Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the ...tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.
Summary
Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways ...are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.
In this work, a series of metabolic engineering targets to improve mcl‐PHA production were combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene (phaZ) and genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted, while phaG, alkK, phaC1, and phaC2 were overexpressed to increase carbon flux from fatty acid biosynthesis to mcl‐PHA production. This strain demonstrated an increase in mcl‐PHA titer (g l−1) and yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and from lignin compared to the wild‐type strain, resulting in a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET ...depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.
•Enabled terephthalate (TPA) and bis(2-hydroxyethyl)TPA (BHET) catabolism in P. putida•Engineered conversion of BHET to β-ketoadipate (βKA), a performance-advantaged bioproduct•Achieved a titer of 15.1 g βKA/L at a 76% molar yield from commercial BHET•Demonstrated βKA production from glycolyzed poly(ethylene TPA) (PET)
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with ...well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.
Corrigendum: In the originally published article by Salvachua et al. (2019), the author Anna Furches was accidentally omitted from the byline of manuscript, ‘Metabolic engineering of Pseudomonas ...putida for increased polyhydroxyalkanoate production from lignin’ Volume 13, Issue 1, Pages 290–298. The byline should read as given above. https://doi.org/10.1111/1751-7915.13481
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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