Metabolic engineering of Pseudomonas putida KT2440 for medium-chain-length fatty alcohol and ester production from fatty acids

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Pseudomonas putida as a platform for medium-chain length α,ω-diol production:Opportunities and challenges

Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.</p

Metabolic engineering of Pseudomonas putida KT2440 for medium-chain-length fatty alcohol and ester production from fatty acids

Medium-chain-length fatty alcohols have broad applications in the surfactant, lubricant, and cosmetic industries. Their acetate esters are widely used as flavoring and fragrance substances. Pseudomonas putida KT2440 is a promising chassis for fatty alcohol and ester production at the industrial scale due to its robustness, versatility, and high oxidative capacity. However, P. putida has also numerous native alcohol dehydrogenases, which lead to the degradation of these alcohols and thereby hinder its use as an effective biocatalyst. Therefore, to harness its capacity as a producer, we constructed two engineered strains (WTΔpedFΔadhP, GN346ΔadhP) incapable of growing on mcl-fatty alcohols by deleting either a cytochrome c oxidase PedF and a short-chain alcohol dehydrogenase AdhP in P. putida or AdhP in P. putida GN346. Carboxylic acid reductase, phosphopantetheinyl transferase, and alcohol acetyltransferase were expressed in the engineered P. putida strains to produce hexyl acetate. Overexpression of transporters further increased 1-hexanol and hexyl acetate production. The optimal strain G23E-MPAscTP produced 93.8 mg/L 1-hexanol and 160.5 mg/L hexyl acetate, with a yield of 63.1%. The engineered strain is applicable for C6–C10 fatty alcohols and their acetate ester production. This study lays a foundation for P. putida being used as a microbial cell factory for sustainable synthesis of a broad range of products based on medium-chain-length fatty alcohols

Microbial production of medium-chain-length α, ω-diols via two-stage process under mild conditions

Medium-chain-length α, ω-diols (mcl-diols) are versatile compounds widely used as building blocks of coating materials and polymers. Mcl-diols are currently synthesized through energy intensive chemical process. Recently, esterified diols have been produced from n-alkanes in E. coli by co-expression of the alkane monooxygenase module (AlkBGTL) and the esterification module (Atf1), thereby establishing the technical feasibility of the process. However, esterified diols need to be hydrolyzed for further applications. In this study, we developed bio-catalysts for mcl-diol production from n-alkanes under mild conditions. The engineered P. putida KT2440 with overexpression of Est12 can efficiently hydrolyze esterified diols (C6-C10). Later, the engineered strain was co-cultured with an E. coli strain (AlkBGTL-Atf1) to produce mcl-diols. In a two-stage approach, 5 mM 1,6-hexanediol was produced, 61.5 times of one-stage test, from n-hexane by biocatalysts for the first time. In conclusion, the present work indicates that bio-catalysis offers a green biobased alternative for synthesis of mcl-diols

When metabolic prowess is too much of a good thing : how carbon catabolite repression and metabolic versatility impede production of esterified α,ω-diols in Pseudomonas putida KT2440

Background: Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols. Results: AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain. Conclusions: This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes

Revisiting Nonresidential Environmental Exposures and Childhood Lead Poisoning in the US: Findings from Kansas, 2000–2005

Although blood lead levels (BLLs) in US children have dramatically declined over the past 40 years, there remain pockets of children living in areas with elevated BLLs. While some increases (≥10 μg/dL) may be associated with legacy lead paint, ambient air lead may be contributing to the problem. A deidentified dataset of information on over 60,000 Kansas children under 3 years of age who were tested for BLL was provided through the Kansas Environmental Public Health Tracking Network for the period 2000–2005. Using ArcGIS, we calculated distance (in miles) from a lead-emitting industry referred to as a toxic release inventory (TRI) site. The USEPA TRI database tracks the management of certain toxic chemicals that may pose a threat to human health. US facilities in different industry sectors must report annually amount of substances like lead into the environment including their exact location. Distance from a TRI site was inversely related to BLL after controlling for area-level poverty and pre-1950 housing. The results of our evaluation indicate there is a significant relationship between proximity to lead industry and childhood BLLs. Proximity to sources of lead emissions should be evaluated as a possible factor when identifying children for targeted BLL testing