Machine-learning from Pseudomonas putida KT2440 transcriptomes reveals its transcriptional regulatory network.

Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida KT2440, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida KT2440 and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.

Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Characterization of a Glycyl Radical Enzyme Bacterial Microcompartment Pathway in Rhodobacter capsulatus.

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

The plastid genome as a chassis for synthetic biology-enabled metabolic engineering: players in gene expression.

Owing to its small size, prokaryotic-like molecular genetics, and potential for very high transgene expression, the plastid genome (plastome) is an attractive plant synthetic biology chassis for metabolic engineering. The plastome exists as a homogenous, compact, multicopy genome within multiple-specialized differentiated plastid compartments. Because of this multiplicity, transgenes can be highly expressed. For coordinated gene expression, it is the prokaryotic molecular genetics that is an especially attractive feature. Multiple genes in a metabolic pathway can be expressed in a series of operons, which are regulated at the transcriptional and translational levels with cross talk from the plant's nuclear genome. Key features of each regulatory level are reviewed, as well as some examples of plastome-enabled metabolic engineering. We also speculate about the transformative future of plastid-based synthetic biology to enable metabolic engineering in plants as well as the problems that must be solved before routine plastome-enabled synthetic circuits can be installed.

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named "LAbrini", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain "LAbrini" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

Transcriptomic analysis of aerobic respiratory and anaerobic photosynthetic states in Rhodobacter capsulatus and their modulation by global redox regulators RegA, FnrL and CrtJ.

Anoxygenicphotosynthetic prokaryotes have simplified photosystems that represent ancient lineages that predate the more complex oxygen evolving photosystems present in cyanobacteria and chloroplasts. These organisms thrive under illuminated anaerobic photosynthetic conditions, but also have the ability to grow under dark aerobic respiratory conditions. This study provides a detailed snapshot of transcription ground states of both dark aerobic and anaerobic photosynthetic growth modes in the purple photosynthetic bacterium Rhodobactercapsulatus. Using 18 biological replicates for aerobic and photosynthetic states, we observed that 1834 genes (53 % of the genome) exhibited altered expression between aerobic and anaerobic growth. In comparison with aerobically grown cells, photosynthetically grown anaerobic cells showed decreased transcription of genes for cobalamin biosynthesis (-45 %), iron transport and homeostasis (-42 %), motility (-32 %), and glycolysis (-34 %). Conversely and more intuitively, the expression of genes involved in carbon fixation (547 %), bacteriochlorophyll biosynthesis (162 %) and carotenogenesis (114 %) were induced. We also analysed the relative contributions of known global redox transcription factors RegA, FnrL and CrtJ in regulating aerobic and anaerobic growth. Approximately 50 % of differentially expressed genes (913 of 1834) were affected by a deletion of RegA, while 33 % (598 out of 1834) were affected by FnrL, and just 7 % (136 out of 1834) by CrtJ. Numerous genes were also shown to be controlled by more than one redox responding regulator.

The RegA regulon exhibits variability in response to altered growth conditions and differs markedly between Rhodobacter species.

The RegB/RegA two-component system from Rhodobacter capsulatus regulates global changes in gene expression in response to alterations in oxygen levels. Studies have shown that RegB/RegA controls many energy-generating and energy-utilizing systems such as photosynthesis, nitrogen fixation, carbon fixation, hydrogen utilization, respiration, electron transport and denitrification. In this report, we utilized RNA-seq and ChIP-seq to analyse the breadth of genes indirectly and directly regulated by RegA. A comparison of mRNA transcript levels in wild type cells relative to a RegA deletion strain shows that there are 257 differentially expressed genes under photosynthetic defined minimal growth medium conditions and 591 differentially expressed genes when grown photosynthetically in a complex rich medium. ChIP-seq analysis also identified 61 unique RegA binding sites with a well-conserved recognition sequence, 33 of which exhibit changes in neighbouring gene expression. These transcriptome results define new members of the RegA regulon including genes involved in iron transport and motility. These results also reveal that the set of genes that are regulated by RegA are growth medium specific. Similar analyses under dark aerobic conditions where RegA is thought not to be phosphorylated by RegB reveal 40 genes that are differentially expressed in minimal medium and 20 in rich medium. Finally, a comparison of the R. capsulatus RegA regulon with the orthologous PrrA regulon in Rhodobacter sphaeroides shows that the number of photosystem genes regulated by RegA and PrrA are similar but that the identity of genes regulated by RegA and PrrA beyond those involved in photosynthesis are quite distinct.