Abstract：
:Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5'-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gfp and xynA constructs were due to differences in 5'-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Sauer C,Ver Loren van Themaat E,Boender LGM,Groothuis D,Cruz R,Hamoen LW,Harwood CR,van Rij Tdoi
10.1021/acssynbio.8b00110subject
Has Abstractpub_date
2018-07-20 00:00:00pages
1773-1784issue
7issn
2161-5063journal_volume
7pub_type
杂志文章abstract：:Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves n...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400089c
更新日期：2014-03-21 00:00:00
abstract：:Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We id...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb500363y
更新日期：2015-07-17 00:00:00
abstract：:Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein pur...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb500341a
更新日期：2015-07-17 00:00:00
abstract：:In response to emergent antibiotic resistance, new strategies are needed to enhance the effectiveness of existing antibiotics. Here, we describe a phagemid-delivered, RNA-mediated system capable of directly knocking down antibiotic resistance phenotypes. Small regulatory RNAs (sRNAs) were designed to specifically inhi...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb500033d
更新日期：2014-12-19 00:00:00
abstract：:A negating functionality is fundamental to information processing of logic circuits within cells and computers. Aiming to adapt unutilized electronic concepts to the interrogation of signaling circuits in cells, we first took a bottom-up strategy whereby we created protein-based devices that perform negating Boolean l...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb500222z
更新日期：2014-09-19 00:00:00
abstract：:In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00003
更新日期：2018-05-18 00:00:00
abstract：:The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologo...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400082j
更新日期：2014-07-18 00:00:00
abstract：:Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic mani...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00038
更新日期：2015-09-18 00:00:00
abstract：:Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysi...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00128
更新日期：2020-07-17 00:00:00
abstract：:A major force shaping form and patterns in biology is based in the presence of amplification mechanisms able to generate ordered, large-scale spatial structures out of local interactions and random initial conditions. Turing patterns are one of the best known candidates for such ordering dynamics, and their existence ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00254
更新日期：2016-07-15 00:00:00
abstract：:The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00041
更新日期：2018-07-20 00:00:00
abstract：:Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00354
更新日期：2020-11-20 00:00:00
abstract：:Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of benef...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00359
更新日期：2018-07-20 00:00:00
abstract：:Engineered genetic systems are commonly unstable; if propagated, they evolve to reverse or modify engineered elements because the elements impair fitness. A goal of synthetic biology is thus to anticipate and avoid detrimental engineering, but little is yet known about which types of elements cause problems in differe...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb300040v
更新日期：2012-09-21 00:00:00
abstract：:Glycolate is an important α-hydroxy acid with a wide range of industrial applications. The current industrial production of glycolate mainly depends on chemical synthesis, but biochemical production from renewable resources using engineered microorganisms is increasingly viewed as an attractive alternative. Crude glyc...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00404
更新日期：2020-09-18 00:00:00
abstract：:2-Pyrone-4,6-dicarboxylic acid (PDC) is a pseudoaromatic dicarboxylic acid and is a promising biobased building block chemical that can be used to make diverse polyesters with novel functionalities. In this study, Escherichia coli was metabolically engineered to produce PDC from glucose. First, an efficient biosynthet...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00281
更新日期：2018-09-21 00:00:00
abstract：:Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such desi...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00294
更新日期：2016-06-17 00:00:00
abstract：:DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00137
更新日期：2020-07-17 00:00:00
abstract：:Strain engineering for synthetic biology and metabolic engineering applications often requires the expression of foreign proteins that can reduce cellular fitness. In order to quantify and visualize the evolutionary stability dynamics in engineered populations of Escherichia coli , we constructed randomized CMY (cyan-...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400055h
更新日期：2013-09-20 00:00:00
abstract：:One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00036
更新日期：2019-05-17 00:00:00
abstract：:Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00040
更新日期：2018-05-18 00:00:00
abstract：:We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inh...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00053
更新日期：2015-10-16 00:00:00
abstract：:To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machiner...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400003y
更新日期：2013-09-20 00:00:00
abstract：:Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cya...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00505
更新日期：2020-04-17 00:00:00
abstract：:Microbial mutualisms play critical roles in a diverse number of ecosystems and have the potential to improve the efficiency of bioproduction for desirable chemicals. We investigate the growth of a photosynthetic cyanobacterium, Synechococcus elongatus PCC 7942, and a diazotroph, Azotobacter vinelandii, in coculture. F...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00107
更新日期：2016-09-16 00:00:00
abstract：:Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segrega...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00008
更新日期：2020-06-19 00:00:00
abstract：:Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb5003136
更新日期：2015-05-15 00:00:00
abstract：:The Synthetic Biology Open Language (SBOL) is an emerging synthetic biology data exchange standard, designed primarily for unambiguous and efficient machine-to-machine communication. However, manual editing of SBOL is generally difficult for nontrivial designs. Here, we describe ShortBOL, a lightweight SBOL scripting ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00470
更新日期：2020-04-17 00:00:00
abstract：:Eukaryotic transcriptional factors (TFs) typically recognize short genomic sequences alone or together with other proteins to modulate gene expression. Mapping of TF-DNA interactions in the genome is crucial for understanding the gene regulatory programs in cells. While chromatin immunoprecipitation followed by sequen...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00358
更新日期：2017-06-16 00:00:00
abstract：:Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent contr...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00345
更新日期：2018-11-16 00:00:00
