Promoter Library Research Articles

Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription-Translation Systems.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Promoters Constrain Evolution of Expression Levels of Essential Genes in Escherichia coli.

Variability in expression levels in response to random genomic mutations varies among genes, influencing both the facilitation and constraint of phenotypic evolution in organisms. Despite its importance, both the underlying mechanisms and evolutionary origins of this variability remain largely unknown due to the mixed contributions of cis- and trans-acting elements. To address this issue, we focused on the mutational variability of cis-acting elements, that is, promoter regions, in Escherichia coli. Random mutations were introduced into the natural and synthetic promoters to generate mutant promoter libraries. By comparing the variance in promoter activity of these mutant libraries, we found no significant difference in mutational variability in promoter activity between promoter groups, suggesting the absence of a signature of natural selection for mutational robustness. In contrast, the promoters controlling essential genes exhibited a remarkable bias in mutational variability, with mutants displaying higher activities than the wild types being relatively rare compared to those with lower activities. Our evolutionary simulation on a rugged fitness landscape provided a rationale for this vulnerability. These findings suggest that past selection created nonuniform mutational variability in promoters biased toward lower activities of random mutants, which now constrains the future evolution of downstream essential genes toward higher expression levels.

Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli

The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10–21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element.Key points• Degenerate synthetic promoters are remarkable tools to produce protocatechuic acid.• The constitutive synthetic promoters did not affect the growth rate of the bacterial host.• The use of constitutive synthetic promoters avoids the need for the costly inducer.

Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria

BackgroundNon-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers.ResultsIn this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter.ConclusionNon-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.Graphical

A promoter-RBS library for fine-tuning gene expression in Methanosarcina acetivorans.

Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

Expanding the synthetic biology repertoire of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. The promoters PcpcB, PpsbA1, and P11770 exhibited consistently high fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for identifying RBSs, using an operon construct comprising fluorescent protein genes eyfp and mTurq under PcpcB (PcpcB:eyfp:RBS:mTurq:TrrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Engineering of the Lrp/AsnC-type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L-cysteine biosynthesis pathway in Escherichia coli.

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD)simulations and electrophoretic mobility shift analysis (EMSA)showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

Exploration of Streptomyces fradiae J1-021 as a Potential Host for the Heterologous Production of Spinosad.

Spinosad is a potent insecticide produced by Saccharopolyspora spinosa. However, it harbors certain limitations of a low growing rate and unfeasible genetic manipulation that can be overcome by adopting a superior platform, such as Streptomyces. Herein, we exploited the industrial tylosin-producing Streptomyces fradiae J1-021 for the heterologous production of spinosad. An engineered strain (HW01) with deletion of the tylosin biosynthetic gene cluster (BGC) was constructed and then transformed with the natural spinosad BGC. The distribution and expression levels of the tylosin BGC operons were assessed to construct a natural promoter library. The rate-limiting steps of spinosad biosynthesis were identified by analyzing the transcriptional expression of the spinosad biosynthetic genes. The stepwise engineering work involved the overexpression of the biosynthetic genes participating in rate-limiting pathways using strong promoters, affording an increase in spinosad production to 112.4 μg/L. These results demonstrate that strain HW01 has the potential to be used as a chassis for the heterologous production of polyketides.

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum.

Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA . Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high-yielding strains AG-6, AG-18, and AG-41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac-∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression.

Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

Characterization and Application of a Stationary Phase Promoter Library from Serratia marcescens.

Serratia marcescens has garnered increasing attention as a promising host for valuable compound production. However, the lack of an efficient gene regulation toolkit severely hampers its applications. Here, a library of stationary phase promoters was screened in S. marcescens HBA7 using RNA-seq and RT-qPCR, revealing a 43-fold regulatory range with the red fluorescent protein mKate2 as the reporter. The β-galactosidase was employed to demonstrate the universality in driving the expression of different proteins. The wide-ranging utility of these promoters in different hosts was demonstrated in Escherichia coli. Moreover, to assess their potential application, the strongest promoter, P2, was employed to express the swrW gene, resulting in a roughly 20-fold increase in serrawettin W1 production in S. marcescens HBQA7ΔswrW. In summary, this study successfully constructed a gradient-strength stationary phase promoter library, providing an effective toolkit for gene regulation and secondary metabolite production in diverse prokaryotes, including S. marcescens and E. coli.

Construction and characterization of a promoter library with varying strengths to enhance acetoin production from xylose in Serratia marcescens.

Serratia marcescens is utilized as a significant enterobacteria in the production of various high-value secondary metabolites. Acetoin serves as a crucial foundational compound of development and finds application in a broad range of fields. Furthermore, S. marcescens HBQA-7 is capable of utilizing xylose as its exclusive carbon source for acetoin production. The objective of this study was to utilize a constitutive promoter screening strategy to enhance both xylose utilization and acetoin production in S. marcescens HBQA-7. By utilizing RNA-seq, we identified the endogenous constitutive promoter P6 that is the most robust, which facilitated the overexpression of the sugar transporter protein GlfL445I , α-acetyl lactate synthase, and α-acetyl lactate decarboxylase, respectively. The resultant recombinant strains exhibited enhanced xylose utilization rates and acetoin yields. Subsequently, a recombinant plasmid, denoted as pBBR1MCS-P6-glfL445I alsSalsD, was constructed, simultaneously expressing the aforementioned three genes. The resulting recombinant strain, designated as S3, demonstrated a 1.89-fold boost in xylose consumption rate compared with the original strain during shake flask fermentation. resulting in the accumulation of 7.14g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75g/L, corresponding to a xylose-to-acetoin conversion yield of 0.375g/g.

