At the molecular level, polymers are long chains in which the emergent material properties are dictated by the movement, arrangement, and interactions of these chains. Key factors that contribute to how the polymer chains move and rearrange are the molecular identity and arrangement, crystallinity, and molecular weight. Generally, the monomer identity influences the final application of the polymer by dictating many properties, such as the glass transition temperature (Tg). 12 As the Tg represents a softening of the material, it is a prime factor in determining the final polymer application. Flexible molecules in the backbone, which can relax faster, may result in low Tg materials with applications such as PE bags or rubber (i.e. polybutadiene). 13 Meanwhile, rigid molecules or molecules that result in stronger interchain interactions (and relax on longer timescales) can result in high Tg materials, ideal for reinforced applications. In general, when materials are at temperatures below the Tg, the polymer chains are kinetically arrested, exhibiting higher strengths. Even though monomer identity is often the largest contribution to Tg, it is not the only factor, as molecular weight, 14 tacticity, 15 and crystallinity 16 also contribute. While nearly all polymers exhibit a Tg characteristic of their amorphous region, semi-crystalline polymers will also exhibit concomitant melting behaviour crystalline in their crystalline regions, making them semi-crystalline. Crystallinity has a direct impact on polymer properties, as increases in crystallinity augment the strength of the final product and reduce the permeability of liquids and gases. Co-monomers (e.g., isophthalic acid in poly(ethylene terephthalate) (PET)) are often used to lower or completely remove crystallinity to make polymers easier to process or more transparent. 17 Finally, molecular weight, and the distributions of molecular weights, have some effect on the thermomechanical polymer properties (e.g., increasing molecular weight leads to higher Tg, modulii, etc.). However, over a critical molecular weight, nearly all thermomechanical polymer properties are constant. The exception to this generalization is the viscosity of a polymer melt, which scales with the molecular weight to the 3-3.5 power (η ~ MW 3-3.5 ) and also encapsulates properties such as diffusivity. These factors together contribute to polymer recalcitrance by limiting polymer mobility and accessibility to chemical linkages, posing a challenge for catalytic plastics deconstruction.

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Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t-cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t-cinnamate with yields of 23 and 39% Cmol Cmol−1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol−1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t-cinnamate.

The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the l-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters β-glucuronidase and mNeonGreen, up to 480-fold induction with 1% l-arabinose, and tunability from 0.1 to 1% l-arabinose. In G. oxydans 621H, l-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in d-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB. Key points • We found the AraC-PBADsystem from E. coli MC4100 was well tunable in G. oxydans. •  In the absence of AraC orl-arabinose, expression from PBADwas extremely low. • This araC-PBADsystem could also be fully functional in other acetic acid bacteria.

Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida . Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida . We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.

Aromatic compounds such as 4-hydroxybenzoic acid are broadly applied in industry for a myriad of applications used in everyday life. However, their industrial production currently relies heavily on fossil resources and involves environmentally unfriendly production conditions, thus creating the need for more sustainable biotechnological alternatives. In this study, synthetic biology was applied to metabolically engineer Pseudomonas taiwanensis VLB120 to produce 4-hydroxybenzoate from glucose, xylose, or glycerol as sole carbon sources. Genes encoding a 4-hydroxybenzoate production pathway were integrated into the host genome and the flux toward the central precursor tyrosine was enhanced by overexpressing genes encoding key enzymes of the shikimate pathway. The flux toward tryptophan biosynthesis was decreased by introducing a P290S point mutation in the trpE gene, and degradation pathways for 4-hydroxybenzoate, 4-hydroxyphenylpyruvate and 3-dehydroshikimate were knocked out. The resulting production strains were tailored for the utilization of glucose and glycerol through the rational modification of central carbon metabolism. In batch cultivations with a completely mineral medium, the best strain produced 1.37 mM 4-hydroxybenzoate from xylose with a C-mol yield of 8% and 3.3 mM from glucose with a C-mol yield of 19.0%. Using glycerol as a sole carbon source, the C-mol yield increased to 29.6%. To our knowledge, this is the highest yield achieved by any species in a fully mineral medium. In all, the efficient conversion of bio-based substrates into 4-hydroxybenzoate by these deeply engineered P. taiwanensis strains brings the renewable production of aromatics one step closer.

Aromatics and their derivatives are valuable chemicals with a plethora of important applications and thus play an integral role in modern society. Their current production relies mostly on the exploitation of petroleum resources. Independency from dwindling fossil resources and rising environmental concerns are major driving forces for the transition towards the production of sustainable aromatics from renewable feedstocks or waste streams. Whole‐cell biocatalysis is a promising strategy that allows the valorization of highly abundant, low‐cost substrates. In the last decades, extensive efforts are undertaken to allow the production of a wide spectrum of different aromatics and derivatives using microbes as biocatalysts. Pseudomonads are intriguing hosts for biocatalysis, as they display unique characteristics beneficial for the production of aromatics, including a distinct tolerance and versatile metabolism. This review highlights biotechnological applications of Pseudomonas as host for the production of aromatics and derived compounds. This includes their de novo biosynthesis from renewable resources, biotransformations in single‐ and biphasic fermentation setups, metabolic funneling of lignin‐derived aromatics, and the upcycling of aromatic monomers from plastic waste streams. Additionally, this review provides insights into unique features of Pseudomonads that make them exceptional hosts for aromatics biotechnology and discusses engineering strategies.

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solventsensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis àla carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.