Are we ready for pig-to-human clinical xenotransplantation trials? / Estamos prontos para ensaios clínicos de xenotransplante porco-para-humanos? / ¿Estamos listos para ensayos clínicos de xenotrasplante del cerdo al humano?

Abstract: The invention and widely use of organ allotransplantation provides effective treatment of some originally fetal diseases such as liver/kidney failure and has saved million of lives around the globe. However, the scarcity of human organs has caused many patients, who could have been treated, to die while waiting for suitable organs around the world. Pig-to human xenotransplantation provides a potential solution to solve this tough problem. Pig organs have been considered as major sources of xenotransplantation because of the sufficient number of donors, the sizes of organs, and physiologically structural similarities. However, xenotransplantation also has some problems, such as the possibility of spreading animal diseases to human, the interspecies immunological barrier, organs of animal origin challenging human nature, and potential informed consent issues. This article will discuss these potential issues and to see whether it is the suitable time to conduct clinical xenotransplantation trials in humans.

Resumen: La invención y el amplio uso de trasplantes alógenos proporciona tratamiento efectivo de algunas enfermedades de origen fetal, como la insuficiencia renal y hepática, y ha salvado a millones de pacientes en el mundo. Sin embargo, la escasez de órganos humanos ha causado que muchos pacientes en el mundo, que podrían haber sido tratados, murieran por esperar un órgano adecuado. El xenotrasplante del cerdo al humano proporciona una solución potencial para resolver este difícil problema. Los órganos de cerdo han sido considerados como fuentes mayores para xenotrasplantes debido al suficiente número de donantes, el tamaño de los órganos y estructuras fisiológicas similares. No obstante, el xenotrasplante también tiene algunos problemas, como la posibilidad de expandir enfermedades animales a humanos, la barrera inmunológica entre especies, el desafío para la naturaleza humana de tener órganos de origen animal y problemas potenciales de consentimiento informado. Este artículo discute estos temas potenciales y plantea si estamos en un momento apropiado para realizar ensayos clínicos de xenotrasplantes en humanos.

Resumo: A invenção e amplo uso de alotransplante de órgãos propicia tratamento efetivo para algumas doenças originalmente fetais tais como falência hepática/renal e tem salvo milhões de vidas em todo o globo. Entretanto, a escassez de órgãos humanos tem causado a morte de muitos pacientes que poderiam ter sido tratados - aguardando por órgãos apropriados em todo o globo. Xenotransplante porco-para-humanos propicia uma solução potencial para resolver este difícil problema. Órgãos de porco tem sido considerados como as principais fontes de xenotransplante por causa do número suficiente de doadores, do tamanho dos órgãos e de similaridades estruturais fisiológicas. Entretanto, xenotransplante também tem alguns problemas, tais como a possibilidade de disseminar doenças animais aos humanos, a barreira imunológica entre espécies, órgão de origem animal desafiando a natureza humana e aspectos potenciais de consentimento informado. Esse artigo discutirá esses aspectos potenciais e verificará se é o momento adequado para conduzir ensaios clínicos de xenotransplante em humanos.

Intervenciones genéticas de Aedes aegypti para el control de arbovirosis / Genetic interventions of Aedes aegypti for the control of arboviruses

La ineficacia de las estrategias actuales para el control químico de los mosquitos vectores plantea la necesidad de desarrollar enfoques novedosos, entre estos están las estrategias genéticas para reducir las poblaciones de mosquitos vectores o sustituirlos por aquellos que no son capaces de transmitir patógenos, esto se logra a través de herramientas moleculares que permiten la manipulación y transgénesis de genes. Las secuencias del genoma de los mosquitos y las bases de datos de marcadores de secuencias expresadas asociadas permiten investigaciones a gran escala para proporcionar nuevos conocimientos sobre las vías evolutivas, bioquímicas, genéticas, metabólicas y fisiológicas. Además, la genómica comparativa revela las bases de los mecanismos evolutivos con especial atención a las interacciones específicas entre vectores y patógenos. Se ha desarrollado tecnología de transgénesis para el mosquito de la fiebre amarilla y dengue, Aedes aegypti. Se ha logrado integración exitosa de ADN exógeno en la línea germinal de este mosquito con los elementos transponibles. La disponibilidad de múltiples elementos y genes marcadores proporciona un poderoso conjunto de herramientas para investigar las propiedades biológicas básicas de este insecto vector, así como los materiales para desarrollar nuevas estrategias de control genético de poblaciones de mosquitos basadas en la técnica del insecto estéril. Una de estas estrategias consiste en liberar a la población machos esterilizados por radiación; otro, de integrar un gen letal dominante bajo el control de un promotor específico en hembras inmaduras. El uso de esta técnica de modificación genética constituirá una herramienta importante para el manejo integrado de vectores(AU)