Biosensors for the detection of chorismate and cis,cis-muconic acid in Corynebacterium glutamicum.

High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.

AI and Knowledge-Based Method for Rational Design of Escherichia coli Sigma70 Promoters.

Expanding sigma70 promoter libraries can support the engineering of metabolic pathways and enhance recombinant protein expression. Herein, we developed an artificial intelligence (AI) and knowledge-based method for the rational design of sigma70 promoters. Strong sigma70 promoters were identified by using high-throughput screening (HTS) with enhanced green fluorescent protein (eGFP) as a reporter gene. The features of these strong promoters were adopted to guide promoter design based on our previous reported deep learning model. In the following case study, the obtained strong promoters were used to express collagen and microbial transglutaminase (mTG), resulting in increased expression levels by 81.4% and 33.4%, respectively. Moreover, these constitutive promoters achieved soluble expression of mTG-activating protease and contributed to active mTG expression in Escherichia coli. The results suggested that the combined method may be effective for promoter engineering.

Fine-Tuning Gene Expression in Bacteria by Synthetic Promoters.

Promoters are key genetic elements in the initiation and regulation of gene expression. A limited number of natural promoters has been described for the control of gene expression in synthetic biology applications. Therefore, synthetic promoters have been developed to fine-tune the transcription for the desired amount of gene product. Mostly, synthetic promoters are characterized using promoter libraries that are constructed via mutagenesis of promoter sequences. The strength of promoters in the library is determined according to the expression of a reporter gene such as gfp encoding green fluorescent protein. Gene expression can be controlled using inducers. The majority of the studies on gram-negative bacteria are conducted using the expression system of the model organism Escherichia coli while that of the model organism Bacillus subtilis is mostly used in the studies on gram-positive bacteria. Additionally, synthetic promoters for the cyanobacteria, which are phototrophic microorganisms, are evaluated, especially using the model cyanobacterium Synechocystis sp. PCC 6803. Moreover, a variety of algorithms based on machine learning methods were developed to characterize the features of promoter elements. Some of these in silico models were verified using in vitro or in vivo experiments. Identification of novel synthetic promoters with improved features compared to natural ones contributes much to the synthetic biology approaches in terms of fine-tuning gene expression.

Tuning of Protein Expression by Promoter Engineering.

The promoter is an essential component of an expression system since it regulates the transcriptional beginning of related genes. The optimal expression level can be achieved by employing a promoter engineering approach. Typically, creating a library of T7 promoters allows for titratable protein expression. In the process of making β-amino acid (sitagliptin intermediate) from β-keto ester, esterase from Pseudomonas stutzeri (Est PS) is used to convert the β-keto ester to β-keto acid. Subsequently, transaminase from Ilumatobacter coccineus (TAIC) transforms the β-keto acid to its corresponding β-amino acid. Here, we describe the optimization of the expression levels of Est PS for the maximum production of sitagliptin intermediate. The different promoter strengths for Est PS were built into the T7 promoters of the pET15b vector. With the help of these new co-expressing entire cells, the expressed enzyme ratio for each enzyme was determined. As the strength of the promoter of Est PS decreases, the expression level also decreases (from 100% to 10%). Conversely, the TAIC expression level is increased. This developed system produced a higher sitagliptin intermediate than enzymes' unoptimized expression level.

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.

Toolbox of Characterized Genetic Parts for Staphylococcus aureus.

Staphylococcus aureus is an important clinical bacterium prevalent in human-associated microbiomes and the cause of many diseases. However, S. aureus has been intractable to synthetic biology approaches due to limited characterized genetic parts for this nonmodel Gram-positive bacterium. Moreover, genetic manipulation of S. aureus has relied on cumbersome and inefficient cloning strategies. Here, we report the first standardized genetic parts toolbox for S. aureus, which includes characterized promoters, ribosome binding sites, terminators, and plasmid replicons from a variety of bacteria for precise control of gene expression. We established a standard relative expression unit (REU) for S. aureus using a plasmid reference and characterized genetic parts in standardized REUs using S. aureus ATCC 12600. We constructed promoter and terminator part plasmids that are compatible with an efficient Type IIS DNA assembly strategy to effectively build multipart DNA constructs. A library of 24 constitutive promoters was built and characterized in S. aureus, which showed a 380-fold activity range. This promoter library was also assayed in Bacillus subtilis (122-fold activity range) to demonstrate the transferability of the constitutive promoters between these Gram-positive bacteria. By applying an iterative design-build-test-learn cycle, we demonstrated the use of our toolbox for the rational design and engineering of a tetracycline sensor in S. aureus using the PXyl-TetO aTc-inducible promoter that achieved 25.8-fold induction. This toolbox greatly expands the growing number of genetic parts for Gram-positive bacteria and will allow researchers to leverage synthetic biology approaches to study and engineer cellular processes in S. aureus.

High-throughput, microscopy-based screening and quantification of genetic elements.

Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions. Microscopy is a powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements. Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution and can be applied to large-scale samples for library screening of genetic elements. However, strategies for high-throughput microscopy experiments remain limited. Here, we present a high-throughput, microscopy-based platform that can simultaneously complete the preparation of an 8 × 12-well agarose pad plate, allowing for the screening of 96 independent strains or experimental conditions in a single experiment. Using this platform, we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions. Additionally, the platform allowed for single-cell measurement of genetic elements over time, enabling the identification of complex and dynamic phenotypes to map genotype in high throughput. We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems, as well as to understand the relationship between cellular phenotypes and internal states, including genotypes and gene expression programs.

Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.