Search

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Cultivated peanut (Arachis hypogaea L.) is a key oil- and protein-providing legume crop of the world. It is full of nutrients, and its nutrient profile is comparable to that of other nuts. Peanut is a unique plant as it showcases a pegging phenomenon, producing flowers above ground, and after fertilization, the developing peg enters the soil and produces seeds underground. This geocarpic nature of peanut exposes its seeds to soil pathogens. Peanut seeds are protected by an inedible pericarp and testa. The pericarp- and testa-specific promoters can be effectively used to improve the seed defense. We identified a pericarp- and testa-abundant expression gene (AhN8DT-2) from available transcriptome expression data, whose tissue-specific expression was further confirmed by the qRT-PCR. The 1827bp promoter sequence was used to construct the expression vector using the pMDC164 vector for further analysis. Quantitative expression of the GUS gene in transgenic Arabidopsis plants showed its high expression in the pericarp. GUS staining showed a deep blue color in the pericarp and testa. Cryostat sectioning of stained Arabidopsis seeds showed that expression is only limited to seed coat (testa), and staining was not present in cotyledons and embryos. GUS staining was not detected in any other tissues, including seedlings, leaves, stems, and roots, except for some staining in flowers. Under different phytohormones, this promoter did not show an increase in expression level. These results indicated that the AhN8DT-2 promoter drives GUS gene expression in a pericarp- and testa-specific manner. The identified promoter can be utilized to drive disease resistance genes, specifically in the pericarp and testa, enhancing peanut seed defense against soil-borne pathogens. This approach has broader implications for improving the resilience of peanut crops and other legumes, contributing to sustainable agricultural practices and food security. [ABSTRACT FROM AUTHOR]

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‐glfL445IalsSalsD, 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 7.14 g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75 g/L, corresponding to a xylose‐to‐acetoin conversion yield of 0.375 g/g. [ABSTRACT FROM AUTHOR]

The brown macroalga Saccharina japonica (Laminariales, Phaeophyta) is the most productive cultured seaweed in the world. In order to improve the biosafety of transgenic kelp, it is necessary to develop endogenous constitutive promoters, replacing those of virus origin. In this study, the housekeeping actin gene from S. japonica (SjACT) was found to contain three exons and two introns, representing a unique actin gene structure pattern in brown algae. Additionally, the 5′ upstream region was obtained using genome walking, and fused to the reporter gene lacZ or EGFP to construct promoter-detective vectors. Using an established genetic transformation system, kelps in different life-cycle stages were transformed. The detection results showed that, in the diploid sporophyte stage, the transient expression from the lacZ gene could be observed in the frond, stipe, or holdfast of kelps, indicating a manner of being non-tissue-specific. And, in the haploid gametophyte stage of S. japonica, the fluorescence of the expressed EGFP were detected in vivo in gametophyte cells of both genders. These results indicate that the promoter of the SjACT gene (pSjACT) functions in a constitutive manner and is expected to be a key endogenous element in the genetic manipulation of kelps. [ABSTRACT FROM AUTHOR]

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a β-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3–10-fold higher compared to those under the CaMV 35S promoter at 10–30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding. [ABSTRACT FROM AUTHOR]

It is important to improve the production of bioactive secondary products for drug development. The Escherichia coli—Streptomyces shuttle vector pSET152 and its derived vector pIB139 containing a strong constitutive promoter ermEp* are commonly used as integrative vectors in actinomycetes. Four new integrative vectors carrying the strong constitutive promoter kasOp*, hrdBp, SCO5768p, and SP44, respectively, were constructed and proven to be functional in different mangrove-derived Streptomyces host strains by using kanamycin resistance gene neo as a reporter. Some biosynthetic genes of elaiophylins, azalomycin Fs, and armeniaspirols were selected and inserted into these vectors to overexpress in their producers including Streptomyces sp. 219807, Streptomyces sp. 211726, and S. armeniacus DSM 43125, resulting in an approximately 1.1–1.4-fold enhancement of the antibiotic yields. [ABSTRACT FROM AUTHOR]

