Hybrid Promoter Research Articles

In Silico Dissection of Regulatory Regions of PHT Genes from Saccharum spp. Hybrid and Sorghum bicolor and Expression Analysis of PHT Promoters under Osmotic Stress Conditions in Tobacco

Phosphorus (P) is the second-most essential macronutrient required for the growth and development of plants. It is involved in a number of cellular processes that contribute to the plant’s growth and development. This study investigated Saccharum spp. hybrid and Sorghum bicolor promoter regions of Phosphate transporters (PHT), viz., PHT1, PHT2, PHT3, PHT4, and PHO1, through in silico analysis. The transcription start sites (TSS), conserved motifs, and CpG islands were studied using various computational techniques. The distribution of TSSs indicated the highest promoter prediction scores (1.0). MSh2 and MSb4 were recognized as the common promoter motifs for PHT promoters, found in with 85 to 100% percentage of distribution. The CpG analysis revealed that the promoter regions of most PHT genes had low CpG density, indicating a possible tissue-specific expression. The PHT promoters were investigated for the presence of biotic- and abiotic-stress-associated transcription factor binding sites (TFbs) that revealed the presence of binding motifs for major transcription factors (TFs), namely, AP2/ERF, bHLH, bZIP, MYB, NAC, and WRKY. Therefore, the in-silico analysis of the promoter regions helps us to understand the regulation mechanism of phosphate transporter promoters and gene expression under stress management. The 5′ regulatory region of the EaPHT gene was isolated from Erianthus, a wild relative of the genus Saccharum. The promoter construct was prepared and transformed in tobacco wherein the promoter drove the expression of GUS. Analysis of GUS expression in transgenic tobacco revealed enhanced expression of GUS under salt-stress conditions. This is the first report of the isolation and characterization of a phosphate transporter gene promoter from Erianthus and is expected to be useful for the development of salt-stress transgenic crop plants.

Strong expression of Cas9 under a new 3′-truncated TEF1α promoter enhances genome editing in Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica is gaining interest in biotechnology as a workhorse for the production of proteins, lipids and other biomolecules. Site-specific genome editing is however limited in this yeast. Although, this was much improved by the recent adaptation of a CRISPR-Cas9 genome editing protocol for Y. lipolytica based on a tRNA-sgRNA fusion, yet, in the latter protocol, Cas9 is under the control of a synthetic hybrid promoter, pUAS1B8-TEF(136) that is associated with some drawbacks. This hybrid promoter contains tandem repeats are suggested to cause in vivo and in vitro inconveniences like polymerase slippage, random genetic rearrangements and cloning difficulties. Here we report a newly designed synthetic TEF promoter to drive Cas9 expression, pTEF(-41–406)-Kozak, which is a rationally 3′-truncated version of the already known 5′-truncated pTEF(406) promoter of Y. lipolytica fused to a synthetic Kozak sequence. Our comparison of the promoters’ strength using hrGFP reporters and RT-qPCR showed that the synthetic pTEF(-41–406)-Kozak has an equivalent expression strength to that of pTEF(406), yet is at least 5 times stronger than the hybrid pUAS1B8-TEF(136). The pTEF(-41–406) promoter mediated high expression of Cas9 and was not associated with any growth defects. Moreover, expression of Cas9 under pTEF(-41–406)-Kozak increased the gene integration efficiency by up to 40 % relative to that when Cas9 is expressed under pUAS1B8-TEF(136). Both pTEF(-41–406) and pUAS1B8-TEF(136) performed equally well as drivers of Cas9 expression with respect to gene deletion as demonstrated both on the genotypic and phenoypic levels. This is the first study conducting rational 3′-truncation in TEF promoter in Y. lipolytica based on in silico analysis of promoter sequence and structure. This approach of promoter engineering can be extended to the engineering of other yeast promoters to generate small-sized synthetic biology parts for convenient engineering of biological systems. This work provides a strong Cas9 expression cassette for more convenient and efficient CRISPR-Cas9-mediated genome editing in Y. lipolytica which will facilitate harnessing the full potential of this industrial strain.

