Exploring the Persistence of Transgenes in Genetically Engineered Cyanobacteria

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Barnes , James W. Lee , Lesley H. Greene doi: https://doi.org/10.1101/2025.11.26.690176 Cherrelle L. Barnes 1 Department of Chemistry and Biochemistry, Old Dominion University , Norfolk, Virginia 23529 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site James W. Lee 1 Department of Chemistry and Biochemistry, Old Dominion University , Norfolk, Virginia 23529 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lesley H. Greene 1 Department of Chemistry and Biochemistry, Old Dominion University , Norfolk, Virginia 23529 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: lgreene{at}odu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Genetically engineered organisms including bacteria, plants, and animals are very commonplace in industry and scientific research where they are designed to perform specific tasks. Cyanobacteria are a group of ubiquitous and ancient microorganisms that have been engineered to produce a range of products such as biofuels. However, the potential ramifications of genetic engineering could have unintended consequences. One key question is how long do foreign genes persist? Thus, we undertook a two-year study that investigates the fate and stability of transgenes in genetically engineered Thermosynechococcus elongatus BP1. The results show transgenes are very persistent within the host genome and begin to become lost slowly over time. Introduction Transgene stability can be defined as the persistent presence or expression of a foreign gene within a host. For commercial and scientific use of genetically engineered (GE) organisms, the goal is for the organisms to possess and express their transgenes for the duration of their lifetime. Transgene stability has been studied in many GE organisms including bacteria, plants, and insects ( Handler, 2004 ; Li et al., 2009 ; Abidin et al., 2021 ). Transgene stability within GE organisms can be affected through gene silencing by epigenetic and transcriptional mechanisms or gene loss because of crossbreeding ( Finnegan and McElroy, 1994 ; Dietz-Pfeilstetter, 2010 ). From a bio-safety perspective, a concern about the development of GE organisms is the fate and stability of transgenes within the host, as it could lead to gene escape and allow transgenes to persist in unintended environments ( Warwick et al., 2008 ; Agga et al., 2019 ). It is important to investigate and understand long-term stability of transgenes, as it could have an impact on the environment as well as human and animal health. The research presented in this study focuses on assessing the fate and stability of transgenes within genetically engineered cyanobacteria, which can be of great concern if they escape containment. It is also of importance to know how long transgenes persist in GE organisms to address general mechanisms in microbial genetics and evolution. To gain a better understanding of the outcome and long-term stability of transgenes inserted into host chromosomes, we conducted an exploratory study ( Barnes, 2022 ). Here cultures of a model cyanobacteria with a cassette of transgenes which included the kanamycin resistance gene as an antibiotic selection marker were incubated with and without antibiotic for a two-year study. Monthly, genomic material was extracted from each culture and used to determine the presence of the transgene cassette which revealed significant stability as evidenced from their persistence in the genome. Results and Discussion The presence of the pUC57-pKA cassette slowly decreases over time without antibiotic pressure Each month genomic DNA was isolated and PCR of the insertion site, the Kan resistance gene and the rpsL gene, which served as a control, was conducted to determine the presence of the integrated transgenes over a two-year period. PCR was used to amplify the insert region ( Figure 1 ) for the transgenes, resulting in a 6.7 kb or 2.4 kb band indicating chromosome copies that have or have not integrated the transgene cassette, respectively. Initially, the 2.4 kb bands are much less intense than the 6.7 kb bands among all cultures, with and without antibiotic selective pressure ( Figure 2 ). This indicated that a higher number of chromosomes within the culture have integrated transgenes. This trend remained the same for cultures with antibiotic selective pressure for the entire duration of the study. However, in the cultures without antibiotic selective pressure, the 2.4 kb band become more intense indicating that a