Background: Metabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly. Despite great progresses in genome editing for the model microorganism Escherichia coli, the integration of large pathway into genome for stabilized chemical production is still challenging compared with small DNA integration. Results: We have developed a λ-Red assisted homology-dependent recombination for large synthetic pathway integration in E. coli. With this approach, we can integrate as large as 12 kb DNA module into the chromosome of E. coli W3110 in a single step. The efficiency of this method can reach 100%, thus markedly improve the integration efficiency and overcome the limitation of the integration size adopted the common method. Furthermore, the limiting step in the methylerythritol 4-phosphate (MEP) pathway and lycopene synthetic pathway were integrated into the W3110 genome using our system. Subsequently, the yields of the final strain were increased 106 and 4.4-fold compared to the initial strain and the reference strain, respectively. Conclusions: In addition to pre-existing method, our system presents an optional strategy for avoiding using plasmids and a valuable tool for large synthetic pathway assembly in E. coli.

Co-utilization of xylose and glucose from lignocellulosic biomass is an economically feasible bioprocess for chemical production. Many strategies have been implemented for efficiently assimilating xylose which is one of the predominant sugars of lignocellulosic biomass. However, there were few reports about engineering Saccharomyces cerevisiae for carotenoid production from xylose-glucose mixtures. Herein, we developed a platform for facilitating carotenoid production in S. cerevisiae by fermentation of xylose-glucose mixtures. Firstly, a xylose assimilation pathway with mutant xylose reductase (XYL1m), xylitol dehydrogenase (XYL2), and xylulokinase (XK) was constructed for utilizing xylose. Then, introduction of phosphoketolase (PK) pathway, deletion of Pho13 and engineering yeast hexose transporter Gal2 were conducted to improve carotenoid yields. The final strain SC105 produced a 1.6-fold higher production from mixed sugars than that from glucose in flask culture. In fed-batch fermentation with continuous feeding of mixed sugars, carotenoid production represented a 2.6-fold higher. To the best of our knowledge, this is the first report that S. cerevisiae was engineered to utilize xylose-glucose mixtures for carotenoid production with a considerable high yield. The present study exhibits a promising advantage of xylose-glucose mixtures assimilating strain as an industrial carotenoid producer from lignocellulosic biomass.