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Additional file 1:Figure S1. The experiment presented in Fig. 2 was repeated, and reproducible titers were obtained at a maximum of a C:N ratio of 40:1 for both urea and ammonium sulfate media, with a dip in TAL titers at a C:N of 8:1 in urea media. Error bars represent standard deviation of biological triplicates.Figure S2. Addition of buffer slightly improves TAL production. All buffers were added at a concentration of 100mM. Error bars represent standard deviation of biological triplicates.Figure S3. Antibiotic addition slows early growth on day 1 in YPD, but does not reduce maximum titers later on. TAL was measured after four days of growth. Antibiotic concentrations are listed in working concentration (µg/mL) under each condition in the top graph, which reflects the identical order in the bottom graph. Error bars represent standard deviation of biological triplicates.Figure S4. Comparison of hydrolysates derived from field-grown sorghum biomass that was stored via ensilage, or was dried prior to storage. Fermentation was performed in bench-scale flask cultures. (A) Titers obtained during fermentation over time. (B) Glucose during fermentation. (C) Xylose during fermentation. (D) Total viable colony forming units (CFUs) obtained at the end of the experiment. All error bars represent standard deviations of biological triplicates.Figure S5. The effect of addition of 1.25% DMSO to enhance TAL titers from ensiled hydrolysates produced from the same batch as in Supplementary Figure S4. Error bars represent standard deviation of biological triplicates.Figure S6. The same experiment performed in parallel to Fig. 4, but using only one day of pre-adaption in the final step of the seed culturing. In this case, a slight delay in growth in both dry and ensiled hydrolysates occurred during the first day. (A) Titers obtained during fermentation on filtered hydrolysates over time. (B) Growth in filtered hydrolysates, as measured by optical densities. (C) Primary carbon sources during fermentation on filtered hydrolysates, at the experiment start and end. (D) Titers obtained during fermentation on unfiltered, pasteurized hydrolysates over time. (E) Growth in unfiltered hydrolysates, as measured by viable colony forming units (CFUs) of R. toruloides on YPD plates. The limit of detectable colony units is indicated (1000 units/mL). (F) Primary carbon sources during fermentation on unfiltered hydrolysates, at the experiment start and end. All error bars represent standard deviations of biological triplicates.Figure S7. Analysis of partition coefficient of TAL between water and various common solvents used as overlays in bioreactor fermentations. Water was mixed at a 1:1 ratio with each solvent, and 6 g/L TAL was added. Subsequently, the aqueous phase was analyzed for TAL titers in five technical replicates. Error bars represent standard deviation. The partition coefficient between dodecane, durasyn164, and oleyl alcohol and water were calculated as 1.11, 0.59, and 2.68 respectively.

Additional file 1: Figure S1. Nitrogen physisorption isotherms of Pd/SiAl. Figure S2. Nitrogen physisorption isotherms of Pd/SiO2. Figure S3. Nitrogen physisorption isotherms of Pd/Al2O3. Figure S4. Nitrogen physisorption isotherms of Al2O3. Figure S5. Nitrogen physisorption isotherms of Ni/Al2O3. Figure S6. Nitrogen physisorption isotherms of Cu/Al2O3. Figure S7. Nitrogen physisorption isotherms of Pt/Al2O3. Figure S8. Mass spectrum of limonene. Figure S9. Mass spectrum of 1,8-cineole. Figure S10. Mass spectrum of p-cymene. Figure S11. GC–MS spectrum of products of Pd/SiO2 catalytic reaction with limonene for 3 h. Figure S12. GC–MS spectrum of products of Pd/SiO2 catalytic reaction with 1,8-cineole for 3 h. Figure S13. GC–MS spectrum of products of γ-Al2O3 catalytic reaction with limonene for 3 h. Figure S14. GC–MS spectrum of products of γ-Al2O3 catalytic reaction with 1,8-cineole for 3 h. Figure S15. Deactivation of 100 mg of 5 wt% Pd/Al2O3 with (A) limonene (B)1,8-cineole as the reactants under 250 °C. Figure S16. Production of 1,8-cineole. (A) production at different C/N ratios; (B) production at different induction conditions. Figure S17. Production of 1,8-cineole with HMGR_Da expressed from different plasmid configurations. (A) Plasmid configurations (HMGR_Da in the plasmid is highlighted); (B) 1, 8-cineole titers; (C) Pyruvate accumulation. Production was done in test tubes using fermentation media supplemented with 1.5% glucose, grown at 30 °C. Figure S18. Fed-batch fermentation for Ferm 3_Da. Figure S19. Glucose consumption comparison for Ferm 2 and Ferm 3.