portion of the chromosomes with integrated transgenes decreases over time ( Figure 2 ). This trend is evident in the two-year stability study. Download figure Open in new tab Figure 1. Schematic of insertion and PCR sites. Primers depicted as red arrows were used to amplify the kanamycin resistance gene and the insertion site within the genome. KIVD = alpha-ketoisovalerate decarboxylase, ADH = NADH-dependent alcohol dehydrogenase, Kan = Kanamycin resistance gene. Download figure Open in new tab Figure 2. Presence of the gene cassette within GE T. elongatus BP1 over 24 months. Primers were used to amplify the insertion site of T. elongatus BP1 genome containing the gene cassette, resulting in either a 2.4 kb band (wild-type size, no integration of genes) or 6.7 kb band (integration of genes) at 1, 6, 12, 18 and 24 months. The lanes are marked as followed: M = molecular weight marker, 1-5 = genomic DNA from the control GE T. elongatus BP1-pKA cultures (+ Kan) and 6-10 = genomic DNA from the experimental GE T. elongatus BP1-pKA cultures (- Kan). To further assess these results, there was a control PCR reaction which amplified the Kan resistance gene resulting in a 0.7 kb band. The Kan resistance gene remained present within the genomic DNA of GE T. elongatus BP1-pKA for the two-year period ( Figure 3 ). Further, the kanamycin resistance gene was shown to still be expressed after one year of study, even in the absence of antibiotic pressure (data not shown). One important aspect of stable transgene expression within GE organisms is the choice for the promoter. The transgene cassette was designed where the expression of the kanamycin resistance gene was under the control of the cpc and slpA continuous promoters, which are known to be very strong ( Hynönen et al., 2010 ; Zhou et al., 2014 ). Download figure Open in new tab Figure 3. Control for the presence of the Kan resistance gene within GE T. elongatus BP1 for 24 months. Primers were used to amplify the Kan resistance gene within the gene cassette, resulting in a 0.7 kb band at 1, 6, 12, 18 and 24 months. The lanes are marked as followed: M = molecular weight marker, 1-5 = genomic DNA from the control GE T. elongatus BP1-pKA cultures (+ Kan) and 6-10 = genomic DNA from the experimental GE T. elongatus BP1-pKA cultures (- Kan). Conclusions A gradual decrease in the population of chromosomes that have the transgene cassette was observed over the two-year study with genomic DNA. One explanation for the loss of genes over time could be genome streamlining. Bacteria, including cyanobacteria, have been known to reduce the size of the chromosomes as an evolutionary mechanism, resulting in the loss of genes not essential for survival of the organism ( Giovannoni et al., 2005 ; Marais et al., 2008 ; Moya et al., 2009 ; Yus et al., 2009 ). Genomic streamlining has been demonstrated within Prochlorococcus marinus and Pelagibacter ubique , which both have genome sizes around or less than 2 Mb but still possesses essential genes for biosynthesis of all amino acids and complete metabolic network ( Giovannoni et al., 2005 ; Moya et al., 2009 ). Genomic studies have also revealed evidence that several strains of the cyanobacterium Phlanktothrix lost the microcystin synthetase gene cluster through evolutionary deletion events, resulting in the strains becoming nontoxic ( Christiansen et al., 2008 ). Although the results in the genomic DNA PCR show there is loss of the transgene cassette within the GE T. elongatus BP1 over time the specific mechanism of this gene loss is uncertain. In general, the genomic stability of the transgene cassette is shown to be very stable for up to two years within the chromosome of T. elongatus BP1, both in the presence and absence of antibiotic pressure. This finding was very surprising as we initially theorized the transgenes would be lost more rapidly without the selective pressure of kanamycin in the media. There have been a few previous studies that investigated long-term transgene stability within plants and fungus ( Weaver et al., 2005 ; Li et al., 2009 ; Zeng et al., 2009 ). One group investigated the long-term stability and expression of the rolC gene within GE aspen trees, which revealed that the transgene was still present within the genome and expression up to 18 years after transformation ( Li et al., 2009 ). A different group examined the stability of transgenes within GE Trichoderma virens over a 250-day experiment and found that the opd gene was still present and express in the absence of antibiotic pressure ( Weaver et al., 2005 ). Another study involved assessing