Programmed cell-lysis system based on hybrid sigma factor-dependent promoters

BackgroundRecognized as the biotechnology workhorse, Escherichia coli has been commonly employed for mass production of recombinant proteins. Target proteins that are overexpressed in E. coli usually accumulate in cell cyctoplasm. This inherent limitation renders the downstream process for protein recovery complicated and costly. This issue was addressed by development of the programmed cell-lysis system. MethodsThe cell-lysis system was constructed with a core element consisting of the cell lesion-incurred module and the regulatory circuit. The module comprises the gene E of bacteriophage ϕX174 (ϕXE) and the gene e of T4 bacteriophage (T4E). The toxin activity of ϕXE and T4E is responsible for disintegration of cell membrane and of cell wall, respectively. The circuit encompasses the thermo-sensitive cI857 and hybrid promoters which are recognized by various sigma factors including σ70, σ32, and σ38. Consequently, the expression of ϕXE and T4E was induced by heat. Significant findingsThe production of amidohydrolase (AHL) by the T7 expression system was investigated in E. coli BL21(DE3) strain equipped with the cell-lysis system. It resulted in 93% of and 76% of the expressed AHL being secreted after administration of thermal induction in the late log phase and the early stationery phase, respectively. The result indicates that the cell-lysis system is programmed in response to heat, which shows a promise for efficient secretion of the recombinant protein.

Landscape of blaNDM genes in Enterobacteriaceae.

The blaNDM-1 gene encodes a carbapenemase, New Delhi metallo-β-lactamase (NDM-1), and the ability to produce NDM-1 is spread among Enterobacteriaceae via horizontal gene transfer of plasmids. It has been widely accepted that blaNDM-1 is regulated by a hybrid promoter (PISAba125) consisting of a -10 box from the original blaNDM-1 and a -35 box from ISAba125. However, the conservation of this promoter and the vertical transmission of blaNDM genes by chromosomal integration have not been comprehensively analyzed. We retrieved the region containing the ORF of blaNDM-1 (>95% translated protein identity) and a region 120 bp upstream of the blaNDM-1 start codon from the complete sequence data of Enterobacteriaceae plasmids (n = 10,914) and chromosomes (n = 4908) deposited in GenBank, and the 310 extracted blaNDM genes were analyzed by an in-silico approach. The results showed that most blaNDM genes (99.0%) utilized the promoter, PISAba125. Interestingly, two blaNDM-1 genes from the genus Citrobacter utilized the ISCR1-derived outward-oriented promoters POUT (PISCR1). Furthermore, the insertion of ISAba125 and ISCR1 occurred upstream of the CCATATTT sequence, which is located upstream of the -10 box. We also confirmed that most of the blaNDM genes were disseminated by horizontal gene transfer of the plasmid, but 10 cases of the blaNDM genes were integrated into the chromosome via mobile genetic elements such as IS26, IS150, ISCR1, ICE, and Tn7-like elements. Thus, plasmid-mediated transmission of the PISAba125-blaNDM genes is predominant in Enterobacteriaceae. However, the spread of blaNDM genes with new promoters and vertical dissemination via chromosomal integrations may pose additional serious clinical problems.

Emergence of a Salmonella Rissen ST469 clinical isolate carrying bla NDM-13 in China.

New Delhi metallo-β-lactamase-13 (NDM-13) is an NDM variant that was first identified in 2015 and has not been detected in Salmonella species prior to this study. Here we describe the first identification of a Salmonella Rissen strain SR33 carrying bla NDM-13. The aim of this study was to molecularly characterize SR33’s antimicrobial resistance and virulence features as well as investigate the genetic environment of bla NDM-13. The Salmonella Rissen SR33 strain was isolated from a patient with fever and diarrhea. SR33 belonged to ST469, and it was found to be multidrug-resistant (MDR) and to carry many virulence genes. Phylogenetic analysis showed that SR33 shared a close relationship with most of the Chinese S. Rissen ST469 strains. bla NDM-13 was located in a transmissible IncI1 plasmid pNDM13-SR33. Sequence analysis of bla NDM-13-positive genomes downloaded from GenBank revealed that a genetic context (ΔISAba125-bla NDM-13-ble MBL-trpF) and a hybrid promoter (consisting of −35 sequences provided by ISAba125 and −10 sequences) were conserved. ISAba125 was truncated by IS1294 in three plasmids carrying bla NDM-13, including pNDM13-SR33. To our knowledge, this is the first report of bla NDM-13 carried by Salmonella. The emergence of bla NDM-13 in a clinical MDR S. Rissen ST469 strain highlights the critical need for monitoring and controlling the dissemination of bla NDM-13. bla NDM-13 carried by a transmissible IncI1 plasmid may result in an increased risk of bla NDM-13 transmission. IS1294 may be involved in the movement of bla NDM-13.

