The plasmids pSB3 of Zygosaccharomyces rouxii and the 2-micron circle of Saccharomyces cerevisiae belong to a small family of multi-copy yeast DNA plasmids. Members are similarly organized, with each encoding a conserved version of Flp, the recombinase required for copy number amplification. In contrast, proteins and loci required for partitioning seemed to differ. For the 2-micron circle, partitioning involves association of plasmid proteins Rep1 and Rep2 with each other and with the plasmid STB locus. Here, updated sequencing revealed all members encode a Rep1 protein more conserved than previously reported with pSB3 Rep1 the largest due to a long internal non-conserved region. pSB3 partitioning protein C, despite not resembling Rep2, was found to associate with pSB3 Rep1 in vivo, with amino-terminal domains of each sufficient for this interaction. Plasmid inheritance assays identified a region upstream of the REP1 gene as the pSB3 partitioning locus (PAR) and showed pSB3 could replicate and partition in several budding yeast species including Lachancea waltii, S. cerevisiae, Torulaspora delbrueckii, and Zygosaccharomyces bailii. In one-hybrid protein-DNA interaction assays, pSB3 Rep1 and C were found to require each other for association with pSB3 PAR and the plasmid gene promoters and did not recognize 2-micron STB. Taken together, these results support the mechanism of partitioning and regulation of plasmid gene expression by partitioning proteins being conserved between pSB3 and 2-micron. Furthermore, molecular tools developed in this study provide the basis for developing new plasmid vectors based on pSB3 that can be effectively inherited in multiple budding yeast species.

Bacterial foodborne contamination poses a dual challenge of chemical preservative risks and antibiotic resistance, drives the need for green production of natural antimicrobial alternatives. The reported cationic antimicrobial peptide (AMP) Spgillcin177-189 derived from the Scylla paramamosain, has strong antimicrobial activity against Staphylococcus aureus and clinical isolation strains. To meet industry demand in future, large-scale production of Spgillcin177- 189 is essential. In the study, Pichia pastoris expression system was established for production of the recombinant Spgillcin177- 189 (rSpgillcin177-189). Then, multicopy strategy was selectively designed by employing the Golden Gate assembly technology to efficiently construct multi-copy plasmids, which significantly enhanced the expression level of Spgillcin177- 189. A yield of 126.1mg/L was harvested with 2.75-fold higher that of the single-copy strain. In addition, the recombinant Spgillcin177 - 189 exhibited potent antibacterial activity against multiple foodborne pathogens within a MIC range of 5.25-84µg/mL. It also showed effective bactericidal activity and anti-biofilm activity against Staphylococcus aureus and Vibrio parahaemolyticus. rSpgillcin177 - 189 exhibited good thermostability, with no obvious cytotoxicity and hemolytic activity. rSpgillcin177 - 189 may interact with microbial surface components via hydrogen bonding, which were vital for peptide activity in combating bacteria. The rSpgillcin177 - 189 specifically targeting the cell membrane, disrupted bacterial membrane integrity and leading to cell death. This study provided a very feasible genetic engineering strategy for large-scale production of rSpgillcin177 - 189, which will be applied at a lower cost in agricultural and food industries in future.

Here, we applied a transcription factor (TF) decoy strategy to enhance avermectin (AVE) production and activate the cryptic lycopene biosynthetic gene cluster (BGC) in industrial species Streptomyces avermitilis. Two decoy systems based on multicopy plasmid pKC1139 for repressors AfsR and AveI significantly increased AVE titers by 526.1% and 417.6%, respectively, compared to the wild-type (WT) strain. We further constructed a series of integrative decoy plasmids harboring varying numbers of AfsR decoys to demonstrate the dose-dependent effect, followed by a pKC1139-based AfsR decoy library to screen for optimal AVE-producing sequences. Three top-performing sequences were identified, increasing AVE titers by up to 1040.2% in the WT strain and 36.1% in an industrial strain. Finally, we successfully activated lycopene production (45.5 μg/mL) by using the crtY-crtE intergenic region as a decoy. These findings demonstrate that the simple, easy-to-implement TF decoy strategy can be applied to Streptomyces metabolic engineering for the overproduction of secondary metabolites.

Multicopy plasmids are widespread in nature and compose a common strategy for spreading beneficial traits across microbes. However, the role of plasmids in supporting the evolution of encoded genes remains underexplored due to challenges in experimentally manipulating key parameters such as plasmid copy number and mutation rate. Here we developed a strategy for controlling copy number in the plasmid-based Saccharomyces cerevisiae continuous evolution system, OrthoRep, and used our resulting capabilities to investigate the evolution of a conditionally essential gene under varying copy number and mutation rate conditions. Our results show that low copy number facilitated the faster enrichment of beneficial alleles whereas high copy number promoted robustness through the maintenance of allelic diversity. High copy number also slowed the removal of deleterious mutations and increased the fraction of non-functional alleles that could hitchhike during evolution. This study highlights the nuanced relationships between plasmid copy number, mutation rate and evolutionary outcomes, providing insights into the adaptive dynamics of genes encoded on multicopy plasmids and nominating OrthoRep as a versatile tool for studying plasmid evolution.

Yersinia pestis has spilled over from wild rodent reservoirs to commensal rodents and humans, causing three historically recorded pandemics. Depletion in the copy number of the plasmid-encoded virulence gene pla occurred in later-dated strains of the first and second pandemics, yet the biological relevance of the pla deletion has been difficult to test. We identified modern Y. pestis strains that independently acquired the same pla depletion as ancient strains and herein show that excision of pla from the multicopy pPCP1 plasmid is accompanied by the integration of a separate full pPCP1 harboring pla into the single-copy pCD1 plasmid, reducing pla dosage. Moreover, we demonstrate that this depletion decreases the mortality of mice in models of bubonic plague but not in the pneumonic and septicemic forms of the disease. We hypothesize that pla depletion may have been selectively advantageous in bubonic plague, owing to rodent fragmentation after pandemic-induced mortality.

Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.

Although bacterial cells typically contain a single chromosome, some species are naturally polyploid and carry multiple copies of their chromosome. Polyploid chromosomes can be identical or heterogeneous, the latter giving rise to bacterial heterozygosity. Although the benefits of heterozygosity are well studied in eukaryotes, its consequences in bacteria are less understood. Here, we examine this question in the context of antibiotic resistance to understand how bacterial genomic heterozygosity affects bacterial survival. Using a cell-wall-deficient model system in the actinomycete Kitasatospora viridifaciens, we found that heterozygous cells that contain different chromosomes expressing different antibiotic resistance markers persist across a broad range of antibiotic concentrations. Recombinant cells containing the same resistance genes on a single chromosome also survive these conditions, but these cells pay a significant fitness cost due to the constitutive expression of these genes. By contrast, heterozygous cells can mitigate these costs by flexibly adjusting the ratio of their different chromosomes, thereby allowing rapid responses in temporally and spatially variable environments. Our results provide evidence that bacterial heterozygosity can increase adaptive plasticity in bacterial cells in a similar manner to the evolutionary benefits provided by multicopy plasmids in bacteria.

We developed a T7 expression system in Bacillus subtilis, incorporating the rhiL promoter responsive to pectin and glucose to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene encoding a mutated green fluorescent protein (EGFP) under the T7 promoter in a multicopy plasmid, we demonstrated that the EGFP expression was robustly induced by pectin and effectively repressed by glucose. These non-toxic and highly soluble effector compounds facilitate homogeneous expression control and large-scale protein production. The modified system, in which the Shine-Dalgarno sequence upstream of T7 pol was replaced with a highly efficient one from the ylbP gene, achieved a 6.1-fold increase in the maximum expression level upon induction while maintaining tight glucose-mediated repression. Moreover, the modified system's applicability to extracellular protein production was validated by the secretory production of B. subtilis cellulase EglS induced by pectin.

Allotopic expression refers to the artificial relocation of an organellar gene to the nucleus. Subunit 2 (Cox2) of cytochrome c oxidase, a subunit with 2 transmembrane domains (TMS1 and TMS2) residing in the inner mitochondrial membrane with a Nout-Cout topology, is typically encoded in the mitochondrial cox2 gene. In the yeast Saccharomyces cerevisiae, the cox2 gene can be allotopically expressed in the nucleus, yielding a functional protein that restores respiratory growth to a Δcox2 null mutant. In addition to a mitochondrial targeting sequence followed by its natural 15-residue leader peptide, the cytosol synthesized Cox2 precursor must carry one or several amino acid substitutions that decrease the mean hydrophobicity of TMS1 and facilitate its import into the matrix by the TIM23 translocase. Here, using a yeast strain that contains a COX2W56R gene construct inserted in a nuclear chromosome, we searched for genes whose overexpression could facilitate import into mitochondria of the Cox2W56R precursor and increase respiratory growth of the corresponding mutant strain. A COX2W56R expressing strain was transformed with a multicopy plasmid genomic library, and transformants exhibiting enhanced respiratory growth on nonfermentable carbon sources were selected. We identified 3 genes whose overexpression facilitates the internalization of the Cox2W56R subunit into mitochondria, namely: TYE7, RAS2, and COX12. TYE7 encodes a transcriptional factor, RAS2, a GTP-binding protein, and COX12, a non-core subunit of cytochrome c oxidase. We discuss potential mechanisms by which the TYE7, RAS2, and COX12 gene products could facilitate the import and assembly of the Cox2W56R subunit produced allotopically.

