Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis.

Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.

Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE).

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.

Engineering 5-hydroxymethylfurfural (HMF) oxidation in Pseudomonas boosts tolerance and accelerates 2,5-furandicarboxylic acid (FDCA) production.

Due to its tolerance properties, Pseudomonas has gained particular interest as host for oxidative upgrading of the toxic aldehyde 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA), a promising biobased alternative to terephthalate in polyesters. However, until now, the native enzymes responsible for aldehyde oxidation are unknown. Here, we report the identification of the primary HMF-converting enzymes of P. taiwanensis VLB120 and P. putida KT2440 by extended gene deletions. The key players in HMF oxidation are a molybdenum-dependent periplasmic oxidoreductase and a cytoplasmic dehydrogenase. Deletion of the corresponding genes almost completely abolished HMF oxidation, leading instead to aldehyde reduction. In this context, two HMF-reducing dehydrogenases were also revealed. These discoveries enabled enhancement of Pseudomonas' furanic aldehyde oxidation machinery by genomic overexpression of the respective genes. The resulting BOX strains (Boosted OXidation) represent superior hosts for biotechnological synthesis of FDCA from HMF. The increased oxidation rates provide greatly elevated HMF tolerance, thus tackling one of the major drawbacks of whole-cell catalysis with this aldehyde. Furthermore, the ROX (Reduced OXidation) and ROAR (Reduced Oxidation And Reduction) deletion mutants offer a solid foundation for future development of Pseudomonads as biotechnological chassis notably for scenarios where rapid HMF conversion is undesirable.

An acetyltransferase controls the metabolic flux in rubromycin polyketide biosynthesis by direct modulation of redox tailoring enzymes.

The often complex control of bacterial natural product biosynthesis typically involves global and pathway-specific transcriptional regulators of gene expression, which often limits the yield of bioactive compounds under laboratory conditions. However, little is known about regulation mechanisms on the enzymatic level. Here, we report a novel regulatory principle for natural products involving a dedicated acetyltransferase, which modifies a redox-tailoring enzyme and thereby enables pathway furcation and alternating pharmacophore assembly in rubromycin polyketide biosynthesis. The rubromycins such as griseorhodin (grh) A are complex bioactive aromatic polyketides from Actinobacteria with a hallmark bisbenzannulated [5,6]-spiroketal pharmacophore that is mainly installed by two flavoprotein monooxygenases. First, GrhO5 converts the advanced precursor collinone into the [6,6]-spiroketal containing dihydrolenticulone, before GrhO6 effectuates a ring contraction to afford the [5,6]-spiroketal. Our results show that pharmacophore assembly in addition involves the acetyl-CoA-dependent acetyltransferase GrhJ that activates GrhO6 to allow the rapid generation and release of its labile product, which is subsequently sequestered by ketoreductase GrhO10 and converted into a stable intermediate. Consequently, the biosynthesis is directed to the generation of canonical rubromycins, while the alternative spontaneous [5,6]-spiroketal hydrolysis to a ring-opened pathway product is thwarted. Presumably, this allows the bacteria to rapidly adjust the biosynthesis of functionally distinct secondary metabolites depending on nutrient and precursor (i.e. acetyl-CoA) availability. Our study thus illustrates how natural product biosynthesis can be enzymatically regulated and provides new perspectives for the improvement of in vitro enzyme activities and natural product titers via biotechnological approaches.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways.

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides.

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO2 become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.