Comprehensive study on Escherichia coli genomic expression: Does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola.

Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing.

Chromosomal integration of biosynthetic pathways for the biotechnological production of high-value chemicals is a necessity to develop industrial strains with a high long-term stability and a low production variability. However, the introduction of multiple transcription units into the microbial genome remains a difficult task. Despite recent advances, current methodologies are either laborious or efficiencies highly fluctuate depending on the length and the type of the construct. Here we present serine integrase recombinational engineering (SIRE), a novel methodology which combines the ease of recombinase-mediated cassette exchange (RMCE) with the selectivity of orthogonal att sites of the PhiC31 integrase. As a proof of concept, this toolbox is developed for Escherichia coli. Using SIRE we were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette. Next to integrating large fragments, the option for multitargeting, for deleting operons, as well as for performing in vivo assemblies further expand and proof the versatility of the SIRE toolbox for E. coli. Finally, the serine integrase PhiC31 was also applied in the yeast Saccharomyces cerevisiae as a marker recovery tool, indicating the potential and portability of this toolbox.

Standardization in synthetic biology: an engineering discipline coming of age.

BACKGROUND: Leaping DNA read-and-write technologies, and extensive automation and miniaturization are radically transforming the field of biological experimentation by providing the tools that enable the cost-effective high-throughput required to address the enormous complexity of biological systems. However, standardization of the synthetic biology workflow has not kept abreast with dwindling technical and resource constraints, leading, for example, to the collection of multi-level and multi-omics large data sets that end up disconnected or remain under- or even unexploited. PURPOSE: In this contribution, we critically evaluate the various efforts, and the (limited) success thereof, in order to introduce standards for defining, designing, assembling, characterizing, and sharing synthetic biology parts. The causes for this success or the lack thereof, as well as possible solutions to overcome these, are discussed. CONCLUSION: Akin to other engineering disciplines, extensive standardization will undoubtedly speed-up and reduce the cost of bioprocess development. In this respect, further implementation of synthetic biology standards will be crucial for the field in order to redeem its promise, i.e. to enable predictable forward engineering.

Tailor-made transcriptional biosensors for optimizing microbial cell factories.

Monitoring cellular behavior and eventually properly adapting cellular processes is key to handle the enormous complexity of today's metabolic engineering questions. Hence, transcriptional biosensors bear the potential to augment and accelerate current metabolic engineering strategies, catalyzing vital advances in industrial biotechnology. The development of such transcriptional biosensors typically starts with exploring nature's richness. Hence, in a first part, the transcriptional biosensor architecture and the various modi operandi are briefly discussed, as well as experimental and computational methods and relevant ontologies to search for natural transcription factors and their corresponding binding sites. In the second part of this review, various engineering approaches are reviewed to tune the main characteristics of these (natural) transcriptional biosensors, i.e., the response curve and ligand specificity, in view of specific industrial biotechnology applications, which is illustrated using success stories of transcriptional biosensor engineering.

One step DNA assembly for combinatorial metabolic engineering.

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity.

Mapping and refactoring pathway control through metabolic and protein engineering: The hexosamine biosynthesis pathway.

Microorganisms possess a plethora of regulatory mechanisms to tightly control the flux through their metabolic network, allowing optimal behaviour in response to environmental conditions. However, these mechanisms typically counteract metabolic engineering efforts to rewire the metabolism with a view to overproduction. Hence, overcoming flux control is key in the development of microbial cell factories, illustrated in this contribution using the strictly controlled hexosamine biosynthesis pathway. The hexosamine biosynthesis pathway has recently garnered attention as gateway for the industrial biotechnological production of numerous mono-, oligo- and polysaccharidic compounds, composed of, i.a., glucosamine, N-acetylglucosamine, and neuraminic acid and with a vast application potential in the health, comsetics, and agricultural sector. First, the various alternative pathways in eukaryotes and prokaryotes are discussed. Second, the main regulatory mechanisms on transcriptional, translational and post-translational control, and the strategies to circumvent these pathway bottlenecks are highlighted. These efforts can serve as an inspiration to tackle regulatory control when optimizing any microbial cell factory.

Direct Combinatorial Pathway Optimization.

Combinatorial engineering approaches are becoming increasingly popular, yet they are hindered by the lack of specialized techniques for both efficient introduction of sequence variability and assembly of numerous DNA parts, required for the construction of lengthy multigene pathways. In this contribution, we introduce a new combinatorial multigene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly, exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accompanying set of well-characterized and ready-to-use genetic parts, the developed workflow allows effective introduction of various libraries and efficient assembly of multigene pathways. It was put to the test by optimizing the lycopene pathway as proof-of-principle. The here constructed libraries yield ample variation in lycopene production. In addition, good-performing transformants with a significantly higher lycopene production were obtained as compared to previously published reference strains. The best selected producer yielded 3-fold improvement in lycopene titers up to 448 mg lycopene/g CDW. The proposed workflow in combination with the accompanying sets of ready-to-use expression and carrier plasmids, will allow the combinatorial assembly of increasingly lengthy product pathways with minimal effort.

Putting RNA to work: Translating RNA fundamentals into biotechnological engineering practice.

Synthetic biology, in close concert with systems biology, is revolutionizing the field of metabolic engineering by providing novel tools and technologies to rationally, in a standardized way, reroute metabolism with a view to optimally converting renewable resources into a broad range of bio-products, bio-materials and bio-energy. Increasingly, these novel synthetic biology tools are exploiting the extensive programmable nature of RNA, vis-à-vis DNA- and protein-based devices, to rationally design standardized, composable, and orthogonal parts, which can be scaled and tuned promptly and at will. This review gives an extensive overview of the recently developed parts and tools for i) modulating gene expression ii) building genetic circuits iii) detecting molecules, iv) reporting cellular processes and v) building RNA nanostructures. These parts and tools are becoming necessary armamentarium for contemporary metabolic engineering. Furthermore, the design criteria, technological challenges, and recent metabolic engineering success stories of the use of RNA devices are highlighted. Finally, the future trends in transforming metabolism through RNA engineering are critically evaluated and summarized.