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Glycolate plays an important role as a platform chemical in both polymeric material and cosmetic industries. However, the microbial production of glycolate often encounters challenges associated with unbalanced metabolic flux, leading to a notably low titer. Additionally, the use of expensive inducers, such as IPTG, contributes to an increase in the overall production cost. To address these issues, the key enzymes involved in the glycolate biosynthetic route, including citrate synthase (gltA), isocitrate lyase (aceA), isocitrate dehydrogenase kinase/phosphatase (aceK) and glyoxylate reductase (ycdW), were overexpressed in E. coli under the control of inducible promoters with varying strengths in order to determine the optimal combination. Subsequently, the glycolate pathway was further modulated by replacing inducible promoters with various constitutive synthetic promoters. Through this systematic optimization, the best strain, named Mgly4T1562, produced 3.02 g/L glycolate with 97.32% theoretical yield in shake-flask cultivation. The titer further increased to 15.53 g/L in a fed-batch experiment. Notably, this study marks the first successful utilization of synthetic promoters in tuning the glycolate biosynthetic pathway for glycolate biosynthesis. The strategy presented in this research holds significant promise for facilitating the cost-effective and industrially viable production of glycolate without the need for expensive inducers. [ABSTRACT FROM AUTHOR]

Cultivated peanut (Arachis hypogaea L.) is a key oil- and protein-providing legume crop of the world. It is full of nutrients, and its nutrient profile is comparable to that of other nuts. Peanut is a unique plant as it showcases a pegging phenomenon, producing flowers above ground, and after fertilization, the developing peg enters the soil and produces seeds underground. This geocarpic nature of peanut exposes its seeds to soil pathogens. Peanut seeds are protected by an inedible pericarp and testa. The pericarp- and testa-specific promoters can be effectively used to improve the seed defense. We identified a pericarp- and testa-abundant expression gene (AhN8DT-2) from available transcriptome expression data, whose tissue-specific expression was further confirmed by the qRT-PCR. The 1827bp promoter sequence was used to construct the expression vector using the pMDC164 vector for further analysis. Quantitative expression of the GUS gene in transgenic Arabidopsis plants showed its high expression in the pericarp. GUS staining showed a deep blue color in the pericarp and testa. Cryostat sectioning of stained Arabidopsis seeds showed that expression is only limited to seed coat (testa), and staining was not present in cotyledons and embryos. GUS staining was not detected in any other tissues, including seedlings, leaves, stems, and roots, except for some staining in flowers. Under different phytohormones, this promoter did not show an increase in expression level. These results indicated that the AhN8DT-2 promoter drives GUS gene expression in a pericarp- and testa-specific manner. The identified promoter can be utilized to drive disease resistance genes, specifically in the pericarp and testa, enhancing peanut seed defense against soil-borne pathogens. This approach has broader implications for improving the resilience of peanut crops and other legumes, contributing to sustainable agricultural practices and food security.

The brown macroalga Saccharina japonica (Laminariales, Phaeophyta) is the most productive cultured seaweed in the world. In order to improve the biosafety of transgenic kelp, it is necessary to develop endogenous constitutive promoters, replacing those of virus origin. In this study, the housekeeping actin gene from S. japonica (SjACT) was found to contain three exons and two introns, representing a unique actin gene structure pattern in brown algae. Additionally, the 5′ upstream region was obtained using genome walking, and fused to the reporter gene lacZ or EGFP to construct promoter-detective vectors. Using an established genetic transformation system, kelps in different life-cycle stages were transformed. The detection results showed that, in the diploid sporophyte stage, the transient expression from the lacZ gene could be observed in the frond, stipe, or holdfast of kelps, indicating a manner of being non-tissue-specific. And, in the haploid gametophyte stage of S. japonica, the fluorescence of the expressed EGFP were detected in vivo in gametophyte cells of both genders. These results indicate that the promoter of the SjACT gene (pSjACT) functions in a constitutive manner and is expected to be a key endogenous element in the genetic manipulation of kelps.