The ineffectiveness of current strategies for the chemical control of vector mosquitoes raises the need to develop novel approaches, among these are genetic strategies to reduce populations of vector mosquitoes or replace them with those that are not capable of transmitting pathogens, this is achieved through molecular tools that allow the manipulation and transgenesis of genes. Mosquito genome sequences and associated expressed sequence marker databases enable large-scale investigations to provide new insights into evolutionary, biochemical, genetic, metabolic, and physiological pathways. Furthermore, comparative genomics reveals the basis of evolutionary mechanisms with special attention to the specific interactions between vectors and pathogens. Transgenesis technology has been developed for the yellow fever and dengue mosquito, Aedes aegypti. Successful integration of exogenous DNA into the germ line of this mosquito with the transposable elements has been achieved. The availability of multiple elements and marker genes provides a powerful set of tools to investigate the basic biological properties of this insect vector, as well as the materials to develop new strategies for genetic control of mosquito populations based on the sterile insect technique. One of this strategy is to release radiation-sterilized males into the population; another, to integrate a dominant lethal gene under the control of a specific promoter in immature females. The use of this genetic modification technique will constitute an important tool for the integrated management of vectors(AU)

Cultivation of college students' innovative and entrepreneurial thinking and ability based on Synthetic Biology and iGEM / 生物工程学报

Synthetic Biology is one of the most promising fields of modern Biology and a frontier interdisciplinary subject in the 21st century. With the rapid development of synthetic biology, the International Genetically Engineered Machine (iGEM) competition has emerged. The iGEM competition, based on the subject foundation of Synthetic Biology, intends to solve the biological problems in our daily life by applying modern biological technology. In recent years, with the continuous increase of participating teams, the iGEM competition has received extensive attention and achieved great progress. On the basis of the development of Synthetic Biology, we analyzed the 2018-2020 award-winning projects of the iGEM competition and illustrated the role and significance of the iGEM competition in cultivating college students' innovative thinking and ability with the participation experience of the iGEM team of Southwest Jiaotong University as an example.

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.

Exploration of an integrated competency development model for undergraduates training by participating the international Genetic Engineering Machine Competition / 生物工程学报

Starting from participating the high-level professional competition, our school has built a talent training system with the spirit of "biomaker" and an innovative practical ability training system. Such system takes the interest of student as the starting point, and relies on the strong scientific research and teaching infrastructure. The programme gives full play to students' initiatives and enhances the scientific research literacy and comprehensive ability of undergraduates majoring in biotechnology. It is an effective exploration of the traditional university education model and meets the urgent demand for innovative talents training in the era of rapid development of life sciences.

Curriculum design and implementation of teaching in experimental genetic engineering blended course under the principle of learning-centered teaching / 生物工程学报

Teaching in experiments of biology is important for the cultivation of life science talents. In view of the rapid development of life science and the increasing demand for research-oriented talent training, teaching in experiments of biology should set up a variety of learning outcomes: to train experimental skill, to cultivate students' experimental design and operation abilities, and to improve students' scientific thinking and innovative consciousness. We have carried out an educational reform on experimental genetic engineering blended course. In this paper, we introduced our methods of organizing online materials, the curriculum design of the blended course, the implementation details, and a preliminary analysis of teaching effects. We found that experimental genetic engineering blended course could support students' active learning and a learning-centered teaching model. Moreover, it could facilitate students' achievement of improving experimental skills, cultivating a rigorous scientific attitude, professional research quality and academic innovation ability.

Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques / 生物工程学报

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.

Reflexiones sobre la ingeniería genética: a propósito del nacimiento de gemelas sometidas a edición génica / Considerations on genetic engineering: regarding the birth of twins subjected to gene edition

Resumen En este ensayo se analizan las implicaciones bioéticas de la reciente manipulación genética en embriones humanos con CRISPR-Cas9 para eliminar el gen CCR5 y el nacimiento de dos gemelas discordantes. El experimento se divulgó en medios sociales. Los principales problemas bioéticos identificados son la justificación del modelo, el proceso de consentimiento informado y la falta de declaración de evidentes conflictos de interés. No se evaluaron apropiadamente las consecuencias del experimento sobre la vida de las gemelas nacidas como la afectación a su autonomía, los supuestos beneficios por recibir y los riesgos futuros de daño durante su vida. Habiendo manipulado la línea celular germinal, no se consideraron los efectos sobre su descendencia futura. Este tipo de acciones tiene un impacto negativo en la forma como la sociedad concibe la ciencia. La ingeniería genética debe reservarse al contexto experimental básico o bien como investigación cínica para la corrección de enfermedades conocidas graves de origen genético, bajo estricta supervisión regulatoria y bioética y de manera gradualista de acuerdo con el progreso de las técnicas de edición genética.

