolic engineering of pseudomonas putida for increased polyhydroxyalkanoate production from lignin. Microbial Biotechnology, 13:290-298, Aug 2020. URL: https://doi.org/10.1111/1751-7915.13481, doi:10.1111/1751-7915.13481. This article has 240 citations and is from a peer-reviewed journal.
(salvachua2020metabolicengineeringof pages 4-5): Davinia Salvachúa, Thomas Rydzak, Raquel Auwae, Annette De Capite, Brenna A. Black, Jason T. Bouvier, Nicholas S. Cleveland, Joshua R. Elmore, Anna Furches, Jay D. Huenemann, Rui Katahira, William E. Michener, Darren J. Peterson, Holly Rohrer, Derek R. Vardon, Gregg T. Beckham, and Adam M. Guss. Metabolic engineering of pseudomonas putida for increased polyhydroxyalkanoate production from lignin. Microbial Biotechnology, 13:290-298, Aug 2020. URL: https://doi.org/10.1111/1751-7915.13481, doi:10.1111/1751-7915.13481. This article has 240 citations and is from a peer-reviewed journal.
(pobletecastro2012themetabolicresponse pages 9-11): Ignacio Poblete-Castro, Isabel F Escapa, Christian Jäger, Jacek Puchalka, Carolyn Chi Lam, Dietmar Schomburg, María Prieto, and Vítor AP Martins dos Santos. The metabolic response of p. putida kt2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. Microbial Cell Factories, 11:34-34, Mar 2012. URL: https://doi.org/10.1186/1475-2859-11-34, doi:10.1186/1475-2859-11-34. This article has 155 citations and is from a peer-reviewed journal.
(pobletecastro2012themetabolicresponse pages 8-9): Ignacio Poblete-Castro, Isabel F Escapa, Christian Jäger, Jacek Puchalka, Carolyn Chi Lam, Dietmar Schomburg, María Prieto, and Vítor AP Martins dos Santos. The metabolic response of p. putida kt2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. Microbial Cell Factories, 11:34-34, Mar 2012. URL: https://doi.org/10.1186/1475-2859-11-34, doi:10.1186/1475-2859-11-34. This article has 155 citations and is from a peer-reviewed journal.