Abstract Background Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. Results A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. Conclusions The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

It is important to improve the production of bioactive secondary products for drug development. The Escherichia coli—Streptomyces shuttle vector pSET152 and its derived vector pIB139 containing a strong constitutive promoter ermEp* are commonly used as integrative vectors in actinomycetes. Four new integrative vectors carrying the strong constitutive promoter kasOp*, hrdBp, SCO5768p, and SP44, respectively, were constructed and proven to be functional in different mangrove-derived Streptomyces host strains by using kanamycin resistance gene neo as a reporter. Some biosynthetic genes of elaiophylins, azalomycin Fs, and armeniaspirols were selected and inserted into these vectors to overexpress in their producers including Streptomyces sp. 219807, Streptomyces sp. 211726, and S. armeniacus DSM 43125, resulting in an approximately 1.1–1.4-fold enhancement of the antibiotic yields.

Background: Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. Results: A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. Conclusions: The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species. [ABSTRACT FROM AUTHOR]

SUMMARY: Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high‐quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology. Significance Statement: Hornworts, despite their key phylogenetic position and their unique biology, have been widely overlooked by the plant biology community. Here we report a greatly optimized transformation technique applicable for several hornwort species (Anthoceros agrestis, Anthoceros punctatus, Leiosporoceros dussi, and Phaeoceros carolinianus) as well as provide subcellular localization signals, a new selection marker, and additional constitutive promoters to be used. [ABSTRACT FROM AUTHOR]

Transcriptional regulation has been adopted for developing metabolic engineering tools. The regulatory promoter is a crucial genetic element for strain optimization. In this study, a gene set of Aspergillus oryzae with highly constitutive expression across different growth stages was identified through transcriptome data analysis. The candidate promoters were functionally characterized in A. oryzae by transcriptional control of β-glucuronidase (GUS) as a reporter. The results showed that the glyceraldehyde triphosphate dehydrogenase promoter (PgpdA1) of A. oryzae with a unique structure displayed the most robust strength in constitutively controlling the expression compared to the PgpdA2 and other putative promoters tested. In addition, the ubiquitin promoter (Pubi) of A. oryzae exhibited a moderate expression strength. The deletion analysis revealed that the 5' untranslated regions of gpdA1 and ubi with the length of 1028 and 811 nucleotides, counted from the putative translation start site (ATG), respectively, could efficiently drive the GUS expression. Interestingly, both promoters could function on various carbon sources for cell growth. Glucose was the best fermentable carbon source for allocating high constitutive expressions during cell growth, and the high concentrations (6–8% glucose, w/v) did not repress their functions. It was also demonstrated that the secondary metabolite gene coding for indigoidine could express under the control of PgpdA1 or Pubi promoter. These strong and moderate promoters of A. oryzae provided beneficial options in tuning the transcriptional expression for leveraging the metabolic control towards the targeted products. [ABSTRACT FROM AUTHOR]

Glycolate plays an important role as a platform chemical in both polymeric material and cosmetic industries. However, the microbial production of glycolate often encounters challenges associated with unbalanced metabolic flux, leading to a notably low titer. Additionally, the use of expensive inducers, such as IPTG, contributes to an increase in the overall production cost. To address these issues, the key enzymes involved in the glycolate biosynthetic route, including citrate synthase (gltA), isocitrate lyase (aceA), isocitrate dehydrogenase kinase/phosphatase (aceK) and glyoxylate reductase (ycdW), were overexpressed in E. coli under the control of inducible promoters with varying strengths in order to determine the optimal combination. Subsequently, the glycolate pathway was further modulated by replacing inducible promoters with various constitutive synthetic promoters. Through this systematic optimization, the best strain, named Mgly4T1562, produced 3.02 g/L glycolate with 97.32% theoretical yield in shake-flask cultivation. The titer further increased to 15.53 g/L in a fed-batch experiment. Notably, this study marks the first successful utilization of synthetic promoters in tuning the glycolate biosynthetic pathway for glycolate biosynthesis. The strategy presented in this research holds significant promise for facilitating the cost-effective and industrially viable production of glycolate without the need for expensive inducers.