Tuning Geraniol Biosynthesis via a Novel Decane-Responsive Promoter in Candida glycerinogenes.

Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of Candida glycerinogenes as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter PGAP derived from C. glycerinogenes itself. The hybrid promoter was able to be induced by n-decane with 3.6 times higher transcriptional intensity than the natural promoter PGAP. In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.

Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha inmetabolic engineeringare expounded. • Threepromoter engineering strategies are introduced in order to expand the promoter toolbox.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation

BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium’s composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host.ResultsCommonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28—which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM—which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)—RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels.ConclusionsThe use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Controlled Decomposable Hydrogel Triggered with a Specific Enzyme.

In this study, superabsorbent polyelectrolyte hydrogels were synthesized by cross-linking a nondegradable poly (allylamine hydrochloride) (PAH) and a recombinant protein with a specific enzymatic cleavage site. The recombinant protein was produced by E. coli with the pET-32b(+) plasmid, which is featured with the thioredoxin (Trx) gene containing a thrombin recognition site and a T7/lac hybrid promoter for high expression of recombinant protein. The swelling test shows that the composite hydrogel still maintained a high swelling ratio to 900% when 15% recombinant protein was cross-linked with PAH. The degradation test shows that such a PAH composite hydrogel could be decomposed by the addition of specific enzyme thrombin, which might lead to new biomedical applications of hydrogels needed to be decomposable by specific time not determined by the time period.

Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.ResultsIn this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.ConclusionsThe synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (PPDH, PPYK, PFBA, PPGM, PGLK, PTRI, PGPI, PADH1, PTEF1 and PGCW14) were obtained with the activity range of 13%–130% of the common promoter PGAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which PPDH and PGCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced PICL1, rhamnose-induced PLRA3 and PLRA4, and a bidirectional promoter (PMal-PPer) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter PLRA3 by 4.7–10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments.

Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassisEscherichia coliNissle 1917 and can detect endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.

CRISPR/Cas9-mediated genome editing directed by a 5S rRNA\u2013tRNAGly hybrid promoter in the thermophilic filamentous fungus Humicola insolens

BackgroundHumicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism.ResultsHerein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation.ConclusionsThe established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis.

ABSTRACTIn Anabaena variabilis, the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co2+-inducible coaT promoter, activated the expression of both nifB1 and nifB2. Activation of nifB2, but not nifB1, by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2. Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2::nifB1::nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity.IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system.

Exploring the Tunability and Dynamic Properties of MarR-PmarO Sensor System in Escherichia coli.

Transcriptional factor-based biosensors (TFBs) have been widely used in dynamic pathway control or high-throughput screening. Here, we systematically explored the tunability of a salicylic acid responsive regulator MarR from Escherichia coli aiming to explore its engineering potential. The effect of endogenous MarR in E. coli on the MarR-PmarO biosensor system was investigated. Furthermore, to investigate the function of marO binding boxes in this biosensor system, a series of hybrid promoters were constructed by placing the marO binding boxes in the strong constitutive pL promoter. The engineered hybrid promoters became responsive to MarR and salicylic acid. To further study the influence of each nucleotide in the marO box on MarR binding, we employed dynamic modeling to simulate the interaction and binding energy between each nucleotide in the marO boxes with the corresponding residues on MarR. Guided by the results of the simulation, we introduced mutations to key positions on the hybrid promoters and investigated corresponding dynamic performance. Two promoter variants I12AII4T and I12AII14T that exhibited improved responsive strengths and shifted dynamic ranges were obtained, which can be beneficial for future metabolic engineering research.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

BackgroundIn biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.ResultsIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.ConclusionWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.

A combinatorial cis-regulatory logic restricts color-sensing Rhodopsins to specific photoreceptor subsets in Drosophila.

Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. The underlying cis-regulatory logic remains poorly understood, but it has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins.

Substrate-activated expression of a biosynthetic pathway in Escherichia coli.

Microbes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of d-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Activation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production d-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13±0.03g L-1 d-glyceric acid within 6h of galacturonate substrate addition. This work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.

Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.