Abstract In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.

A general view of CD33⁺ leukemic stem cells and CAR-T cells as interesting targets in acute myeloblatsic leukemia therapy

Acute myeloblastic leukemia (AML) is the most frequent acute leukemia in adulthood with very poor overall survival rates. In the past few decades, significant progresses had led to the findings of new therapeutic approaches and the better understanding of the molecular complexity of this hematologic malignancy. Leukemic stem cells (LSCs) play a key role in the initiation, progression, regression, and drug resistance of different types of leukemia. The cellular and molecular characteristics of LSCs and their mechanism in the development of leukemia had not yet been specified. Therefore, determining their cellular and molecular characteristics and creating new approaches for targeted therapy of LSCs is crucial for the future of leukemia research. For this reason, the recognition of surface maker targets on the cell surface of LSCs has attracted much attention. CD33 has been detected on blasts in most AML patients, making them an interesting target for AML therapy. Genetic engineering of T cells with chimeric antigen receptor (CAR-T cell therapy) is a novel therapeutic strategy. It extends the range of antigens available for use in adoptive T-cell immunotherapy. This review will focus on CAR-T cell approaches as well as monoclonal antibody (mAB)-based therapy, the two antibody-based therapies utilized in AML treatment.

Construction of an engineered Saccharomyces cerevisiae expressing endoglucanase efficiently / 生物工程学报

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.

Construction of CAR-T cells targeting CS1 and analysis of their antitumor activity in vitro / 生物工程学报

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.

Progress in the genetic modification of blue flowers based on anthocyanin metabolism / 生物工程学报

As water-soluble, natural pigments, anthocyanins are responsible for the red, purple and blue colors of many flowers, which attract pollinators to spread pollen. The colors of flowers are also essential for plants to survive in the nature and become one of the most significant characteristics of ornamental plants. In the booming floriculture industry, to produce various flower colors could increase the richness of natural colors, but it is still difficult to breed flowers with coveted blue color. The diversity of flower color is mainly determined by the types and contents of anthocyanins and their derivatives. The synthesis of delphinidin pigments is the key factor for breeding blue flowers. However, there are no structural genes in many plants to biosynthesize delphinidin pigments. Blue flowers are successfully created by genetic engineering in recent years. In this paper, using common ornamental plants as examples, we review the mechanism of plant flower coloration from the aspects of the key factors affecting the synthesis of delphinidin pigment and the production strategies of blue flowers based on the regulation of anthocyanin metabolism. Different strategies of molecular breeding could provide opportunities to improve colors of other floriculture plants and to develop anthocyanin-rich economic crops, such as colored cotton with blue fibers.

Unnatural amino acid orthogonal translation: a genetic engineering technology for the development of new-type live viral vaccine / 生物工程学报

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.

Progress in engineering application of human adenovirus / 生物工程学报

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.

An update on the tools for creating transgenic animal models of human diseases - focus on atherosclerosis

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.

Therapeutic genome engineering: Implications for South Africa

South Africa encompasses extraordinary genetic diversity, frequently revealing unique mutations and variations associated with disease. Despite the advances of traditional gene therapy, our understanding of causative mutations in the South African population has, for the most part, contributed to diagnostic rather than therapeutic interventions. Recent developments in genome engineering and its ease of use have released a powerful tool with which to intervene in otherwise untreatable disease. In addition, harnessing this tool for discrete genetic edits provides a mechanism by which screening of new drugs specific to our population's diversity can be accomplished. Here, we use examples of some of the most advanced genome engineering approaches to develop therapeutic strategies that would specifically affect South African individuals

Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21–42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.

Functionalized genetic engineered silk-based biomaterials and their applications / 生物工程学报

Silk-based biomaterials are featured with excellent mechanical properties, good biocompatibility and biodegradability, which contribute to their potential applications in biomedical field. The current recognition of silk protein materials in structure and function provides a basic theory for the transformation of silk protein into new types of biomaterials. In addition, exogenous sequences encoding new peptide or structural domain can be inserted into the maternal gene sequences encoding silk proteins through genetic engineering technology to synthesize novel silk-based biomaterials with unique functions. This review summarizes the current trend and development perspective of genetically engineered functional silk-based materials for biomedical applications.

Effect of fcl gene for butenyl-spinosyn biosynthesis and growth of Saccharopolyspora pogona / 生物工程学报

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.