Simple Summary: With the advancement of synthetic biology and metabolic engineering, regulatory elements applied for the accurate expression of target genes have become more important. Among them, due to their important role in regulating gene expression at the transcription level, a number of homologous or heterologous promoters have been used to improve the yield of target metabolites in different microorganisms. However, the method to isolate strong constitutive promoters in different microorganisms is still limited. Our work describes a novel approach to identify strong constitutive promoters in Escherichia coli and Serratia marcescens. The identified promoters were further used for fine-tuning gene expression and reprogramming the metabolic flux of L-valine and prodigiosin in E. coli and S. marcescens, respectively, and finally, the higher-level L-valine synthesis strain and prodigiosin production strain were isolated. The method shown in our study can also be a useful strategy to identify strong constitutive promoters in other bacteria and isolate other effective genetic regulatory elements, such as ribosome binding sites, terminators, and N-terminal coding sequences (NCS), for tuning gene expression in different microorganisms. Promoters serve as the switch of gene transcription, playing an important role in regulating gene expression and metabolites production. However, the approach to screening strong constitutive promoters in microorganisms is still limited. In this study, a novel method was designed to identify strong constitutive promoters in E. coli and S. marcescens based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of E. coli, Bacillus subtilis, and Corynebacterium glutamicum were randomly interrupted and inserted into the upstream of reporter gene gfp to construct three promoter libraries, and a potential strong constitutive promoter (PBS) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (PBS76) of the screened promoter was identified by sequence truncation. Third, a promoter library of PBS76 was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter PBS76-100 was 59.56 times higher than that of the promoter PBBa_J23118. Subsequently, promoters PBBa_J23118, PBS76, PBS76-50, PBS76-75, PBS76-85, and PBS76-100 with different strengths were applied to enhance the metabolic flux of L-valine synthesis, and the L-valine yield was significantly improved. Finally, a strong constitutive promoter suitable for S. marcescens was screened by a similar method and applied to enhance prodigiosin production by 34.81%. Taken together, the construction of a promoter library based on random genomic interruption was effective to screen the strong constitutive promoters for fine-tuning gene expression and reprogramming metabolic flux in various microorganisms. [ABSTRACT FROM AUTHOR]

To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future. [ABSTRACT FROM AUTHOR]

To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.

Promoter-centric genetic tools play a crucial role in controlling gene expression for various applications, such as strain engineering and synthetic biology studies. Hence, a critical need persists for the development of robust gene expression tools. Streptomyces are well-known prolific producers of natural products and exceptional surrogate hosts for the production of high-value chemical compounds and enzymes. In this study, we reported a straightforward and effective strategy for the creation of potent gene expression tools. This was primarily achieved by introducing an additional -35-like motif upstream of the original -35 region of the promoter, coupled with the integration of a palindromic cis -element into the 5'-UTR region. This approach has generated a collection of robust constitutive and inducible gene expression tools tailored for Streptomyces . Of particular note, the fully activated o xytetracycline-inducible gene expression system containing an engineered k asO p* promoter ( OK ) exhibited nearly an order of magnitude greater activity compared to the well-established high-strength promoter kasO p* under the tested conditions, establishing itself as a powerful gene expression system for Streptomyces . This strategy is expected to be applicable in modifying various other promoters to acquire robust gene expression tools, as evidenced by the enhancement observed in the other two promoters, PL and P21 in this study. Moreover, the effectiveness of these tools has been demonstrated through the augmented production of transglutaminase and daptomycin. The gene expression tools established in this study, alongside those anticipated in forthcoming research, are positioned to markedly advance pathway engineering and synthetic biology investigations in Streptomyces and other microbial strains.

Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering. [ABSTRACT FROM AUTHOR]

Acetobacter pasteurianus is an excellent cell factory for production of highly-strength acetic acid, and attracts an increasing attention in metabolic engineering. However, the available well-characterized constitutive and inducible promoters are rather limited to adjust metabolic fluxes in A. pasteurianus. In this study, we screened a panel of constitutive and acid stress-driven promoters based on time-series of RNA-seq data and characterized in A. pasteurianus and Escherichia coli. Nine constitutive promoters ranged in strength from 1.7-fold to 100-fold that of the well-known strong promoter P adh under non-acetic acid environment. Subsequently, an acetic acid-stable red fluorescent visual reporting system was established and applied to evaluate acid stress-driven promoter in A. pasteurianus during highly-acidic fermentation environment. P groES was identified as acid stress-driven strong promoters, with expression outputs varied from 100% to 200% when acetic acid treatment. To assess their application potential, ultra-strong constitutive promoter P tuf and acid stress-driven strong promoter P groES were selected to overexpress acetyl-CoA synthase and greatly improved acetic acid tolerance. Notably, the acid stress-driven promoter displayed more favorable for regulating strain robustness against acid stress by overexpressing tolerance gene. In summary, this is the first well-characterized constitutive and acid stress-driven promoter library from A. pasteurianus , which could be used as a promising toolbox for metabolic engineering in acetic acid bacteria and other gram-negative bacteria. [Display omitted] ● A panel of strong constitutive and acid stress-driven promoter is firstly identified in A. pasteurianus. ● A red fluorescent protein mRFP1 with low pKa value is more suitable as visual reporter under acetic acid environment. ● Stress-driven promoter is more favorable for regulating acetic acid tolerance by overexpressing tolerance gene. [ABSTRACT FROM AUTHOR]

