The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization. In the current study on taking lycopene biosynthesis as an example, an integrated Escherichia coli system has been generated successfully, which resulted into stable and high lycopene production. In this process, two modules of mevalonate (MVA) pathway and one module of lycopene expression pathway were completely integrated in the chromosome. Firstly, the copy number and integrated position of three modules of heterologous pathways were rationally optimized. Later, a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411. The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L (49.9 mg/g DCW) in a 5 L fermenter with mean productivity of 61.0 mg/L/h. Additionally, the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.
Genome engineering is the extensive and intentional genetic modification of a replicating system for a specific purpose. Escherichia coli, one of the most common cell factories, is widely used in the domains of biological manufacturing and becomes a major subject in synthetic biology. Its application fields could be further expanded by genome engineering. This paper reviews the latest technology development of genome engineering, such as genome integration of heterogenous pathway, chromosomal evolution, multiplex automated genome engineering and trackable multiplex recombineering, and their application in improving the productivity, the stability and the tolerance of Escherichia coli as cell factories.
We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts: cma (which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.
We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination.
The cat cassette was divided into two parts: cma and cmb (has a homologous sequence
with cma ). Each of them has no resistance separately
unless the two parts are fused together. The cmb fragment
was first integrated into a side of the target cloning region, then
a linear cma -containing plasmid vector was electroporated
into the cells to directly capture the target region. Based on this
strategy, we successfully cloned a DNA fragment about 48 kb from E. coli DH1-Z chromosome with positive frequency of about
80%. Then, combined with double-strand breakage-stimulated homologous
recombination, we applied this strategy to replace the corresponding
region of E. coli DH36 chromosome and knock out four
nonessential genomic regions at one time. This strategy provides a
powerful tool for the heterologous expression of microbial natural
product biosynthetic pathways, for genome assembly and for the function
study of large DNA sequence.
Abstract Background Thymosin α1 (Tα1), a 28-amino acid N α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N α -acetylation. In this study, we describe a novel production process for N α -acetylated Tα1 in Escherichia coli . Results To obtain recombinant N α -acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis ), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic N α -acetyltransferase) in Escherichia coli . After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols β-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. Conclusions The present data demonstrate that N α -acetylated Tα1 can be efficiently produced in recombinant E. coli . This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides.
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