Aug 2026· Applied Microbiology and Biotechnology· 0 citations
TL;DR
Results identify P SD13 as a phage-derived promoter exhibiting strong activity in both Streptomyces and E. coli, suggesting its potential as a useful genetic element for Streptomyces engineering and heterologous gene expression.
Abstract
Efficient and tunable promoters are essential tools for metabolic engineering and synthetic biology in
Streptomyces
. In this study, the lytic bacteriophage phiSASD1 was exploited as a source of novel regulatory elements. Fifteen candidate promoter fragments were identified through bioinformatic prediction and systematically evaluated using a catechol 2,3-dioxygenase (
xylE
) reporter system in
Streptomyces lividans
TK54 and
Escherichia coli
JM109. Among these candidates, seven fragments exhibited measurable promoter activity, with P
SD13
showing the highest transcriptional strength. In
S. lividans
, P
SD13
displayed up to a 9.79
-
fold higher activity than the widely used strong promoter P
ermE
*, while retaining detectable activity in
E. coli
, indicating excellent cross-host compatibility. Sequence analysis combined with 5′ RACE revealed that P
SD13
possesses a typical σ⁷⁰
-
dependent promoter architecture, with its core functional region located between − 70 to + 9 bp relative to the transcription start site. Furthermore, P
SD13
efficiently drove the soluble expression of phage endolysin in
E. coli
, potentially reducing inclusion body formation compared with conventional T7-based expression systems. Collectively, these results identify P
SD13
as a phage-derived promoter exhibiting strong activity in both
Streptomyces
and
E. coli
, suggesting its potential as a useful genetic element for
Streptomyces
engineering and heterologous gene expression.
Key points
•
Strong transcriptional promoters were systematically identified from Streptomyces phages.
•
The novel SD13 promoter shows higher activity than PermE* and works across hosts.
•
SD13 promoter efficiently expresses soluble heterologous proteins, advancing Streptomyces synthetic biology.
This review systematically summarizes the features of Bacillus subtilis expression systems, the regulatory effects of various promoters on heterologous expression efficiency, and rational modification strategies of promoter core regions. B. subtilis WB600 or WB800 are the preferred hosts for autonomous plasmid-based expression systems due to their multiple protease deletions, which reduce recombinant protein degradation. In contrast, wild-type strains are more suitable for chromosomal integration systems because of native genomic background and physiological characteristics, which are advantageous for long-term stable expression. Constitutive promoters are suitable for stable and large-scale industrial enzyme production, while inducible promoters realize accurate and tunable control of gene expression. Dual promoters can effectively enhance transcription and protein yield, but are not universally superior to single promoters, and the final expression efficiency mainly depends on the downstream promoter adjacent to the target gene. Core region modification, such as optimizing the consensus sequences of the -35 and - 10 regions and adjusting the spacer length, can significantly improve promoter strength and consequently protein expression. The coordinated optimization of host-promoter-critical elements is critical to achieve highly efficient expression of target products. This review provides a theoretical basis for the further development and application of B. subtilis expression systems, offering valuable references for the rational development of high-efficiency promoter engineering strategies and heterologous gene expression optimization.
Man-Fei Li, Meng Zheng, Peng-Yuan Wang et al.· Journal of Microbiological M...· 0 citations
ABSTRACT Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust, well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance, and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. IMPORTANCE The use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. The use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications.
Kevin P. Trieu, Bryan Bishé, Arnaud Taton et al.· Applied and Environmental Mi...· 0 citations
Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. Importance The use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. Research Areas Microbial genetics, plasmids and other genetic constructs, biotechnology Journal Secction Biotechnology
Kevin P. Trieu, Bryan Bishé, Arnaud Taton et al.· bioRxiv· 0 citations
The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.
Qin Fan, Stevanie Stevanie, S. Frielingsdorf et al.· Microbial Cell Factories· 0 citations
This study investigates the effects of pBHR1’s native mobilization protein, MobV, on the retention of pBBR1 origin plasmids in R. palustris, and provides design principles for constructing stable, high-performing vectors in non-model gram-negative hosts.
M. Kathol, Quin Barton, Cheryl M. Immethun et al.· Microbiology spectrum· 0 citations
The yeast Komagataella phaffii (formerly Pichia pastoris) is a robust host for recombinant protein production, capable of growing at high cell densities, performing post-translational modifications, and efficiently secreting heterologous proteins. However, gene expression still relies mainly on the methanol-inducible AOX1 promoter (PAOX1) and the constitutive GAPDH promoter (PGAP), highlighting the need for alternative promoters with distinct regulatory properties. This study aimed to identify and characterize promoters from the most highly transcribed endogenous genes across diverse culture conditions as strong promoter candidates to expand the genetic toolbox for this yeast. Seven promoters were identified through RNA-seq analysis from genes showing transcript levels comparable to or higher than those of genes regulated by PAOX1 or PGAP under specific culture conditions. These promoters were further transcriptionally characterized by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) under different carbon sources, growth phases, and temperatures through the measurement of transcript levels of their corresponding genes, revealing distinct transcriptional profiles. Two major clusters were identified: one associated with genes upregulated during the stationary phase and the other with genes upregulated during the exponential phase. These transcriptional profiles provided a basis for the rational design of fed-batch strategies tailored to each promoter when driving heterologous gene expression. The 0208 and HSP12 promoters showed particular potential at low specific growth rates, favoring high recombinant protein titers with reduced biomass accumulation. In silico analyses suggested that 0208 encodes a mitochondrial membrane-associated protein with a possible functional relationship to Hsp12. Overall, this work provides well-characterized K. phaffii strong promoter candidates, supporting future biotechnological applications.
Karla B. Fernández-Cano, J. M. Viader-Salvadó, M. Guerrero-Olazarán· PeerJ· 0 citations