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Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

Producing scalable, economically viable, low-carbon biofuels or biochemicals hinges on more efficient bioconversion processes. While microbial conversion can offer robust solutions, the native microbial growth process often redirects a large fraction of carbon to CO2 and cell mass. By integrating genome-scale metabolic models with techno-economic and life cycle assessment models, this study analyzes the effects of converting cell mass lipids to hydrocarbon fuels, and CO2 to methanol on the facility’s costs and life-cycle carbon footprint. Results show that upgrading microbial lipids or both microbial lipids and CO2 using renewable hydrogen produces carbon-negative bisabolene. Additionally, on-site electrolytic hydrogen production offers a supply of pure oxygen to use in place of air for bioconversion and fuel combustion in the boiler. To reach cost parity with conventional jet fuel, renewable hydrogen needs to be produced at less than $2.2 to $3.1/kg, with a bisabolene yield of 80% of the theoretical yield, along with cell mass and CO2 yields of 22 wt% and 54 wt%, respectively. The economic combination of cell mass, CO2, and bisabolene yields demonstrated in this study provides practical insights for prioritizing research, selecting suitable hosts, and determining necessary engineered production levels.

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino-refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin-utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino-chemical industry.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

We explore when and why large classes of proteins expand into new sequence space. We used an unsupervised machine learning approach to observe the sequence landscape of REC domains of bacterial response regulator proteins. We find that within-gene recombination can switch effector domains and, consequently, change the regulatory context of the duplicated protein.

Maximizing the production of heterologous biomolecules is a complex problem that can be addressed with a systems-level understanding of cellular metabolism and regulation. Specifically, growth-coupling approaches can increase product titers and yields and also enhance production rates. However, implementing these methods for non-canonical carbon streams is challenging due to gaps in metabolic models. Over four design-build-test-learn cycles, we rewire Pseudomonas putida KT2440 for growth-coupled production of indigoidine from para-coumarate. We explore 4,114 potential growth-coupling solutions and refine one design through laboratory evolution and ensemble data-driven methods. The final growth-coupled strain produces 7.3 g/L indigoidine at 77% maximum theoretical yield in para-coumarate minimal medium. The iterative use of growth-coupling designs and functional genomics with experimental validation was highly effective and agnostic to specific hosts, carbon streams, and final products and thus generalizable across many systems.

Building a stronger bioeconomy requires production capabilities that are largely generated through microbial genetic engineering. Plant feedstocks can additionally be genetically engineered to generate desirable feedstock traits and provide precursors for direct microbial conversion into desired products. The oleaginous yeast Rhodosporidium toruloides is a promising organism for this type of conversion as it can grow on a wide range of deconstructed biomass and consume a variety of carbon sources. Here, we leveraged R. toruloides native p-coumaric acid consumption pathway to accumulate protocatechuate (PCA) from 4-hydroxybenzoate (4HBA) released from a sorghum feedstock line genetically engineered to overproduce 4HBA. We did so by generating and evaluating an R. toruloides strain that accumulates PCA, RSΔ12623. We then show that at two scales a cholinium lysinate pretreatment with enzymatic saccharification successfully extracts 95% of the 4HBA from the engineered sorghum biomass while producing deconstructed lignin that can be more efficiently depolymerized in a subsequent thermochemical reaction. We also demonstrate that strain RSΔ12623 can convert more than 95% of 4HBA to PCA while consuming >95% of the glucose and >80% of the xylose present in sorghum hydrolysates. Finally, to evaluate the scalability of such fermentations, we conducted the conversion of 4HBA to PCA in a 2 L bioreactor under controlled conditions. This work demonstrates the potential of purposefully producing aromatic precursors in planta that can be liberated during biomass deconstruction for direct microbial conversion to desirable bioproducts.

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

Corynebacterium glutamicum, a natural glutamate-producing bacterium adopted for industrial production of amino acids, has been extensively explored recently for high-level biosynthesis of amino acid derivatives, bulk chemicals such as organic acids and short-chain alcohols, aromatics, and natural products, including polyphenols and terpenoids. Here, we review the recent advances with a focus on biosystem design principles, metabolic characterization and modeling, omics analysis, utilization of nonmodel feedstock, emerging CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools for Corynebacterium strain engineering, biosensors, and novel strains of C. glutamicum. Future research directions for developing C. glutamicum cell factories are also discussed.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6-fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2-fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5-fold and 1.8-fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.

