Engineering of industrially important microorganisms for assimilation of cellulosic biomass: towards consolidated bioprocessing.

Conversion of cellulosic biomass (non-edible plant material) to products such as chemical feedstocks and liquid fuels is a major goal of industrial biotechnology and an essential component of plans to move from an economy based on fossil carbon to one based on renewable materials. Many microorganisms can effectively degrade cellulosic biomass, but attempts to engineer this ability into industrially useful strains have met with limited success, suggesting an incomplete understanding of the process. The recent discovery and continuing study of enzymes involved in oxidative depolymerisation, as well as more detailed study of natural cellulose degradation processes, may offer a way forward.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

PaperClip: rapid multi-part DNA assembly from existing libraries.

Assembly of DNA 'parts' to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides-'Clips'. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Joint Universal Modular Plasmids: A Flexible Platform for Golden Gate Assembly in Any Microbial Host.

Modular cloning standards based on Golden Gate DNA assembly allow for construction of complex DNA constructs over several rounds of assembly. Despite being reliable and automation-friendly, each standard uses a specific set of vectors, requiring researchers to generate new tool kits for novel hosts and cloning applications. JUMP vectors (Valenzuela-Ortega and French, bioRxiv 799585, 2019) combine the robustness of modular cloning standards with the Standard European Vector Architecture and a flexible design that allows researchers to easily modify the vector backbone via secondary cloning sites. This flexibility allows for JUMP vectors to be used in a wide variety of applications and hosts.

PaperClip DNA Assembly: Reduce, Reuse, Recycle.

Creating DNA constructs is a basic and fundamental step in molecular and synthetic biology. While prices for gene synthesis are decreasing, it is still more economical in most cases to assemble constructs from a library of components (Parts). Many methods for DNA assembly are available, but most require either a fixed and inflexible format for the construct, with all Parts first being cloned in specific donor plasmids, or remaking Parts with new homology ends for each specific assembly reaction, requiring large numbers of single-use oligonucleotides. PaperClip assembly allows Parts stored in any format (linear PCR products or synthetic DNA, or cloned in any plasmid) to be used in totally flexible assembly reactions; up to 11 parts can be assembled in a single reaction, in any order, to give a linear or circular construct, and the oligonucleotides required in the assembly process can be reused in any subsequent assembly. In addition to constructing plasmids for bacterial transformation, PaperClip is also well suited to generate linear products for direct transfection of yeast, mammalian, or cyanobacterial cell lines. Thus, PaperClip offers a simple, flexible, and economical route to multipart assembly of constructs for a wide variety of purposes.

Hetero-trans-ß-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage ß-d-glucan (MLG). However, Equisetum hetero-trans-ß-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) - and, we now show, xyloglucan polysaccharide - in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose-xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

ParAlleL: A Novel Population-Based Approach to Biological Logic Gates.

In vivo logic gates have proven difficult to combine into larger devices. Our cell-based logic system, ParAlleL, decomposes a large circuit into a collection of small subcircuits working in parallel, each subcircuit responding to a different combination of inputs. A final global output is then generated by a combination of the responses. Using ParAlleL, for the first time a completely functional 3-bit full adder and full subtractor were generated using Escherichia coli cells, as well as a calculator-style display that shows a numeric result, from 0 to 7, when the proper 3 bit binary inputs are introduced into the system. ParAlleL demonstrates the use of a parallel approach for the design of cell-based logic gates that facilitates the generation and analysis of complex processes, without the need for complex genetic engineering.

Diversity and distribution of hemerythrin-like proteins in prokaryotes.

Hemerythrins are oxygen-binding proteins found in the body fluids and tissues of certain invertebrates. Oxygen is bound at a nonheme iron centre consisting of two oxo-bridged iron atoms bound to a characteristic set of conserved histidine: aspartate and glutamate residues with the motifs H-HxxxE-HxxxH-HxxxxD. It has recently been demonstrated biochemically that two bacterial proteins bearing the same motifs do in fact possess similar iron centres and bind oxygen in the same way. The recent profusion of prokaryotic genomic sequence data has shown that proteins bearing hemerythrin motifs are present in a wide variety of bacteria, and a few archaea. Some of these are short proteins as in eukaryotes; others appear to consist of a hemerythrin domain fused to another domain, generally a putative signal transduction domain such as a methyl-accepting chemotaxis protein, a histidine kinase, or a GGDEF protein (cyclic di-GMP synthase). If, as initial evidence suggests, these are in fact hemerythrin-like oxygen-binding proteins, then their diversity in prokaryotes far exceeds that seen in eukaryotes. Here, a survey is presented of prokaryotic protein sequences bearing hemerythrin-like motifs, for which the designation 'bacteriohemerythrins' is proposed, and their functions are speculated.

Characterisation of novel biomass degradation enzymes from the genome of Cellulomonas fimi.

Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format. Six novel genes showed activity in E. coli. Four interesting enzymes were characterised further. α-l-arabinofuranosidase AfsB, ß-xylosidases BxyF and BxyH and multi-functional ß-cellobiosidase/xylosidase XynF were partially purified to determine their optimum pH, temperature and kinetic parameters. One of these enzymes, BxyH, was unexpectedly found to be highly active at strong alkaline pH and at temperatures as high as 100 °C. This report demonstrates a simple method of quickly screening and characterising putative genes as BioBricks.

Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems.

Monitoring bacterial growth is an important technique required for many applications such as testing bacteria against compounds (e.g. drugs), evaluating bacterial composition in the environment (e.g. sewage and wastewater or food suspensions) and testing engineered bacteria for various functions (e.g. cellulose degradation). T?=1,^FigItem(1) ^ReloadFigure=Yesraditionally, rapid estimation of bacterial growth is performed using spectrophotometric measurement at 600nm (OD600) but this estimation does not differentiate live and dead cells or other debris. Colony counting enumerates live cells but the process is laborious and not suitable for large numbers of samples. Enumeration of live bacteria by flow cytometry is a more suitable rapid method with the use of dual staining with SYBR I Green nucleic acid gel stain and Propidium Iodide (SYBR-I/PI). Flow cytometry equipment and maintenance costs however are relatively high and this technique is unavailable in many laboratories that may require a rapid method for evaluating bacteria growth. We therefore sought to adapt and evaluate the SYBR-I/PI technique of enumerating live bacterial cells for a cheaper platform, a fluorimeter. The fluorimetry adapted SYBR-I/PI enumeration of bacteria in turbid growth media had direct correlations with OD600 (p>0.001). To enable comparison of fluorescence results across labs and instruments, a fluorescence intensity standard unit, the equivalent fluorescent DNA (EFD) was proposed, evaluated and found useful. The technique was further evaluated for its usefulness in enumerating bacteria in turbid media containing insoluble particles. Reproducible results were obtained which OD600 could not give. An alternative method based on the assessment of total protein using the Pierce Coomassie Plus (Bradford) Assay was also evaluated and compared. In all, the SYBR-I/PI method was found to be the quickest and most reliable. The protocol is potentially useful for high-throughput applications such as monitoring of growth of live bacterial cells in 96-well microplates and in assessing in vivo activity of cellulose degrading enzyme systems.

Data for discriminating dead/live bacteria in homogenous cell suspensions and the effect of insoluble substrates on turbidimetric measurements.

Estimation of bacterial growth by rapid traditional methods such as spectrophometric measurements at 600 nm (OD600) is not applicable for cultures containing insoluble particles in the growth media. Colony counts are the only suitable alternative but these are laborious and not high-throughput. The data presented in this article is related to the research article entitled "Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems" (Duedu and French, 2017) [1]. This data article presents original primary data describing the discrimination of dead/live bacteria in homogenous cell suspensions and how the presence of insoluble substrates affect the turbidity of the suspensions.

PaperClip: A Simple Method for Flexible Multi-Part DNA Assembly.

Joining DNA sequences to create linear and circular constructs is a basic requirement in molecular biology. Here we describe PaperClip, a recently developed method, which enables assembly of multiple DNA sequences in one reaction in a combinatorial manner. In contrast to other homology-based multi-part assembly methods currently available, PaperClip allows assembly of a given set of parts in any order without requiring specific single-use oligonucleotides for each assembly order.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome.

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass.

Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields.

Acute respiratory distress in Pena-Shokeir syndrome.

Pena-Shokeir syndrome is a rare, autosomal-recessive disorder that usually affects newborns. Its etiology is poorly understood. Pena-Shokeir syndrome is defined by camptodactyly, multiple ankyloses, pulmonary hypoplasia, and various facial anomalies. These manifestations are usually severe, and death generally occurs at birth or shortly thereafter. We describe a case of Pena-Shokeir syndrome in a 9-year-old girl of above-normal intelligence who presented with life-threatening airway distress. To the best of our knowledge, she is the oldest living individual with Pena-Shokeir syndrome, and the only such patient whose intelligence was not impaired. We discuss the acute management and subsequent care of this patient, who not only survived, but maintained excellent grades in school.

Enhanced resistance to nanoparticle toxicity is conferred by overproduction of extracellular polymeric substances.

The increasing production and use of engineered nanoparticles, coupled with their demonstrated toxicity to different organisms, demands the development of a systematic understanding of how nanoparticle toxicity depends on important environmental parameters as well as surface properties of both cells and nanomaterials. We demonstrate that production of the extracellular polymeric substance (EPS), colanic acid by engineered Escherichia coli protects the bacteria against silver nanoparticle toxicity. Moreover, exogenous addition of EPS to a control strain results in an increase in cell viability, as does the addition of commercial EPS polymer analogue xanthan. Furthermore, we have found that an EPS producing strain of Sinorhizobium meliloti shows higher survival upon exposure to silver nanoparticles than the parent strain. Transmission electron microscopy (TEM) observations showed that EPS traps the nanoparticles outside the cells and reduces the exposed surface area of cells to incoming nanoparticles by inducing cell aggregation. Nanoparticle size characterization in the presence of EPS and xanthan indicated a marked tendency towards aggregation. Both are likely effective mechanisms for reducing nanoparticle toxicity in the natural environment.

Synthetic biology and biomass conversion: a match made in heaven?

To move our economy onto a sustainable basis, it is essential that we find a replacement for fossil carbon as a source of liquid fuels and chemical industry feedstocks. Lignocellulosic biomass, available in enormous quantities, is the only feasible replacement. Many micro-organisms are capable of rapid and efficient degradation of biomass, employing a battery of specialized enzymes, but do not produce useful products. Attempts to transfer biomass-degrading capability to industrially useful organisms by heterologous expression of one or a few biomass-degrading enzymes have met with limited success. It seems probable that an effective biomass-degradation system requires the synergistic action of a large number of enzymes, the individual and collective actions of which are poorly understood. By offering the ability to combine any number of transgenes in a modular, combinatorial way, synthetic biology offers a new approach to elucidating the synergistic action of combinations of biomass-degrading enzymes in vivo and may ultimately lead to a transferable biomass-degradation system. Also, synthetic biology offers the potential for assembly of novel product-formation pathways, as well as mechanisms for increased solvent tolerance. Thus, synthetic biology may finally lead to cheap and effective processes for conversion of biomass to useful products.

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane.

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.