The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual-inducible duet-expression vectors for P. putida KT2440. A number of inducible promoters (Plac , Ptac , PtetR/tetA and Pbad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

Studies on bacterial physiology are incomplete without knowledge of the signalling and regulatory systems that a bacterium uses to sense and respond to its environment. Two-component systems (TCSs) are among the most prevalent bacterial signalling systems, and they control essential and secondary physiological processes; however, even in model organisms, we lack a complete understanding of the signals sensed, the phosphotransfer partners and the functions regulated by these systems. In this review, we discuss several tools to map the genes targeted by transcriptionally acting TCSs. Many of these tools have been used for studying individual TCSs across diverse species, but systematic approaches to delineate entire signalling networks have been very few. Since genome sequences and high-throughput technologies are now readily available, the methods presented here can be applied to characterize the entire DNA-binding TCS signalling network in any bacterial species and are especially useful for non-model environmental bacteria.

Additional file 1. Supplementary figures and tables.

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under selected nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, 2 microbial isolates were able to inhibit the growth of 6 out of 11 other microbes tested. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida , to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients with implications for growth and development of the plant.

Advances in engineering biology have expanded the list of renewable compounds that can be produced at scale via biological routes from plant biomass. In most cases, these chemical products have not been evaluated for effects on biological systems, defined in the present study as bioactivity, that may be relevant to their manufacture. For sustainable chemical and fuel production, the industry needs to transition from fossil to renewable carbon sources, resulting in unprecedented expansion in the production and environmental distribution of chemicals used in biomanufacturing. Further, although some chemicals have been assessed for mammalian toxicity, environmental and agricultural hazards are largely unknown. We assessed 6 compounds that are representative of the emerging biofuel and bioproduct manufacturing process for their effect on model plants (Arabidopsis thaliana, Sorghum bicolor) and show that several alter plant seedling physiology at submillimolar concentrations. However, these responses change in the presence of individual bacterial species from the A. thaliana root microbiome. We identified 2 individual microbes that change the effect of chemical treatment on root architecture and a pooled microbial community with different effects relative to its constituents individually. The present study indicates that screening industrial chemicals for bioactivity on model organisms in the presence of their microbiomes is important for biologically and ecologically relevant risk analyses. Environ Toxicol Chem 2019;38:1911-1922. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Systems biology and synthetic biology are increasingly used to examine and modulate complex biological systems. As such, many issues arising during scaling-up microbial production processes can be addressed using these approaches. We review differences between laboratory-scale cultures and larger-scale processes to provide a perspective on those strain characteristics that are especially important during scaling. Systems biology has been used to examine a range of microbial systems for their response in bioreactors to fluctuations in nutrients, dissolved gases, and other stresses. Synthetic biology has been used both to assess and modulate strain response, and to engineer strains to improve production. We discuss these approaches and tools in the context of their use in engineering robust microbes for applications in large-scale production.

© 2018 John Wiley & Sons Ltd Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn2+efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.

The trace elements molybdenum and tungsten are essential components of cofactors of many metalloenzymes. However, in sulfate-reducing bacteria, high concentrations of molybdate and tungstate oxyanions inhibit growth, thus requiring the tight regulation of their homeostasis. By a combination of bioinformatic and experimental techniques, we identified a novel regulator family, tungstate-responsive regulator (TunR), controlling the homeostasis of tungstate and molybdate in sulfate-reducing deltaproteobacteria. The effector-sensing domains of these regulators are similar to those of the known molybdate-responsive regulator ModE, while their DNA-binding domains are homologous to XerC/XerD site-specific recombinases. Using a comparative genomics approach, we identified DNA motifs and reconstructed regulons for 40 TunR family members. Positional analysis of TunR sites and putative promoters allowed us to classify most TunR proteins into two groups: (i) activators of modABC genes encoding a high-affinity molybdenum and tungsten transporting system and (ii) repressors of genes for toluene sulfonate uptake (TSUP) family transporters. The activation of modA and modBC genes by TunR in Desulfovibrio vulgaris Hildenborough was confirmed in vivo, and we discovered that the activation was diminished in the presence of tungstate. A predicted 30-bp TunR-binding motif was confirmed by in vitro binding assays. A novel TunR family of bacterial transcriptional factors controls tungstate and molybdate homeostasis in sulfate-reducing deltaproteobacteria. We proposed that TunR proteins participate in protection of the cells from the inhibition by these oxyanions. To our knowledge, this is a unique case of a family of bacterial transcriptional factors evolved from site-specific recombinases.