Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective

doi:10.12211/2096-8280.2024-079

Abstract

Abstract:

Synthetic biology, as a discipline that designs, constructs, and modifies biological systems to achieve specific functions, is widely applied in biomanufacturing, the biodegradation of environmental pollutants, and drug synthesis. Systematic exploration of gene functions and construction of libraries for engineered strains are driving forces of the development of synthetic biology. These libraries serve as foundational tools for understanding complex biological processes and engineering microorganisms for potential applications. This review focuses on the construction methods and application prospects of various yeast libraries in synthetic biology. With the rapid advancement of genome sequencing and high-throughput screening technologies, microbial libraries, such as those of Saccharomycescerevisiae and Schizosaccharomycespombe, play a pivotal role in systematic research. Yeast libraries, including gene knockout libraries, overexpression libraries, and transposon insertion libraries, provide valuable tools for optimizing gene combinations and designing metabolic pathways, thus promoting applications in metabolic engineering and synthetic biology. These libraries facilitate the development of robust industrial strains, driving improvements in biofuel production, chemical synthesis, and other biotechnological processes. In the environmental field, the screening of modified genes generates strains with pollutant degradation capabilities, contributing to ecological restoration. In drug synthesis, these libraries aid in constructing strains for the efficient production of pharmaceutical compounds, advancing the development of biopharmaceuticals. Despite these successes, there remain challenges in library construction and application, such as the high cost of library generation, difficulty in precise genome editing, and limitation in screening efficiency. In the future, advances in automation, digitization, and novel screening technologies are expected to overcome these barriers, facilitating the rapid construction and efficient screening of yeast libraries. No doubt, synthetic biology holds immense promise, with improvements in library construction and screening processes expected to accelerate the development of sustainable solutions in industrial production, environmental protection, and healthcare, thereby driving innovations in biotechnology.

Key words: yeast, library, functional genomics, directed evolution, synthetic biology

摘要：

合成生物学作为一门通过设计、构建和改造生物系统来实现其特定功能的学科，被广泛应用于生物制造、环境保护和药物合成等领域。基因功能的系统性探索和工程菌株文库的构建是推动合成生物学发展的重要手段。本文重点介绍了不同酵母文库在合成生物学中的构建方法及其应用前景。随着基因组测序和高通量技术的快速进展，酿酒酵母和裂殖酵母等微生物文库在系统性研究中发挥了关键作用。基因缺失文库、过表达文库、转座子插入文库等多种类型的酵母文库为基因组合优化和代谢路径设计提供了重要工具，促进了代谢工程和合成生物学的创新应用。这些文库在工业生产中支持高产菌株的构建，如用于生物燃料和化学品的高效生产；在环境领域，通过基因改造筛选，生成具备污染物降解能力的菌株，为生态修复提供解决方案；在药物合成方面，文库帮助构建高效合成药用化合物的菌株，推动生物制药的发展。然而，当前文库构建和应用仍面临诸如构建成本、基因组编辑的精确度及筛选效率等技术瓶颈。未来，自动化、数字化和新型筛选技术的进步有望突破这些瓶颈，推动酵母文库的快速构建和高效筛选，从而加速合成生物学在可持续发展和生态工程中的应用。

关键词: 酵母, 文库, 功能基因组学, 定向进化, 合成生物学

CLC Number:

Q812

ZHANG Yiqing, LIU Gaowen. Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective[J]. Synthetic Biology Journal, 2025, 6(3): 685-700.

章益蜻, 刘高雯. 合成生物学视角下的基因功能探索与酵母工程菌株文库构建[J]. 合成生物学, 2025, 6(3): 685-700.

Figures/Tables 3

Fig. 1 Schematics for constructing classical libraries (created by biorender)[(a) In the gene deletion library, the ORF (orange fragment) of target gene is replaced by the KanMX (kanamycin resistance selection marker, yellow fragment), flanked by unique barcodes (blue fragments). Chr.DNA: chromosomal DNA.(b) Schematic diagram of the Bar-seq principle. Under specific treatment conditions, the target strain (red) shows reduced growth (light pink), which is reflected by a lower read count of its corresponding barcode during sequencing (fewer red lines compared to others).(c) In the gene overexpression library, the ORF (orange fragment) of target gene is not only expressed from its endogenous locus but is also additionally expressed from a plasmid, driven by a constitutive promoter (light green fragment).(d) In the ORF-Tag library, the C-terminus of the target gene ORF (orange fragment), with its stop codon removed, is fused to the fluorescent protein GFP (dark green fragment) and the selection marker KanMX (yellow fragment).]

Fig. 2 Schematics for SGA and PEM methods (created by biorender)[(a) Double mutant selection strategy. Two mating-type cells each carry different resistance markers at their respective mutation sites: KanMX (green hollow circle) and NatMX (pink hollow circle). These allow for the selection of double mutant progeny (outlined in black). YFG (your favorite gene): target gene of interest.(b) Counter-diploid selection strategy. Haploid cells carrying a wild-type CAN1 gene (blue, a-type) and diploid cells (purple) can uptake the toxic analog canavanine and are killed, while can1Δ mutants cannot transport canavanine and thus survive (outlined in black).(c) Haploid selection strategy. A mating-type-specific promoter is used to drive the expression of a nutritional selection marker in the opposite mating type, allowing selection of haploid progeny of a specific mating type. For example, when an a-type parent cell (blue), harboring a gene cassette expressing STE3pr-LEU2 (red solid circle) — which is only active in α-type cell — mates with another cell, only the α-type progeny that inherit this construct (outlined in red with a red solid circle) will survive.(d) Combined counter-diploid and haploid selection strategy. A dominant-lethal resistance gene, cyhS (brown solid square), is inserted near the mat1 mating-type locus to link its expression with a specific haploid phenotype. For example, insertion near matP (blue square) in h- cells (blue) leads to the death of cyhS -containing h- haploids and diploids, enabling selection of h+ haploids.]

Fig. 3 Construction of conditional allele libraries (created by biorender)[(a) In the temperature-sensitive (TS) allele strategy, the ORF (orange fragment) of target gene is replaced with a corresponding TS mutant (dark orange fragment) and tagged with a KanMX resistance marker (yellow fragment). Chr.DNA: chromosomal DNA.(b) In the promoter replacement strategy, an inducible promoter (green fragment) and a KanMX resistance marker (yellow fragment) is inserted upstream of the start codon of the target gene ORF (orange fragment). Chr.DNA: chromosomal DNA.(c) In the DAmP (Decreased Abundance by mRNA Perturbation) strategy, the 3′UTR of the target gene ORF (orange fragment) is disrupted by insertion of a KanR resistance cassette (yellow fragment) to reduce transcript stability and protein abundance. Chr.DNA: chromosomal DNA.]

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