The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.

The strategy of synergistic application of biological and chemical catalysis is an important approach for efficiently converting renewable biomass into chemicals and fuels. In particular, the method of determining the appropriate intermediate between the two catalytic methods is critical. In this work, we demonstrate p-cymene production through the integration of biosynthesis and heterogenous catalysis and show how a preferred biologically derived precursor could be determined. On the biological side, we performed the limonene and 1,8-cineole production through the mevalonate pathway. Titers of 0.605 g/L and a 1.052 g/L were achieved, respectively. This difference is in agreement with the toxicity of these compounds toward the producing microorganisms, which has implications for subsequent development of the microbial platform. On the heterogeneous catalysis side, we performed the reaction with both biological precursors to allow for direct comparison. Using hydrogenation/dehydrogenation metals on supports with acid sites, both limonene and 1,8-cineole were converted to p-cymene with similar yields under equivalent reaction conditions. Thus, we could determine that the most promising strategy would be to target 1,8-cineole, the higher titer and lower toxicity bio-derived precursor with subsequent catalytic conversion to p-cymene. We further optimized the biological production of 1,8-cineole via fed-batch fermentation and reached the titer of 4.37 g/L which is the highest known 1,8-cineole titer from microbial production. This work provides a valuable paradigm for early stage considerations to determine the best route for the high-efficiency production of a target biobased molecule using an integration of biology and chemistry.

With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C5) and epi-isozizaene (C15) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing L-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass.

Engineered cyanobacterium Synechococcus elongatus can use light and CO2 to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem where S. elongatus is freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either β-carotene-producing Yarrowia lipolytica or indigoidine-producing Pseudomonas putida) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and β-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a β-carotene titer of 1.3 g/L hydrogel, amounts 15-22-fold higher than in a comparable co-culture without encapsulation. Moreover, 13C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

BackgroundRhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R. toruloides to produce the polyketide triacetic acid lactone (TAL) directly from unfiltered hydrolysate deconstructed from biomass with minimal unit process operations.ResultsIntroduction of the 2-pyrone synthase gene into R. toruloides enabled the organism to produce 2.4 g/L TAL from simple media or 2.0 g/L from hydrolysate produced from sorghum biomass. Both of these titers are on par with titers from other better-studied microbial hosts after they had been heavily engineered. We next demonstrate that filtered hydrolysates produced from ensiled sorghum are superior to those derived from dried sorghum for TAL production, likely due to the substantial organic acids produced during ensiling. We also demonstrate that the organic acids found in ensiled biomass can be used for direct synthesis of ionic liquids within the biomass pretreatment process, enabling consolidation of unit operations of in-situ ionic liquid synthesis, pretreatment, saccharification, and fermentation into a one-pot, separations-free process. Finally, we demonstrate this consolidation in a 2 L bioreactor using unfiltered hydrolysate, producing 3.9 g/L TAL.ConclusionMany steps involved in deconstructing biomass into fermentable substrate can be combined into a distinct operation, and directly fed to cultures of engineered R. toruloides cultures for subsequent valorization into gram per liter titers of TAL in a cost-effective manner.

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system's capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.

Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels is biofuels produced by engineered microorganisms that use a renewable carbon source. Two biofuels, ethanol and biodiesel, have made inroads in displacing petroleum-based fuels, but their uptake has been limited by the amounts that can be used in conventional engines and by their cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure but have had limited uptake due to costs. In this Review, we discuss engineering metabolic pathways to produce advanced biofuels, challenges with substrate and product toxicity with regard to host microorganisms and methods to engineer tolerance, and the use of functional genomics and machine learning approaches to produce advanced biofuels and prospects for reducing their costs.

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.

Near-term decarbonization of aviation requires energy-dense, renewable liquid fuels. Biomass-derived 1,4-dimethylcyclooctane (DMCO), a cyclic alkane with a volumetric net heat of combustion up to 9.2% higher than Jet A, has the potential to serve as a low-carbon, high-performance jet fuel blendstock that may enable paraffinic bio-jet fuels to operate without aromatic compounds. DMCO can be produced from bio-derived isoprenol (3-methyl-3-buten-1-ol) through a multistep upgrading process. This study presents detailed process configurations for DMCO production to estimate the minimum selling price and life-cycle greenhouse gas (GHG) footprint considering three different hydrogenation catalysts and two bioconversion pathways. The platinum-based catalyst offers the lowest production cost and GHG footprint of $9.0/L-Jet-Aeq and 61.4 gCO2e/MJ, given the current state of technology. However, when the supply chain and process are optimized, hydrogenation with a Raney nickel catalyst is preferable, resulting in a $1.5/L-Jet-Aeq cost and 18.3 gCO2e/MJ GHG footprint if biomass sorghum is the feedstock. This price point requires dramatic improvements, including 28 metric-ton/ha sorghum yield and 95-98% of the theoretical maximum conversion of biomass-to-sugars, sugars-to-isoprenol, isoprenol-to-isoprene, and isoprene-to-DMCO. Because increased gravimetric energy density of jet fuels translates to reduced aircraft weight, DMCO also has the potential to improve aircraft efficiency, particularly on long-haul flights.

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. In this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (

Pseudomonas putida KT2440 is an emerging biomanufacturing host amenable for use with renewable carbon streams including aromatics such as para-coumarate. We used a pooled transposon library disrupting nearly all (4,778) non-essential genes to characterize this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays. Assessing differential fitness values by monitoring changes in mutant strain abundance identified 33 gene mutants with improved fitness across multiple stirred-tank bioreactor formats. Twenty-one deletion strains from this subset were reconstructed, including GacA, a regulator, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Thirteen deletion strains with roles in varying cellular functions were evaluated for conversion of para-coumarate, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved fitness in a bioreactor by 35 fold and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g, 23% of maximum theoretical yield) from para-coumarate as the carbon source.

Viruses are ubiquitous microbiome components, shaping ecosystems via strain-specific predation, horizontal gene transfer and redistribution of nutrients through host lysis. Viral impacts are important in groundwater ecosystems, where microbes drive many nutrient fluxes and metabolic processes; however, little is known about the diversity of viruses in these environments. We analyzed four groundwater plasmidomes (the entire plasmid content of an environment) and identified 200 viral sequences, which clustered into 41 genus-level viral clusters (approximately equivalent to viral genera) including 9 known and 32 putative new genera. We used publicly available bacterial whole-genome sequences (WGS) and WGS from 261 bacterial isolates from this groundwater environment to identify potential viral hosts. We linked 76 of the 200 viral sequences to a range of bacterial phyla, the majority associated with Proteobacteria, followed by Firmicutes, Bacteroidetes, and Actinobacteria. The publicly available WGS enabled mapping bacterial hosts to several viral sequences. The WGS of groundwater isolates increased the depth of host prediction by allowing host identification at the strain level. The latter included 4 viruses that were almost entirely (>99% query coverage, >99% identity) identified as integrated in the genomes of Pseudomonas, Acidovorax, and Castellaniella strains, resulting in high-confidence host assignments. Lastly, 21 of these viruses carried putative auxiliary metabolite genes for metal and antibiotic resistance, which might drive their infection cycles and/or provide selective advantage to infected hosts. Exploring the groundwater virome provides a necessary foundation for integration of viruses into ecosystem models where they are key players in microbial adaption to environmental stress. IMPORTANCE To our knowledge, this is the first study to identify the bacteriophage distribution in a groundwater ecosystem shedding light on their prevalence and distribution across metal-contaminated and background sites. Our study is uniquely based on selective sequencing of solely the extrachromosomal elements of a microbiome followed by analysis for viral signatures, thus establishing a more focused approach for phage identifications. Using this method, we detected several novel phage genera along with those previously established. Our approach of using the whole-genome sequences of hundreds of bacterial isolates from the same site enabled us to make host assignments with high confidence, several at strain levels. Certain phage genes suggest that they provide an environment-specific selective advantage to their bacterial hosts. Our study lays the foundation for future research on directed phage isolations using specific bacterial host strains to further characterize groundwater phages, their life cycles, and their effects on groundwater microbiome and biogeochemistry.

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs . absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

Machine learning provides researchers a unique opportunity to make metabolic engineering more predictable. In this review, we offer an introduction to this discipline in terms that are relatable to metabolic engineers, as well as providing in-depth illustrative examples leveraging omics data and improving production. We also include practical advice for the practitioner in terms of data management, algorithm libraries, computational resources, and important non-technical issues. A variety of applications ranging from pathway construction and optimization, to genetic editing optimization, cell factory testing, and production scale-up are discussed. Moreover, the promising relationship between machine learning and mechanistic models is thoroughly reviewed. Finally, the future perspectives and most promising directions for this combination of disciplines are examined.

Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para -coumarate ( p CA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of p CA, to a heterologous bioproduct, indigoidine. Several mutants, such as the Δ gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g p CA, 23% MTY) from p CA as the carbon source.

BackgroundNext generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.ResultsIn this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data.ConclusionsWe successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

BackgroundDespite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance.ResultsWe find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses.ConclusionsOur results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

BackgroundIn an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass.ResultsWe developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A.ConclusionOur findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) individually and in combination (pCA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L pCA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of pCA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in pCA ​+ ​FA TALE-derived strains but did not affect growth in pCA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

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.

The genomic revolution ushered in an era of discovery and characterization of enzymes from novel organisms that fueled engineering of microbes to produce commodity and high-value compounds. Over the past decade advances in synthetic biology tools in recent years contributed to significant progress in metabolic engineering efforts to produce both biofuels and bioproducts resulting in several such related items being brought to market. These successes represent a burgeoning bio-economy; however, significant resources and time are still necessary to progress a system from proof-of-concept to market. In order to fully realize this potential, methods that examine biological systems in a comprehensive, systematic and high-throughput manner are essential. Recent success in synthetic biology has coincided with the development of systems biology and analytical approaches that kept pace and scaled with technology development. Here, we review a selection of systems biology methods and their use in synthetic biology approaches for microbial biotechnology platforms.

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.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

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.

Corynebacterium glutamicum ATCC 13032 is an established and industrially-relevant microbial host that has been utilized for the expression of many desirable bioproducts. Tetra-methylpyrazine (TMP) is a naturally occurring alkylpyrazine with broad applications spanning fragrances to resins. We identified an engineered strain of C. glutamicum which produces 5 ​g/L TMP and separately, a strain which can co-produce both TMP and the biofuel compound isopentenol. Ionic liquids also stimulate TMP production in engineered strains. Using a fed batch-mode feeding strategy, ionic liquid stimulated strains produced 2.2 ​g/L of tetra-methylpyrazine. We show that feedback from a specific heterologous gene pathway on host physiology leads to acetoin accumulation and the production of TMP.

We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl-radical-enzyme reaction types known, and a promising biotechnological tool for first-time biochemical synthesis of toluene from renewable resources. Here, we used experimental and computational data to evaluate the plausibility of three candidate PhdB mechanisms, involving either attack at the phenylacetate methylene carbon or carboxyl group [via H-atom abstraction from COOH or single-electron oxidation of COO- (Kolbe-type decarboxylation)]. In vitro experimental data included assays with F-labeled phenylacetate, kinetic studies, and tests with site-directed PhdB mutants; computational data involved estimation of reaction energetics using density functional theory (DFT). The DFT results indicated that all three mechanisms are thermodynamically challenging (beyond the range of many known enzymes in terms of endergonicity or activation energy barrier), reflecting the formidable demands on PhdB for catalysis of this reaction. Evidence that PhdB was able to bind α,α-difluorophenylacetate but was unable to catalyze its decarboxylation supported the enzyme's abstraction of a methylene H atom. Diminished activity of H327A and Y691F mutants was consistent with proposed proton donor roles for His327 and Tyr691. Collectively, these and other data most strongly support PhdB attack at the methylene carbon.