DNA assembly technologies: a review / 生物工程学报

DNA assembly is the core technology of synthetic biology. With the development of synthetic biology, researchers have developed different DNA assembly technologies that rely on DNA polymerase or DNA ligase, and also have developed some non-enzyme-dependent DNA assembly techniques to facilitate the automation of DNA assembly. The assembly of large fragments of DNA from a few hundred kb to Mb is mostly dependent on microbial recombination. In this paper, the three types of DNA assembly technologies, including enzyme-dependent, non-enzymatic and in vivo homologous recombination, are reviewed.

Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production / 生物工程学报

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

Synthetic genomics: the art of design and synthesis / 生物工程学报

Benefited from the rapid development of high-throughput sequencing, genome editing, DNA synthesis and functional genomics, synthetic genomics gains the momentum in this century. The entire genomes of several viruses and one prokaryote have been chemically synthesized and applied to drive normal cellular processes. The first eukaryotic genome synthesis project (Sc2.0) is on-going and about half of the genome has been synthesized and functionally tested. The Human Genome Project-Write (HGP-Write) was proposed in 2016, which pushes the tide of synthetic genomics to a position we have never seen before. Technologies on genome-scale design and DNA synthesis have been rapidly developed, aiming to construct a more predictable and controllable genome at reasonable cost. The generation of synthetic organisms not only has promising applications for industry, environment, healthy and basic researches, but also raises ethic and policy concerns. This review presents the development of synthetic genomics, with emphasis on technologies for whole genome design, synthesis and assembly. We also discussed ethics, prospective and challenge in synthetic genomics. As one of the major branches in synthetic biology, synthetic genomics is still at its infant stage. A lot of excitement will come in the next few years.

Enabling technologies in synthetic biology--DNA synthesis, assembly and editing / 生物工程学报

Synthetic biology is an emerging discipline, which aims at creating artificial lives or remolding the present organisms to generate new features. To achieve these goals, synthetic biologists need to design and synthesize new genes, pathways, modules or even whole genomes. As these enabling technologies (e.g. gene synthesis, DNA assembly and genome editing) are very important for the progress of synthetic biology, we will focus on the development of these technologies in this review.

Key technologies in synthetic biology of natural products / 中国中药杂志

Natural products with complex and diverse structures are the major sources of new drugs. The biosynthesis of natural products is considered to be one of the best ways to solve the problems of complex and scarce natural products. DNA assembly technology and genome editing technology are two key technologies in the emerging interdisciplinary field of synthetic biology. A number of novel DNA assembly methods developed in the last few years have paved the way for the engineering of high molecular weight DNA molecules, including whole genomes, hence, it can realize the reconstruction of the metabolic pathways and speed up optimization process. A wide variety of new tools for microbial genome editing will be applied widely to modify the chassis genome to increase its adaptation with the exogenetic pathways. This article summarized the latest advance with respect to DNA assembly and genome editing, which aims to provide help for reconstruction and optimization of the synthetic biological systems of natural products.

A facile method for the construction of synthetic genes.

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA.