High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism.

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum.

Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields. IMPORTANCE Improving the ethanol yield of C. thermocellum is important for the industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data that are relevant for training genome-scale metabolic models and for improving the validity of their predictions, especially considering the reduced degree-of-freedom in the redox metabolism of the strains generated here. In addition, this study advances the fundamental understanding on the mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as on the regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid in the development of C. thermocellum for the sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.

Simple and Rapid Site-Specific Integration of Multiple Heterologous DNAs into the Escherichia coli Chromosome.

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP-containing "cargo" plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 107 CFU/µg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 105 CFU/µg DNA or higher when the recombinase-expressing plasmid and "cargo" plasmid were co-transformed. The "cargo" plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example of the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species. IMPORTANCE More rapid genetic tools can help accelerate strain engineering, even in advanced hosts like Escherichia coli. Here, we adapt a suite of site-specific recombinases to enable simple, rapid, and highly efficient site-specific integration of heterologous DNA into the chromosome. This utility of this system was demonstrated by sequential insertion of eight DNA methyltransferases into the E. coli chromosome, allowing plasmid DNA to be protected from restriction in Clostridium clariflavum and enabling genetic transformation of this organism. This integration system should also be highly portable into non-model organisms.

Mixed plastics waste valorization through tandem chemical oxidation and biological funneling.

Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of these materials, however, present major barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium, Pseudomonas putida, to funnel these oxygenated compounds into a single exemplary chemical product, either ß-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products.

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering.

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H+ symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L-1 muconate at 0.18 g L-1 h-1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.

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.

Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q.

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator tR'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to ß-ketoadipic acid by Pseudomonas putida KT2440.

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, ß-ketoadipic acid (ßKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L ßKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to ßKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems.

Zymomonas mobilis has emerged as a promising candidate for production of high-value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr-like type IV restriction modification (RM) system, a type I-F CRISPR system, a chromosomal type I RM system (hsdMSc), and a previously uncharacterized type I RM system, located on an endogenous plasmid (hsdRMSp). The DNA recognition motif of HsdRMSp was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMSc and hsdRMSp systems to that of the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMSc and HsdRMSp recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction-negative strains that we constructed and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. IMPORTANCE Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study, we developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identifying a previously unknown type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis.

Identification and characterization of proteins of unknown function (PUFs) in Clostridium thermocellum DSM 1313 strains as potential genetic engineering targets.

BACKGROUND: Mass spectrometry-based proteomics can identify and quantify thousands of proteins from individual microbial species, but a significant percentage of these proteins are unannotated and hence classified as proteins of unknown function (PUFs). Due to the difficulty in extracting meaningful metabolic information, PUFs are often overlooked or discarded during data analysis, even though they might be critically important in functional activities, in particular for metabolic engineering research. RESULTS: We optimized and employed a pipeline integrating various "guilt-by-association" (GBA) metrics, including differential expression and co-expression analyses of high-throughput mass spectrometry proteome data and phylogenetic coevolution analysis, and sequence homology-based approaches to determine putative functions for PUFs in Clostridium thermocellum. Our various analyses provided putative functional information for over 95% of the PUFs detected by mass spectrometry in a wild-type and/or an engineered strain of C. thermocellum. In particular, we validated a predicted acyltransferase PUF (WP_003519433.1) with functional activity towards 2-phenylethyl alcohol, consistent with our GBA and sequence homology-based predictions. CONCLUSIONS: This work demonstrates the value of leveraging sequence homology-based annotations with empirical evidence based on the concept of GBA to broadly predict putative functions for PUFs, opening avenues to further interrogation via targeted experiments.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid.

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (kcat/KM) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O2 consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.

Approaches to genetic tool development for rapid domestication of non-model microorganisms.

Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, "domestication" of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

Complete Genome Sequences of Four Natural Pseudomonas Isolates That Catabolize a Wide Range of Aromatic Compounds Relevant to Lignin Valorization.

Many soil microorganisms have evolved catabolic strategies to utilize phenolic compounds arising from depolymerized lignin. We report the complete genome sequences of four Pseudomonas sp. isolates that demonstrated robust growth on a wide range of aromatic monomers and dimers that are relevant to the valorization of lignin into value-added chemicals.

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid.

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h-1 and 0.38 h-1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.

Methylome and Complete Genome Sequence of Parageobacillus toebii DSM 14590T, a Thermophilic Bacterium.

Here, we present the first complete genome assembly of the thermophilic bacterium Parageobacillus toebii DSM 14590T The P. toebii DSM 14590T genome consists of a 3,270,071-bp circular chromosome and a 52,989-bp native plasmid.