RESUMEN
Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47 mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.
Asunto(s)
Escherichia coli/metabolismo , Hemiterpenos/biosíntesis , Microbiología Industrial/métodos , Ácido Mevalónico/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Butadienos , Carboxiliasas/genética , Carboxiliasas/metabolismo , Enterococcus faecalis/genética , Escherichia coli/genética , Fermentación , Regulación Bacteriana de la Expresión Génica , Hemiterpenos/genética , Ingeniería Metabólica/métodos , Microorganismos Modificados Genéticamente , Compuestos Organofosforados , Populus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution in Escherichia coli (E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.