the expression of a chimeric gene, including an insecticidal peptide gene and the C-peptide of Bt gene, within Betula platyphylla for up to 15 subcultures and although they found the gene was silenced, it was still present within the chromosome for the duration of the study ( Zeng et al., 2009 ). There have been no long-term studies of transgene stability in cyanobacteria previously published based on extensive literature searches. From a bio-risk perspective, the longer transgenes remain intact within the cyanobacterial chromosome, the more opportunity it may have to transfer nonnative genes and persist in unintended environments, thus leading to possible ecological and human/animal health risks. For example, a study assessed the persistence of an escaped herbicide resistance gene from GE Brassica rapa to wild-type relatives through hybridization and the transgene was shown to be persistent in these hybrids for up to 6 years ( Warwick et al., 2008 ). Another study demonstrated that antibiotic resistance genes of bacteria within beef cattle is shown to be transferred and persists for up to two years in feeding areas where they were housed ( Agga et al., 2019 ). If transgenic cyanobacteria are to be used for the commercial production of bioproducts, it is of paramount importance to understand the fate and stability of transgenes as an avenue to prevent any bio-risk concerns. Thus, it is critical that science looks ahead and seeks to understand the outcome of foreign genes. Materials and Methods Growth studies A GE Thermosynechococcus elongatus BP1 (called GE T. elongatus BP-pKA) carrying a cassette of synthetic transgenes including the kanamycin resistance gene in its chromosome ( Nguyen et al., 2019 ) was incubated in the presence and absence of antibiotic selection pressure. PCR was performed to monitor the inserted transgenes for up to 104 weeks (2 years). GE T. elongatus BP1 cells were grown in BG-11 liquid media with a pH ∼7.75. One triplicate set of cultures was supplemented with kanamycin 40 µg/mL to represent the presence of selective pressure and will act as the control for this study, the other triplicate set did not contain any antibiotic to represent absence of selective pressure and will act as the experimental cultures. Growth occurred in a Percival environmental chamber at ∼42 °C with continuous illumination at actinic light intensity of 30 µE m -2 s -1 . The cultures were re-inoculated bi-weekly into fresh BG-11 media (3 mL of culture into 75 mL of media), and 2 mL aliquots of each culture were collected and cryopreserved monthly. The pH of the cultures was measured average at the start of incubation and following 2 weeks of growth prior to reinoculation into fresh media and the pH was ∼7.91 and ∼9.49, respectively. Detection of transgene cassette by PCR Genomic DNA was extracted monthly using the Qiagen QiaAMP DNA Mini Kit and used to amplify two genetic regions: the insertion site and the Kan resistance gene via PCR using 2 ng of purified genomic DNA. For the insertion site, primers were designed to amplify from within the upstream recombination site of designer transgene cassette (homologous to T. elongatus BP1) to outside the downstream recombination site on the chromosome (not part of transgene cassette) ( Figure 1 ). T. elongatus BP1 possess multiple copies of their chromosome, resulting at the onset of the study in some copies that contain the transgenes while possibly others do not. Because of this, there are two expected band sizes for the PCR amplified insert and are located at 2.4 kb and 6.7 kb on an agarose gel, which represents no integration and integration of the transgenes, respectively. The Kan resistance gene was also amplified and were detectable as 0.7 kb. The presence of genomic DNA was tested monthly over a two-year period to monitor any changes over time. Competing Interest Statement The authors declare no competing interests. Acknowledgements This work is supported by Biotechnology Risk Assessment Grant Program competitive grant award no. 2016-33522-25624 and 2023-33522-40974 from the U.S. Department of Agriculture to JWL and LHG and support from Old Dominion University to LHG. 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Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria . Sci Rep 4 : 4500 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 29, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Exploring the Persistence of Transgenes in Genetically Engineered Cyanobacteria Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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