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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.

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.

Terpenoids constitute a class of compounds with remarkable potential for pharmaceutical, fragrance, specialty chemical, and biofuel applications. However, their industrial production is limited by their rarity within their native plant hosts, creating considerable interest in microbial hosts capable of manufacturing terpenoids. To reduce production costs, nondestructive product recovery from these microbial hosts is preferred, and is achievable using a hydrophobic organic overlay. Our prior research has indicated that oxidized fatty acyl products may permeate faster through host membranes, increasing overall biorefinery productivity. To test this hypothesis for terpenoids, we computed membrane permeabilities of conventional terpenoid target products (e.g., limonene, bisabolene, farnesene) and related oxidized compounds through molecular dynamics simulations. These simulations indicate that terpenoid product permeabilities from cytosol to overlay are oxidation independent, as increases in membrane extraction efficiency due to product oxidation are proportionally offset by decreases in the membrane crossing rate if the membrane and organic phase are in close contact. However, if aqueous extraction is required, oxidation will accelerate the slow product extraction from the membrane. Experimental toxicity assays performed indicated that most terpenoids tested were tolerated by microbial hosts, although exposure to oxidized terpenes often retarded microbial growth compared with conventional terpenes. Thus, terpenoid oxidation is not expected to significantly increase or decrease the extraction productivity in an industrial setting where cells are in close contact, unlike the previously studied fatty acyl products.

Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids. Biotechnol. Bioeng. 2015;112: 1613–1622. © 2015 Wiley Periodicals, Inc.

The intrinsic structural properties of branched long-chain fatty alcohols (BLFLs) in the range of C12 to C18 make them more suitable as diesel fuel replacements and for other industrial applications than their straight-chain counterparts. While microbial production of straight long-chain fatty alcohols has been achieved, biosynthesis of BLFLs has never been reported. In this work, we engineered four different biosynthetic pathways in Escherichia coli to produce BLFLs. We then employed a modular engineering approach to optimize the supply of α-keto acid precursors and produced either odd-chain or even-chain BLFLs with high selectivity, reaching 70 and 75% of total fatty alcohols, respectively. The acyl-ACP and alcohol-producing modules were also extensively optimized to balance enzyme expression level and ratio, resulting in a 6.5-fold improvement in BLFL titers. The best performing strain overexpressed 14 genes from 6 engineered operons and produced 350 mg/L of BLFLs in fed-batch fermenter. The modular engineering strategy successfully facilitated microbial production of BLFLs and allowed us to quickly optimize new BLFL pathway with high titers and product specificity. More generally, this work provides pathways and knowledge for the production of BLFLs and BLFL-related, industry-relevant chemicals in high titers and yields. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0936-4) contains supplementary material, which is available to authorized users.

Microbial production of chemicals has provided an attractive alternative to chemical synthesis. A key to make this technology economically viable is to improve titers, productivities, and strain robustness, all of which are often limited by metabolic imbalances. To overcome this problem, dynamic regulation is desired to adjust gene expression and metabolite concentrations to desirable levels, thus reducing cellular stress and avoiding waste of carbon and energy. This chapter will focus on current technologies used to design and build dynamic regulatory systems. This chapter starts from the basics of dynamic regulation to provide a general introduction and rationale of the technologies being used. Next, we will discuss current progress in experimental design of dynamic pathways using various design principles at transcriptional, translational, and posttranslational levels. Besides the experimental approaches, recent progress on mathematical models will also be covered. In addition, we will summarize available tools for the construction and fine-tuning of dynamic pathways, and available models for simulation, with the aim to provide a general guide to design dynamic pathways. Finally, we will discuss future directions in the field of dynamic regulation. With the development of more sophisticated synthetic biology tools and deeper understanding of metabolic dynamics, we expect dynamic regulated pathways to become mainstream in future metabolic engineering, improving productivities, yields, and robustness of the engineered microbial cell factories.

Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

Knee osteoarthritis (OA) is a common degenerative joint disorder and a major cause of pain and disability. The hypothesis tested in this study was that the passion fruit peel extract (PFP), a flavonoid-rich dietary supplement, would reduce symptoms due to knee OA. Thirty-three OA patients were enrolled in a randomized, double-blind, placebo-controlled trial with parallel-group design. Patients received either placebo or PFP pills (150 mg, daily) in a double-blinded fashion for 2 months. The OA clinical symptoms were evaluated monthly with Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index. In the PFP group, there was a significant improvement in total WOMAC score and WOMAC subscale score of physical function after 30 days and pain after 60 days. At 60 days, reductions of 18.6%, 18%, 19.6%, and 19.2% in pain, stiffness, physical function, and composite WOMAC score, respectively, were self-reported in the PFP group. Whereas, in the placebo group, the self-reported WOMAC scores increased in every category. The results of this study show that PFP substantially alleviated osteoarthritis symptoms. This beneficial effect of PFP may be due to its antioxidant and antiinflammatory properties.

Emerging antibiotic resistance threatens human health. Gut microbes are an epidemiologically important reservoir of resistance genes (resistome), yet prior studies indicate that the true diversity of gut-associated resistomes has been underestimated. To deeply characterize the pediatric gut-associated resistome, we created metagenomic recombinant libraries in an Escherichia coli host using fecal DNA from 22 healthy infants and children (most without recent antibiotic exposure), and performed functional selections for resistance to 18 antibiotics from eight drug classes. Resistance-conferring DNA fragments were sequenced (Illumina HiSeq 2000), and reads assembled and annotated with the PARFuMS computational pipeline. Resistance to 14 of the 18 antibiotics was found in stools of infants and children. Recovered genes included chloramphenicol acetyltransferases, drug-resistant dihydrofolate reductases, rRNA methyltransferases, transcriptional regulators, multidrug efflux pumps, and every major class of beta-lactamase, aminoglycoside-modifying enzyme, and tetracycline resistance protein. Many resistance-conferring sequences were mobilizable; some had low identity to any known organism, emphasizing cryptic organisms as potentially important resistance reservoirs. We functionally confirmed three novel resistance genes, including a 16S rRNA methylase conferring aminoglycoside resistance, and two tetracycline-resistance proteins nearly identical to a bifidobacterial MFS transporter (B. longum s. longum JDM301). We provide the first report to our knowledge of resistance to folate-synthesis inhibitors conferred by a predicted Nudix hydrolase (part of the folate synthesis pathway). This functional metagenomic survey of gut-associated resistomes, the largest of its kind to date, demonstrates that fecal resistomes of healthy children are far more diverse than previously suspected, that clinically relevant resistance genes are present even without recent selective antibiotic pressure in the human host, and that cryptic gut microbes are an important resistance reservoir. The observed transferability of gut-associated resistance genes to a gram-negative (E. coli) host also suggests that the potential for gut-associated resistomes to threaten human health by mediating antibiotic resistance in pathogens warrants further investigation.