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80–100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. Key points: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus - Escherichia coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of the egfp gene under the control of different promoters was confirmed and quantified by Western blotting and qPCR, which suggested that the activity of these promoters varied from 18 to 650% of the activity of P sarA , a widely used promoter for gene expression. In addition, our constructed vectors were verified to be highly compatible with gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used to overexpress two endogenous proteins in S. aureus , namely, catalase and the transcriptional repressor of purine biosynthesis (PurR). Meanwhile, the physiological functions and phenotypes of overexpressed PurR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This set of vectors carrying different constitutive promoters developed here will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-Escherichia coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of the egfp gene under the control of different promoters was confirmed and quantified by Western blotting and qPCR, which suggested that the activity of these promoters varied from 18 to 650% of the activity of PsarA, a widely used promoter for gene expression. In addition, our constructed vectors were verified to be highly compatible with gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used to overexpress two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (PurR). Meanwhile, the physiological functions and phenotypes of overexpressed PurR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This set of vectors carrying different constitutive promoters developed here will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.

Abstract Background Docosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp. Results Here, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1α p , TEF-1 p , ccg1 p , and ubiquitin p ) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1 p > TEF-1 p > AOX1 p (induced) > EF-1α p > ubiquitin p . With the strong constitutive promoter ccg1 p , Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively. Conclusions Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp.

Promoters serve as the switch of gene transcription, playing an important role in regulating gene expression and metabolites production. However, the approach to screening strong constitutive promoters in microorganisms is still limited. In this study, a novel method was designed to identify strong constitutive promoters in E. coli and S. marcescens based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of E. coli, Bacillus subtilis, and Corynebacterium glutamicum were randomly interrupted and inserted into the upstream of reporter gene gfp to construct three promoter libraries, and a potential strong constitutive promoter (PBS) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (PBS76) of the screened promoter was identified by sequence truncation. Third, a promoter library of PBS76 was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter PBS76-100 was 59.56 times higher than that of the promoter PBBa_J23118. Subsequently, promoters PBBa_J23118, PBS76, PBS76-50, PBS76-75, PBS76-85, and PBS76-100 with different strengths were applied to enhance the metabolic flux of L-valine synthesis, and the L-valine yield was significantly improved. Finally, a strong constitutive promoter suitable for S. marcescens was screened by a similar method and applied to enhance prodigiosin production by 34.81%. Taken together, the construction of a promoter library based on random genomic interruption was effective to screen the strong constitutive promoters for fine-tuning gene expression and reprogramming metabolic flux in various microorganisms.

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Goswami, Singer, Simmons and Awasthi.)

Synthetic dsRNA are valuable tools for reverse genetics research and virus silencing applications. Its synthesis can be performed both in vivo or in vitro. Whilst the latter presents the drawback of high production cost, the former has the advantage of being less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the gene for the T7 RNA polymerase inducible by IPTG. The (ds)RNAs for gene of interest are then synthesized under the T7 promoter. In this work, we present a reliable vector system that includes the insulated promoter proD for the constitutive expression of dsRNA in E. coli that does not require any inducer and that renders elevated dsRNA yield. In tandem, the T7 and proD promoters render the highest dsRNA yield. The accumulation of dsRNA in this system entails a high metabolic cost for the cell. Bacterial RNA extractions that included dsRNAs homologous to the m5GFPer gene and derived from both the synthetic and constitutive promoters induce silencing of GFP expression in Nicotiana benthamiana 16c. Key points • A vector system that includes a constitutive promoter and a T7 promoter in tandem for maximizing dsRNA synthesis. • The metabolic cost for bacteria is maximum when the two promoters are operating simultaneously and results from the accumulation of dsRNA. • Bacterial RNA extractions from both the induced and constitutive systems that include a mGFP5er-derived dsRNA are capable of silencing the GFP expression in Nicotiana benthamiana 16c plants [ABSTRACT FROM AUTHOR]

Attenuated Listeria monocytogenes could be a potential vaccine vector for the immunotherapy of tumors or pathogens. However, the lack of reliable promoters has limited its ability to express foreign antigens. In the present study, 21 promoters were identified from Listeria monocytogenes through RNA-seq analysis under two pH conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters exhibited higher strength than Phelp (1.8-fold to 5.4-fold), a previously reported strong promoter. Furthermore, the selected 5 constitutive promoters exhibited higher UreB production activity than Phelp (1.1-fold to 8.3-fold). Notably, a well-characterized constitutive promoter P18 was found with the highest activity of fluorescence intensity and UreB production. In summary, the study provides a panel of strong constitutive promoters for Listeria monocytogenes and offers a theoretical basis for mining constitutive promoters in other organisms. Key points: • Twenty-one promoters were identified from L. monocytogenes through RNA-seq. • Fluorescent tracer of L. monocytogenes (P18) was performed in vitro and in vivo. • A well-characterized constitutive promoter P18could improve the expression level of a foreign antigen UreB in L. monocytogenes [ABSTRACT FROM AUTHOR]

To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100 mg/L of linolenic acid reached 10.12 mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids. [ABSTRACT FROM AUTHOR]

Biobased production of 5-aminovalerate (5AVA) from biomass can support a sustainable and economic biorefinery process to produce bio-based nylon 5 for food packaging materials. Cost-competitive production of 5AVA from biomass is a key factor in the successful commercialization of nylon 5. Bioproduction of 5AVA is a promising candidate for the industrial process to the current petrochemical route. In this study, we developed an artificial 2-keto-6-aminocaproate-mediated pathway for cost-competitive and high efficiency production of 5AVA in engineered Escherichia coli. Firstly, the combination of native l -lysine α-oxidase (RaiP) from Scomber japonicas , α-ketoacid decarboxylase (KivD) from Lactococcus lactis and aldehyde dehydrogenase (PadA) from Escherichia coli could efficiently convert l -lysine into 5AVA. Moreover, the engineered strains ML03-P nirB -RKP, ML03-P PL-PR -RKP, ML03-P M1-93 -RKP induced by anaerobic condition, temperature-induced, constitutive expression instead of expensive isopropyl β-D-thiogalactoside were constructed, respectively. The use of nirB promoter induced by anaerobic condition not only could attain a higher titer of 5AVA than PL-PR and M1-93 promoters, but omit cost of expensive exogenous inducers. After the replacement of industrial materials, 5AVA titer successfully reached 33.68 g/L in engineered strain ML03-P nirB -RKP via biotransformation. This biotransformation process conduces to the cosmically industrial 5AVA bioproduction. • An artificial synthetic pathway with promoter replacement was proposed to produce 5AVA. • Strain ML03-P nirB -RKP can achieve a high 5AVA titer of 33.68 g/L. • The anaerobically-induced strategy conduces to the cosmically industrial 5AVA production. [ABSTRACT FROM AUTHOR]

Abstract Background Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production. Results Two strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS). Conclusion This work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain. [ABSTRACT FROM AUTHOR]

The cauliflower mosaic virus (CaMV) 35S promoter is known as the most frequently used promoter in plant biotechnology. Although it is widely considered to be a strong constitutive promoter exhibiting high transcriptional activity, the transcriptional stability of CaMV 35S has not been extensively studied. Using the model plant species Arabidopsis thaliana, this study aimed for a comprehensive expression analysis of two widely used plant transgenes, neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (EGFP), regulated by a double CaMV 35S promoter depending on the organ type, time of day, plant age, and in response to abiotic stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the NPTII and EGFP transcript levels were markedly higher in the cotyledons, young leaves, and roots than in the inflorescences, stems, and adult leaves of three independent transgenic A. thaliana lines. The expression of NPTII and EGFP varied during the day and was elevated with the plant age. Drought and cold stress considerably affected the expression of the transgenes, while heat, high salinity, and wounding had no significant effect. This study shows that transgenes driven by a common constitutive promoter can exhibit marked variations in transcriptional activity depending on plant organ, physiological conditions, and in response to abiotic stress. Therefore, to ensure high and stable transgene activity, considerable attention should be given to the transgenic plant material and incubation conditions before harvesting the plant material. [ABSTRACT FROM AUTHOR]

