Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates.

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

Small-molecule inducible transcriptional control in mammalian cells.

Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies.

Biosensor-guided improvements in salicylate production by recombinant Escherichia coli.

BACKGROUND: Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict. Here, a salicylate-responsive transcription factor was used to optimize the expression levels of shikimate/salicylate pathway genes in recombinant E. coli, and to screen a chromosomal transposon insertion library for improved salicylate production. RESULTS: A high-throughput colony screen was first developed based on a previously designed salicylate-responsive variant of the E. coli AraC regulatory protein ("AraC-SA"). Next, a combinatorial library was constructed comprising a series of ribosome binding site sequences corresponding to a range of predicted protein translation initiation rates, for each of six pathway genes (> 38,000 strain candidates). Screening for improved salicylate production allowed for the rapid identification of optimal gene expression patterns, conferring up to 123% improved production of salicylate in shake-flask culture. Finally, transposon mutagenesis and screening revealed that deletion of rnd (encoding RNase D) from the host chromosome further improved salicylate production by 27%. CONCLUSIONS: These results demonstrate the effectiveness of the salicylate sensor-based screening platform to rapidly identify beneficial gene expression patterns and gene knockout targets for improving production. Such customized high-throughput tools complement other cell factory engineering strategies. This approach can be generalized for the production of other shikimate-derived compounds.

Level of Fimbriation Alters the Adhesion of Escherichia coli Bacteria to Interfaces.

Adhesion of bacteria to interfaces is the first step in pathogenic infection, in biofilm formation, and in bioremediation of oil spills and other pollutants. Bacteria use a variety of surface structures to promote interfacial adhesion, with the level of expression of these structures varying in response to local conditions and environmental signals. Here, we investigated how overexpression of type 1 fimbriae, one such appendage, modifies the ability of Escherichia coli to adhere to solid substrates, via biofilm formation and yeast agglomeration, and to oil/water interfaces, via a microbial adhesion to hydrocarbon assay. A plasmid that enables inducible expression of E. coli MG1655 type 1 fimbriae was transformed into fimbriae-deficient mutant strain MG1655ΔfimA. The level of fimH gene expression in the engineered strain, measured using quantitative real-time PCR, could be tuned by changing the concentration of inducer isopropyl ß-d-1-thiogalactopyranoside (IPTG), and was higher than that in strain MG1655. Increasing the degree of fimbriation only slightly modified the surface energy and zeta potential of the bacteria, but enhanced their ability to agglomerate yeast cells and to adhere to solid substrates (as measured by biofilm formation) and to oil/water interfaces. We anticipate that the tunable extent of fimbriation accessible with this engineered strain can be used to investigate how adhesin expression modifies the ability of bacteria to adhere to interfaces and to actively self-assemble there.

Design and characterization of new ß-glucuronidase active site variants with altered substrate specificity.

OBJECTIVE: To isolate and characterize the kinetics of variants of E. coli ß-glucuronidase (GUS) having altered substrate specificity. RESULTS: Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-ß-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-ß-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant. CONCLUSIONS: The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.

Engineering Escherichia coli to increase triacetic acid lactone (TAL) production using an optimized TAL sensor-reporter system.

Triacetic acid lactone (TAL) (4-hydroxy-6-methyl-2-pyrone) can be upgraded into a variety of higher-value products, and has potential to be developed into a renewable platform chemical through metabolic engineering. We previously developed an endogenous TAL sensor based on the regulatory protein AraC, and applied it to screen 2-pyrone synthase (2-PS) variant libraries in E. coli, resulting in the identification of variants conferring up to 20-fold improved TAL production in liquid culture. In this study, the sensor-reporter system was further optimized and used to further improve TAL production from recombinant E. coli, this time by screening a genomic overexpression library. We identified new and unpredictable gene targets (betT, ompN, and pykA), whose plasmid-based expression improved TAL yield (mg/L/OD595) up to 49% over the control strain. This work further demonstrates the utility of customized transcription factors as molecular reporters in high-throughput engineering of biocatalytic strains.

Subnanometric Roughness Affects the Deposition and Mobile Adhesion of Escherichia coli on Silanized Glass Surfaces.

We investigate the deposition and transient adhesion of Escherichia coli on alkyl and fluoroalkyl silanized glass surfaces of different carbon chain lengths. The rate at which bacteria deposit onto these surfaces decreases as the shear stress is increased from 3 to 67 mPa, but trends in the deposition rate across all surfaces cannot be predicted from extended DLVO calculations of the interaction potential. As the surface root-mean-square (rms) roughness increases, the deposition rate increases and the percentage of motile tethered cells decreases. Furthermore, on surfaces of root-mean-square roughness of less than 0.2 nm, bacteria exhibit mobile adhesion, for which surface-associated cells linearly translate distances greater than approximately 1.5 times their average body length along the flow direction. E. coli bacteria with and without flagella exhibit mobile adhesion, indicating that this behavior is not driven by these appendages. Cells that express fimbriae do not exhibit mobile adhesion. These results suggest that even subnanoscale roughness can influence the deposition and transient adhesion of bacteria and imply that strategies to reduce frictional interactions by making cells or surfaces smoother may help to control the initial fouling of surfaces by E. coli bacteria.

Recent advances in engineering proteins for biocatalysis.

Protein engineers are increasingly able to rely on structure-function insights, computational methods, and deeper understanding of natural biosynthesis processes, to streamline the design and applications of enzymes. This review highlights recent successes in applying new or improved protein engineering strategies toward the design of improved enzymes and enzymes with new activities. We focus on three approaches: structure-guided protein design, computational design, and the use of novel scaffolding and compartmentalization techniques to improve performance of multienzyme systems. Examples described address problems relating to enzyme specificity, stability, and/or activity, or aim to balance sequential reactions and route intermediates by co-localizing multiple enzymes. Specific applications include improving production of biofuels using enzymes with altered cofactor specificity, production of high-value chiral compounds by enzymes with tailored substrate specificities, and accelerated cellulose degradation via multi-enzyme scaffold assemblies. Collectively, these studies demonstrate a growing variety of computational and molecular biology tools. Continued advances on these fronts coupled with better mindfulness of how to apply proteins in unique ways offer exciting prospects for future protein engineering and biocatalysis research.

The efficient synthesis and purification of 2'3'- cGAMP from Escherichia coli.

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/µg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.

Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins.

Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal ß-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal ß-barrel and the threaded α-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering.

Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter.

Triacetic acid lactone (TAL) is a signature byproduct of polyketide synthases (PKSs) and a valuable synthetic precursor. We have developed an endogenous TAL reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The reporter enabled in vivo directed evolution of Gerbera hybrida 2-pyrone synthase activity in E. coli . Two rounds of mutagenesis and high-throughput screening yielded a variant conferring ~20-fold increased TAL production. The catalytic efficiency (kcat/Km) of the variant toward the substrate malonyl-CoA was improved 19-fold. This study broadens the utility of engineered AraC variants as customized molecular reporters. In addition, the TAL reporter can find applications in other basic PKS activity screens.

Design and characterization of a salicylic acid-inducible gene expression system for Jurkat cells.

With continued progress in cell and gene therapies, there is an immediate need for exogenously tunable gene expression systems with safe and predictable behavior in specific human cell types. Here, we demonstrate the ability of the salicylic acid (SA)-inducible MarR repressor protein from Escherichia coli to regulate target gene expression in a human T lymphocyte cell line. Two lentiviral vectors, one encoding an enhanced green fluorescent protein (EGFP) reporter cassette and the other a repressor cassette, were sequentially transduced into Jurkat cells, using fluorescence-activated cell sorting (FACS) to isolate stable Jurkat progeny. As a result, EGFP expression was repressed by MarR and was inducible upon the addition of SA (~1.3 fold). This represents the first example of functional expression of bacterial MarR in mammalian cells, and opens the possibility for further development of regulated, SA-tunable gene expression system for T-cells.

Anaerobic obligatory xylitol production in Escherichia coli strains devoid of native fermentation pathways.

Anaerobic glucose oxidation was coupled to xylose reduction in a nonfermentative Escherichia coli strain expressing NADPH-dependent xylose reductase. Xylitol production serves as the primary means of NAD(P)(+) regeneration, as glucose is converted primarily to acetate and CO(2). The membrane-bound transhydrogenase PntAB is required to achieve the maximum theoretical yield of four moles of xylitol per mole of glucose consumed.

Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli.

P450-dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450-catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product-per-glucose yield (Y(PPG)) in whole-cell reactions involving a proficient NADPH-dependent P450 propane monooxygenase prepared by directed evolution [P450(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed 46:8414-8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol: glucose molar ratio (YPPG ~ 0.5) under aerobic, nongrowing conditions. By altering key processes involved in NAD(P)H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Δndh) combined with removal of two fermentation pathways (ΔadhE, Δldh) provided the highest Y(PPG) (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450(PMO)R2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in Y(PPG). Kinetic analyses suggest, however, that much more favorable parameters (K(M), k(cat)) for the NADH-driven reaction are required to effectively compete with the host's endogenous NADH-utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H-dependent whole-cell biotransformations in E. coli.

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid.

We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Heterologous expression of D-xylulokinase from Pichia stipitis enables high levels of xylitol production by engineered Escherichia coli growing on xylose.

Deletion of the Escherichia coli xylulokinase gene (xylB) is essential for achieving high xylitol titers from xylitol-producing E. coli strains growing on glucose in the presence of xylose. Our study suggests that this is due to XylB-catalyzed toxic synthesis of xylitol-phosphate. This activity prohibits the use of xylose as the sole carbon source during xylitol production by E. coli. To overcome this limitation we turned to the yeast Pichia stipitis, which naturally produces xylitol, as a source of xylulokinase (Xyl3). We examined the effects of plasmid-based expression of Xyl3 versus XylB on growth and xylitol production by engineered E. coli strains. Xylulokinase activity assays show similar levels of functional expression of both enzymes (determined as activity on xylulose), and reveal significantly more activity on xylitol by XylB compared to Xyl3. (31)P NMR confirms the production of xylitol-phosphate from in vitro reactions with XylB. Lastly, the replacement of xylB with XYL3 results in drastically enhanced xylitol titers from E. coli strains co-expressing xylose reductase during growth on xylose.

Analysis of NADPH supply during xylitol production by engineered Escherichia coli.

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.

AraC regulatory protein mutants with altered effector specificity.

The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to D-arabinose and not in response to its native effector L-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM L-arabinose does not influence the response of the reported mutants to D-arabinose, and the mutants are not induced by other sugars tested (D-xylose, D-fucose, D-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by D-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.

Engineering motility as a phenotypic response to LuxI/R-dependent quorum sensing in Escherichia coli.

The repertoire of functional outputs interfaced with the LuxI/LuxR quorum sensing system in engineered Escherichia coli has been expanded to include motility via inducible expression of motB. Appropriate choice of ribosome binding site controlling MotB translation was crucial to achieving control over motility.

Role of xylose transporters in xylitol production from engineered Escherichia coli.

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.