Small RNAs (sRNAs) play a crucial role in modulating target gene expression through short base-pairing interactions and serve as integral components of many stress response pathways and regulatory circuits in bacteria. Transcriptome analyses have facilitated the annotation of dozens of sRNA candidates in the ubiquitous environmental model bacterium Caulobacter crescentus, but their physiological functions have not been systematically investigated so far. To address this gap, we have established CauloSOEP, a multi-copy plasmid library of C. crescentus sRNAs, which can be studied in a chosen genetic background and under select conditions. Demonstrating the power of CauloSOEP, we identified sRNA AbnZ to impair cell viability and morphology. AbnZ is processed from the 3′ end of the polycistronic abn mRNA encoding the tripartite envelope-spanning efflux pump AcrAB-NodT. A combinatorial approach revealed the essential membrane translocation module TamAB as a target of AbnZ, implying that growth inhibition by AbnZ is linked to repression of this system.

Stable production of value-added products using a microbial chassis is pivotal for determining the industrial suitability of the engineered biocatalyst. Microbial cells often lose the multicopy expression plasmids during long-term cultivations. Owing to the advantages related to titers, yields, and productivities when using a multicopy expression system compared with genomic integrations, plasmid stability is essential for industrially relevant biobased processes. Cupriavidus necator H16, a facultative chemolithoautotrophic bacterium, has been successfully engineered to convert inorganic carbon obtained from CO2 fixation into value-added products. The application of this unique capability in the biotech industry has been hindered by C. necator H16 inability to stably maintain multicopy plasmids. In this study, we designed and tested plasmid addiction systems based on the complementation of essential genes. Among these, implementation of a plasmid addiction tool based on the complementation of mutants lacking RubisCO, which is essential for CO2 fixation, successfully stabilized a multicopy plasmid. Expressing the mevalonate pathway operon (MvaES) using this addiction system resulted in the production of ∼10 g/L mevalonate with carbon yields of ∼25%. The mevalonate titers and yields obtained here using CO2 are the highest achieved to date for the production of C6 compounds from C1 feedstocks.

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

To enhance the de novo synthesis of SAM, the effects of several key genes on SAM synthesis were examined based on modular strategy, and the key genes were manipulated to obtain an engineered strain with high SAM production. In Bacillus amyloliquefaciens HSAM6, the deletion of argG gene to block aspartic acid branching degradation increased SAM titer to 254.78 ± 15.91 mg/L, up 18% from HSAM6. Subsequently, deleting the moaA gene to boost the supply of 5-methyltetrahydrofolate led to the stunted growth and the plummeting yield of SAM. Further improvement of strain growth by overexpression of the citA gene, while SAM synthesis was not significantly enhanced. Finally, the maximum SAM titer (452.89 ± 13.42mg/L) was obtained by overexpression SAM2 gene using the multicopy plasmid. The deletion of argG gene and the overexpression of SAM2 gene significantly improved SAM synthesis in B. amyloliquefaciens.

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgo) and the D NA D efense M odule DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe thatthe helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to amonomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Despite the high energetic cost of the reduction of sulfate to H2S, required for the synthesis of sulfur-containing amino acids, some wine Saccharomyces cerevisiae strains have been reported to produce excessive amounts of H2S during alcoholic fermentation, which is detrimental to wine quality. Surprisingly, in the presence of sulfite, used as a preservative, wine strains produce more H2S than wild (oak) or wine velum (flor) isolates during fermentation. Since copper resistance caused by the amplification of the sulfur rich protein Cup1p is a specific adaptation trait of wine strains, we analyzed the link between copper resistance mechanism, sulfur metabolism and H2S production. We show that a higher content of copper in the must increases the production of H2S, and that SO2 increases the resistance to copper. Using a set of 51 strains we observed a positive and then negative relation between the number of copies of CUP1 and H2S production during fermentation. This complex pattern could be mimicked using a multicopy plasmid carrying CUP1, confirming the relation between copper resistance and H2S production. The massive use of copper for vine sanitary management has led to the selection of resistant strains at the cost of a metabolic tradeoff: the overproduction of H2S, resulting in a decrease in wine quality.

The rise of antibiotic resistance is a critical public health concern, requiring an understanding of mechanisms that enable bacteria to tolerate antimicrobial agents. Bacteria use diverse strategies, including the amplification of drug-resistance genes. In this paper, we showed that multicopy plasmids, often carrying antibiotic resistance genes in clinical bacteria, can rapidly amplify genes, leading to plasmid-mediated phenotypic noise and transient antibiotic resistance. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression in Escherichia coli MG1655, we showed that plasmid copy number variability stably maintains populations composed of cells with both low and high plasmid copy numbers. This diversity in plasmid copy number enhances the probability of bacterial survival in the presence of antibiotics, while also rapidly reducing the burden of carrying multiple plasmids in drug-free environments. Our results further support the tenet that multicopy plasmids not only act as vehicles for the horizontal transfer of genetic information between cells but also as drivers of bacterial adaptation, enabling rapid modulation of gene copy numbers. Understanding the role of multicopy plasmids in antibiotic resistance is critical, and our study provides insights into how bacteria can transiently survive lethal concentrations of antibiotics.

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, ‘noisy’, gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast Saccharomyces cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been no attempts to construct the highest strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with ~1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by estradiol is maintained. When combined with a multicopy plasmid, it can express up to ~50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.