Genes involved in the transport and catabolism of carbohydrates are usually controlled through the binding of the catabolite control protein A (CcpA) to the catabolite-responsive elements (cre) of target genes in Gram-positive bacteria. In this work, we show how the elimination of the cre sites in Lactobacillus casei BL23 promoters induced by sorbitol (PgutF), maltose (PmalL), and myo-inositol (PiolT) allowed the induction of gene expression in media supplemented with sorbitol, maltose, and myo-inositol, respectively, even in the presence of glucose. This was studied using plasmids encoding the anaerobic fluorescent protein evoglow-Pp1 as a reporter. In addition, gutF cre site was introduced into a bile inducible promoter (P16090) and into the constitutive promoter of the elongation factor P (PEf-P) of L. casei BL23. The existence of the cre site blocked gene expression in the P16090 inducible promoter in the presence of glucose, while it had no influence on the expression of the PEf-P constitutive one. These results demonstrated that the introduction or elimination of cre sites in inducible promoters allows the control and modification of their heterologous genetic expression, showing how the cre site, the transcriptional regulator, and CcpA interact to control gene expression in inducible genes. Key points: • Cre sequences regulate gene expression in inducible promoters in L. casei BL23. • Cre sites do not affect gene expression in constitutive promoters in L. casei BL23. • Cre sequences could control heterologous genic expression in lactobacilli. [ABSTRACT FROM AUTHOR]

Lactococcus cremoris (homotypic synonym: Lactococcus lactis ) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris , and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA , encoding β-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

Abstract Background Transgenic technology has become an important technique for crop genetic improvement. The application of well-characterized promoters is essential for developing a vector system for efficient genetic transformation. Therefore, isolation and functional validation of more alternative constitutive promoters to the CaMV35S promoter is highly desirable. Results In this study, a 2093-bp sequence upstream of the translation initiation codon ATG of AtSCPL30 was isolated as the full-length promoter (PD1). To characterize the AtSCPL30 promoter (PD1) and eight 5′ deleted fragments (PD2-PD9) of different lengths were fused with GUS to produce the promoter::GUS plasmids and were translocated into Nicotiana benthamiana. PD1-PD9 could confer strong and constitutive expression of transgenes in almost all tissues and development stages in Nicotiana benthamiana transgenic plants. Additionally, PD2-PD7 drove transgene expression consistently over twofold higher than the well-used CaMV35S promoter under normal and stress conditions. Among them, PD7 was only 456 bp in length, and its transcriptional activity was comparable to that of PD2-PD6. Moreover, GUS transient assay in the leaves of Nicotiana benthamiana revealed that the 162-bp (− 456~ − 295 bp) and 111-bp (− 294~ − 184 bp) fragments from the AtSCPL30 promoter could increase the transcriptional activity of mini35S up to 16- and 18-fold, respectively. Conclusions As a small constitutive strong promoter of plant origin, PD7 has the advantage of biosafety and reduces the probability of transgene silencing compared to the virus-derived CaMV35S promoter. PD7 would also be an alternative constitutive promoter to the CaMV35S promoter when multigene transformation was performed in the same vector, thereby avoiding the overuse of the CaMV35S promoter and allowing for the successful application of transgenic technology. And, the 162- and 111-bp fragments will also be very useful for synthetic promoter design based on their high enhancer activities.

Background: Docosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp. Results: Here, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1p> TEF-1p > AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively. Conclusions: Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp. [ABSTRACT FROM AUTHOR]

Abstract Background Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering. Results The limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor l-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (P lacUV5 -aroG fbr -tyrA fbr -aroE) and the lacI gene with module 2 (P trc -glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldh Y52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented. Conclusions The plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.

Summary: The expression of insecticidal proteins under constitutive promoters in transgenic plants is fraught with problems like developmental abnormalities, yield drag, expression in unwanted tissues, and seasonal changes in expression. RbPCD1pro, a rapid, early acting wound‐inducible promoter from rose that is activated within 5 min of wounding, was isolated and characterized. Wounding increased transcript levels up to 150 and 500 folds within 5 and 20 min coupled with high translation as seen by histochemical GUS enzyme activity within 5–20 min. RbPCD1pro was activated by both sucking and chewing insects and showed wound‐inducible expression in various aerial tissues of plants representing commercially important dicot and monocot families. The promoter showed no expression in any vegetative tissue except upon wounding. Functionality of RbPCD1pro was tested by its ability to drive expression of the insecticidal protein gene cryIAc in transgenic Arabidopsis and tomato. Strong wound‐inducible CryIAc expression was observed in both plants that increased 100–350 fold (Arabidopsis) and 280–600 fold (tomato) over the unwounded background within 5 min and over 1000–1600 fold within 20 min. The unwounded background level was just 3–6% of the CaMV35S promoter while wound‐induced expression was 5–27 folds higher than the best CaMV35S line in just 5 min and 80‐fold higher in 20 min. Transgenic plants showed strong resistance even to larger fourth instar larvae of H. armigera and no abnormalities in development and general plant growth. This is one of the earliest acting promoters with wide biotechnological application across monocot and dicot plants. [ABSTRACT FROM AUTHOR]

Highlights • The constitutive expression of GshF from Streptococcus sanguinis (GshFss) was constructed under four constitutive promoters. • The enzyme activity of GshFss was improved under the regulation of constitutive promoter Pdel1. • The deletions of tanA, ggt, and pepT in JM109 enhanced the yield of GSH production. • The GSH yield based on cysteine was achieved 0.8 mol/mol without any inducer in fed-batch fermentation. Abstract Glutathione (GSH) is an important bioactive tripeptide and widely used in food, medicine and other industries. Recently, the bifunctional glutathione synthetase, GshF, has been applied for efficient GSH production under inducible promoter. In this study, the constitutive expression of GshF from Streptococcus sanguinis (GshFss) was investigated under four different constitutive promoters. Based on previous study, five genes in Escherichia coli JM109 were deleted to eliminate the degradation of precursors and GSH. The effects of gene knockout on the constitutive expression of GshFss and GSH production were evaluated by whole cell catalysis. Finally, the engineered strain JM03Pdel1 produced 24 mM glutathione with addition of 30 mM precursors in 5-L bioreactor fed-batch fermentation. The yield of GSH based on cysteine in JM03Pdel1was reached 80% without any inducer, which was improved by 17.3% than that in the control strain. [ABSTRACT FROM AUTHOR]

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1a promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions. [ABSTRACT FROM AUTHOR]

Highlights • Conversion of recinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid. • BVMO-ADH-FadL expressing E. coli biotransformation system. • Induction-free biotransformation system development. • Waste by-product crude glycerol utilization as sole carbon source. Abstract Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from recinoleic acid was achieved by whole-cell biotransformation by Escherichia coli, utilizing crude glycerol as the sole carbon source. Whole-cell biotransformation resulted in ∼93% conversion of the substrate ricinoleic acid to (Z)-11-(heptanoyloxy)undec-9-enoic acid. We replaced the inducer-dependent promoter system (T7 and Rhm promotors) with a constitutive promoter system. This resulted in successful expression of ADH, FadL, and E6-BVMO, without costly inducer addition. Efficacy evaluation of the whole-cell biotransformation by inducer-free system by five different E. coli strains revealed that the highest product titer was accumulated in E. coli BW25113 strain. The engineered inducer-free system using crude glycerol as the sole carbon source showed competitive performance with induction systems. Optimized conditions resulted in the accumulation of 7.38 ± 0.42 mM (Z)-11-(heptanoyloxy)undec-9-enoic acid, and when 10 mM substrate was used as feed concentration, the product titer reached 2.35 g/L. The inducer-free construct with constitutive promoter system that this study established, which utilizes the waste by-product crude glycerol, will pave the way for the economic synthesis of many industrially important chemicals, like (Z)-11-(heptanoyloxy)undec-9-enoic acid. [ABSTRACT FROM AUTHOR]

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering. [ABSTRACT FROM AUTHOR]