Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care

Pérez-Piñera, Pablo; Han, Ningren; Cleto, Sara; Cao, Jicong; Purcell, Oliver; Shah, Kartik; Lee, Kevin; Ram, Rajeev J.; Lu, Timothy K.

doi:10.1038/ncomms12211

Cited by 77 publications

(79 citation statements)

References 41 publications

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“…As the applications of metabolic engineering expand beyond traditional chemical production, tight control of output in response to the external environment will be necessary. Cells are already being engineered to specifically deliver drugs to tumors (Forbes 2010, Din et al 2016, Hosseinidoust et al 2016) and to produce different drugs based on what is needed in low-resource settings (Choi et al 2014, Perez-Pinera et al 2016). For both of these applications, it is necessary that cells produce the desired compound only in the desired condition—specificity of response based on environment is critical.…”

Section: Discussionmentioning

confidence: 99%

Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor

McNerney

Styczynski

2017

Metabolic Engineering

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Pigmented metabolites have great potential for use in biosensors that target low-resource areas, since sensor output can be interpreted without any equipment. However, full repression of pigment production when undesired is challenging, as even small amounts of enzyme can catalyze the production of large, visible amounts of pigment. The red pigment lycopene could be particularly useful because of its position in the multi-pigment carotenoid pathway, but commonly used inducible promoter systems cannot repress lycopene production. In this paper, we designed a system that could fully repress lycopene production in the absence of an inducer and produce visible lycopene within two hours of induction. We engineered Lac, Ara, and T7 systems to be up to 10 times more repressible, but these improved systems could still not fully repress lycopene. Translational modifications proved much more effective in controlling lycopene. By decreasing the strength of the ribosomal binding sites on the crtEBI genes, we enabled full repression of lycopene and production of visible lycopene in 3–4 hours of induction. Finally, we added the mevalonate pathway enzymes to increase the rate of lycopene production upon induction and demonstrated that supplementation of metabolic precursors could decrease the time to coloration to about 1.5 hours. In total, this represents over an order of magnitude reduction in response time compared to the previously reported strategy. The approaches used here demonstrate the disconnect between fluorescent and metabolite reporters, help enable the use of lycopene as a reporter, and are likely generalizable to other systems that require precise control of metabolite production.

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Section: Discussionmentioning

confidence: 99%

Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor

McNerney

Styczynski

2017

Metabolic Engineering

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“…If enzyme or promoter variants are being compared for expression levels, picking outliers from a population may strongly bias the results, leading to an unclear assessment of the effect of the enzyme or promoter variations. To rule out these effects, two strategies appear feasible: (i) confirming specific integration (by demonstrating growth phenotypes, by colony PCR, or by using site-specific molecular "landing pad" recombination systems [55]) or (ii) screening a sufficient number of transformants to obtain a representative population distribution and thus avoid outliers.…”

Section: Discussionmentioning

confidence: 99%

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Vogl

Gebbie

Palfreyman

et al. 2018

Appl Environ Microbiol

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Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of Ͼ700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (Ͻ10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.KEYWORDS integration, Pichia pastoris, protein expression, genome analysis T he yeast Pichia pastoris (syn. Komagataella phaffii) is widely used for heterologous protein p...

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“…Indeed, biomolecular machines capable of dynamic probing and decision‐making in situ could offer new ways to interface biology (Slomovic et al , ; Courbet et al , ) as well as unprecedented versatility in analytical and biomedical applications. For instance, synthetic cell‐based devices can be designed and employed as programmable bioanalytical tools to detect molecular cues for diagnostic purposes (Courbet et al , ; Danino et al , ) or smart therapeutics (Ye & Fussenegger, ; Perez‐Pinera et al , ; Roybal et al , ; Xie et al , ).…”

Section: Introductionmentioning

confidence: 99%

Computer‐aided biochemical programming of synthetic microreactors as diagnostic devices

Courbet

Amar

Fages

et al. 2018

Molecular Systems Biology

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Biological systems have evolved efficient sensing and decision‐making mechanisms to maximize fitness in changing molecular environments. Synthetic biologists have exploited these capabilities to engineer control on information and energy processing in living cells. While engineered organisms pose important technological and ethical challenges, de novo assembly of non‐living biomolecular devices could offer promising avenues toward various real‐world applications. However, assembling biochemical parts into functional information processing systems has remained challenging due to extensive multidimensional parameter spaces that must be sampled comprehensively in order to identify robust, specification compliant molecular implementations. We introduce a systematic methodology based on automated computational design and microfluidics enabling the programming of synthetic cell‐like microreactors embedding biochemical logic circuits, or protosensors, to perform accurate biosensing and biocomputing operations in vitro according to temporal logic specifications. We show that proof‐of‐concept protosensors integrating diagnostic algorithms detect specific patterns of biomarkers in human clinical samples. Protosensors may enable novel approaches to medicine and represent a step toward autonomous micromachines capable of precise interfacing of human physiology or other complex biological environments, ecosystems, or industrial bioprocesses.

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Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care

Cited by 77 publications

References 41 publications

Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor

Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Computer‐aided biochemical programming of synthetic microreactors as diagnostic devices

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