The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering.

Here we introduce the Galaxy-SynBioCAD portal, a toolshed for synthetic biology, metabolic engineering, and industrial biotechnology. The tools and workflows currently shared on the portal enables one to build libraries of strains producing desired chemical targets covering an end-to-end metabolic pathway design and engineering process from the selection of strains and targets, the design of DNA parts to be assembled, to the generation of scripts driving liquid handlers for plasmid assembly and strain transformations. Standard formats like SBML and SBOL are used throughout to enforce the compatibility of the tools. In a study carried out at four different sites, we illustrate the link between pathway design and engineering with the building of a library of E. coli lycopene-producing strains. We also benchmark our workflows on literature and expert validated pathways. Overall, we find an 83% success rate in retrieving the validated pathways among the top 10 pathways generated by the workflows.

Cell-free protein synthesis using Chinese hamster ovary cells.

Cell-free protein synthesis (CFPS) platforms can be used for rapid and flexible expression of proteins. The use of CFPS platforms from mammalian, specifically Chinese hamster ovary (CHO) cells, offers the possibility of a rapid prototyping platform for recombinant protein production with the capabilities of post-translational modifications. In this chapter, we discuss a refined CFPS system based on CHO cells, including: extract preparation, reaction mix composition, and accessory protein supplementation to enhance expression. Specifically, when the CHO cell extract is combined with a truncated version of GADD34 and K3L, stress-induced eIF2 phosphorylation is reduced and inhibition of translation initiation is relieved, increasing yields. A brief summary of the protocol for running the CFPS reactions is also described. Overall, the method is reliable and leads to a highly reproducible expression system. Finally, the advantages and disadvantages of the platform, in addition to expected outcomes, are also discussed.

Systematic Understanding of Recent Developments in Bacterial Cellulose Biosynthesis at Genetic, Bioprocess and Product Levels.

Engineering biological processes has become a standard approach to produce various commercially valuable chemicals, therapeutics, and biomaterials. Among these products, bacterial cellulose represents major advances to biomedical and healthcare applications. In comparison to properties of plant cellulose, bacterial cellulose (BC) shows distinctive characteristics such as a high purity, high water retention, and biocompatibility. However, low product yield and extensive cultivation times have been the main challenges in the large-scale production of BC. For decades, studies focused on optimization of cellulose production through modification of culturing strategies and conditions. With an increasing demand for BC, researchers are now exploring to improve BC production and functionality at different categories: genetic, bioprocess, and product levels as well as model driven approaches targeting each of these categories. This comprehensive review discusses the progress in BC platforms categorizing the most recent advancements under different research focuses and provides systematic understanding of the progress in BC biosynthesis. The aim of this review is to present the potential of 'modern genetic engineering tools' and 'model-driven approaches' on improving the yield of BC, altering the properties, and adding new functionality. We also provide insights for the future perspectives and potential approaches to promote BC use in biomedical applications.

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform.

In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.

Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 µm and a diameter of 10-20 µm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.

More than meets the eye in bacterial cellulose: biosynthesis, bioprocessing, and applications in advanced fiber composites.

Bacterial cellulose (BC) nanofibers are one of the stiffest organic materials produced by nature. It consists of pure cellulose without the impurities that are commonly found in plant-based cellulose. This review discusses the metabolic pathways of cellulose-producing bacteria and the genetic pathways of Acetobacter xylinum. The fermentative production of BC and the bioprocess parameters for the cultivation of bacteria are also discussed. The influence of the composition of the culture medium, pH, temperature, and oxygen content on the morphology and yield of BC are reviewed. In addition, the progress made to date on the genetic modification of bacteria to increase the yield of BC and the large-scale production of BC using various bioreactors, namely static and agitated cultures, stirred tank, airlift, aerosol, rotary, and membrane reactors, is reviewed. The challenges in commercial scale production of BC are thoroughly discussed and the efficiency of various bioreactors is compared. In terms of the application of BC, particular emphasis is placed on the utilization of BC in advanced fiber composites to manufacture the next generation truly green, sustainable and renewable hierarchical composites.