Type IIs restriction based combinatory modulation technique for metabolic pathway optimization

Additional file 1: Table S1. Oligonucleotides used in this study

Combinatory optimization of chromosomal integrated mevalonate pathway for β-carotene production in Escherichia coli

Additional file 1: Table S1. Primers used in this work. Table S2. Modulating genes of mvaS-mvaA-mavD1 operon for improving β-carotene production. Table S3. Modulating genes of Hmg1-erg12 operon for improving β-carotene production. Table S4. Sequences of representative artificial regulatory parts. Table S5. Plasmids used in this work. Table S6. Escherichia coli strains used in this work. Table S7. Calculated strength of mvaS and Hmg1 RBS, RBS sequence and relative β-carotene yield of strains from Re-modulation libraries. Figure S1. Two-step recombination method for inserting Hmg1-erg12 operon in E. coli chromosome. Figure S2. Two-step recombination method for modulating gene expression in E. coli chromosome by different artificial regulatory parts

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

DNAConstructor scripts.zip. Zip file containing files gfp_DNAConstructor.txt and rfp_DNAConstructor.txt, input script files for DNA Constructor. (ZIP 1.7 kb

Engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate

Abstract Background Geranyl acetate is widely used in the fragrance and cosmetic industries, and thus has great economic value. However, plants naturally produce a mixture of hundreds of esters, and geranyl acetate is usually only present in trace amounts, which makes its economical extraction from plant sources practically impossible. As an ideal host for heterologous production of fragrance compound, the Saccharomyces cerevisiae has never been engineered to produce the esters, such as geranyl acetate. Results In this study, a heterologous geranyl acetate synthesis pathway was constructed in S. cerevisiae for the first time, and a titer of 0.63 mg/L geranyl acetate was achieved. By expressing an Erg20 mutant to divert carbon flux from FPP to GPP, the geranyl acetate production increased to 2.64 mg/L. However, the expression of heterologous GPP had limited effect. The highest production of 13.27 mg/L geranyl acetate was achieved by additional integration and expression of tHMG1, IDI1 and MAF1. Furthermore, through optimizing fermentation conditions, the geranyl acetate titer increased to 22.49 mg/L. Conclusions We constructed a monoterpene ester producing cell factory in S. cerevisiae for the first time, and demonstrated the great potential of this system for the heterologous production of a large group of economically important fragrance compounds

Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique

Abstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. Results An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion