Systems engineering N-glycans of recombinant therapeutic proteins

Protein N-glycosylation reactions form a distributed reaction network spanning over different compartments of Golgi apparatus. The resulting glycan structures are influenced by glycosylation enzymes, the supply rate of nucleotide sugars, as well as competition among extending glycan substrates for a common enzyme and among different enzymes for a common substrate. Controlling the glycan profile of a therapeutic protein product is important for product quality for both innovative drugs as well as biosimilars. Metabolic engineering of the glycosylation pathway offers a venue for modulating the glycan profile. We have taken a systems engineering approach to identify, through model assisted design, the genetic manipulations that may steer the glycan flux to the desired path. However, unlike the energy metabolism pathway for which a small number of enzymes play pivotal roles in controlling the flux, the glycosylation pathway lacks key regulated steps as easily identifiable targets for genetic alteration to re-direct the flux. The model prediction thus serves only as a imprecise guide rather than a clear beacon. Furthermore, very likely multiple genetic alterations are needed in order to steer glycan flux distribution. A scheme of rapid construction of gene combinations to facilitate genetic engineering of the cell is necessary. We establish a golden gate assembly workflow for production of multi-gene constructs for engineering the glycan biosynthesis pathway. Libraries containing promoters of varying strengths, terminators, and glycosylation related coding sequences of interest, all refactored to be devoid of type IIS restriction sites, were synthesized. In the first level of assembly, an additional library of single gene constructs were formed from these base components with single reactions. In the second level of assembly, these monocistrons were then combinatorically combined to form a multi-gene cassette library. In an application of this approach, the N-glycosylation pattern of a recombinant IgG produced in CHO cells was examined with a stoichiometric network visualization tool (GlycoVis) to track the reaction paths which lead to the product glycans and identify galactosylation as potentially limiting glycan maturation. Cassettes consisting of sequences coding for nucleotide sugar synthesis enzymes, nucleotide sugar transporters, and glycosyltransferases were then selected to engineer the IgG producing cell. Multiple cassettes successfully directed the glycosylation to produce antibody with desirable glycoforms. These results served to refine our model parameters and sharpen its predictive capabilities. This combination of systems analysis and synthetic glycoengineering can be broadly applied and enhances our capability to steer N-Glycan patterns and control the quality of therapeutic proteins

A synthetic biology based cell line engineering pipeline

An ideal host cell line for deriving cell lines of high recombinant protein production should be stable, predictable, and amenable to rapid cell engineering or other forms of phenotypical manipulation. In the past few years we have employed genomic information to identify “safe harbors” for exogenous gene integration in CHO cells, deployed systems modeling and optimization to design pathways and control strategies to modify important aspects of recombinant protein productivity, and established a synthetic biology approach to implement genetic changes, all with the goal of creating a pipeline to produce “designer” cell lines. Chinese hamster ovary (CHO) cells are the preferred platform for protein production. However, the Chinese hamster genome is unstable in its ploidy, is subject to long and short deletions, duplications, and translocations. In addition, gene expression is subject to epigenetic changes including DNA methylation, histone modification and heterochromatin invasion, thus further complicating transgene expression for protein production in cell lines. With these issues in mind, we set out to engineer a CHO cell line highly amenable to stable protein production using a synthetic biology approach. We compiled karyotyping and chromosome number data of several CHO cell lines and sublines, identified genomic regions with high a frequency of gain and loss of copy number using comparative genome hybridization (CGH), and verified structural variants using sequencing data. We further used ATAC (Assay for Transposase-Accessible Chromatin) sequencing to study chromatin accessibility and epigenetic stability within the CHO genome. RNA-seq data from multiple cell lines were also used to identify regions with high transcriptional activity. Analysis of these data allowed the identification of several “safe harbor” loci that could be used for cell engineering. Based on results of the data analysis and identification of “safe harbors”, we engineered an IgG producing cell line with a single copy of the product transgene as a template cell line. This product gene site is flanked by sequences for recombinase mediated cassette exchange, therefore allowing easy substitution of the IgG producing gene for an alternative product gene. Furthermore, a “landing pad” for multi-gene cassette insertion was integrated into the genome at an additional site. Together, these sites allowed engineering of new cell lines producing a fusion protein and Erythropoietin to be generated from the template cell line. To enable rapid assembly of product transgenes and genetic elements for engineering cell attributes into multi-gene cassettes, we adopted a golden-gate based synthetic biology approach. The assembly of genetic parts into multi-gene cassettes in a LEGO-like fashion allowed different combinations of genes under the control of various promoters to be generated quickly for introduction into the template cell line. Using this engineered CHO cell line, we set out to study metabolism and product protein glycosylation for cell engineering. To guide the selection of genetic elements for cell engineering, we developed a multi-compartment kinetic model, as well as a flux model of energy metabolism and glycosylation. The transcriptome meta-data was used extensively to identify genes and isoforms expressed in the cell line and to estimate the enzyme levels in the model. The flux model was used to identify and the LEGO-like platform was used to implement the genetic changes that can alter the glycosylation pattern of the IgG produced by the template cell line. Concurrently we employed a systems optimization approach to identify the genetic alterations in the metabolic pathway to guide cell metabolism toward a favorable state. The model prediction is being implemented experimentally using the synthetic biology approach. In conclusion, we have illustrated a pipeline of rational cell line engineering that integrates genomic science, systems engineering and synthetic biology approaches. The promise, the technical challenges and possible limitations will be discussed in this presentation

A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co‐expressed to function. The DNA‐binding loop is encoded in a C‐terminal 285‐aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601‐aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67‐aa N‐terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.United States. Office of Naval Research (N00014‐13‐1‐0074)National Institutes of Health (U.S.) (5R01GM095765)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center (SA5284‐11210))United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship (NDSEG) Program))Hertz Foundation (Fellowship

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products

Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms

Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation. Among well-established techniques (codon optimization and improvement of mRNA secondary structure), we describe SINEUPs, a family of antisense long non-coding RNAs that are able to increase translation of partially overlapping protein-coding mRNAs. By exploiting their modular structure, SINEUP molecules can be designed to target virtually any mRNA of interest, and thus to increase the production of secreted proteins. Thus, synthetic SINEUPs represent a new versatile tool to improve the production of secreted proteins in biomanufacturing processes. \ua9 2016 The Author

Auxotrophic Selection Strategy for Improved Production of Coenzyme B-12 in Escherichia coli

The production of coenzyme B-12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B-12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B-12 using a previously engineered coenzyme B-12-producing E. coli strain. To select a high producer, the coenzyme B-12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B-12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B-12 validated the coenzyme B-12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B-12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B-12-producing strains