Abstract
BackgroundAs an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes.ResultsFirstly, screening of key enzymes, IDI, MK and IspS, from other organisms and then RBS optimization of the key enzymes were carried out. The strain utilized IDIsa was firstly detected to produce more isoprene than other IDIs. IDIsa expression was improved after RBS modification, leading to 1610-fold increase of isoprene production. Secondly, RBS sequence optimization was performed to reduce translation initiation rate value of non-key enzymes, ERG19 and MvaE. Decreased ERG19 and MvaE expression and increased isoprene production were detected. The final strain showed 2.6-fold increase in isoprene production relative to the original strain. Furthermore, for the first time, increased key enzyme expression and decreased non-key enzyme expression after RBS sequence optimization were obviously detected through SDS-PAGE analysis.ConclusionsThis study prove that desired enzyme expression and increased isoprene production were obtained after RBS sequence optimization. RBS optimization of genes could be a powerful strategy for metabolic engineering of strain. Moreover, to increase the production of engineered strain, attention should not only be focused on the key enzymes, but also on the non-key enzymes.
Highlights
As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum
In our previous study, an isoprene-producing strain was constructed through overexpressing the hybrid MVA pathway and IspSpa from P. alba in E. coli. 287 mg/L isoprene production was achieved under shake flask condition [7]
It was speculated that the decreased isoprene production resulted from the weak expression of isopentenyl-diphosphate isomerase (IDI), which was reflected by the low T.I.R. value
Summary
As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. 800,000 tons of isoprene monomer are produced annually from cracking petroleum, and over 95% of isoprene is used for rubber manufacture [3]. Isoprene is synthesized by isoprene synthase (IspS) from dimethylallyl diphosphate (DMAPP), final product of mevalonate (MVA) or the methylerythritol phosphate (MEP) pathway (Fig. 1a) [6]. In order to produce isoprene in cell factory such as Escherichia coli or yeast, exogenous whole MVA or/and MEP pathway and IspS were overexpressed and isoprene production was detected [7, 8].
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