Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene

Background: alpha-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, alpha-Pinene used in industry is mainly produced either by tapping trees (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, the extraction of it from trees is tedious and inefficient and requires substantial expenditure of natural resources. Therefore, it is necessary to seek sustainable technologies for alpha-pinene production

Enhancing Production of Bio-Isoprene Using Hybrid MVA Pathway and Isoprene Synthase in E. coli

The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the “upper pathway” which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation

Production of isoprene, one of the high-density fuel precursors, from peanut hull using the high-efficient lignin-removal pretreatment method

Abstract Background Isoprene as the feedstock can be used to produce renewable energy fuels, providing an alternative to replace the rapidly depleting fossil fuels. However, traditional method for isoprene production could not meet the demands for low-energy consumption and environment-friendliness. Moreover, most of the previous studies focused on biofuel production out of lignocellulosic materials such as wood, rice straw, corn cob, while few studies concentrated on biofuel production using peanut hull (PH). As is known, China is the largest peanut producer in the globe with an extremely considerable amount of PH to be produced each year. Therefore, a novel, renewable, and environment-friendly pretreatment strategy to increase the enzymatic hydrolysis efficiency of cellulose and reduce the inhibitors generation was developed to convert PH into isoprene. Results The optimal pretreatment conditions were 100 °C, 60 min, 10% (w/v) solid loading with a 2:8 volume ratio of phosphoric acid and of hydrogen peroxide. In comparison with the raw PH, the hemicellulose and lignin were reduced to 85.0 and 98.0%, respectively. The cellulose–glucose conversion of pretreated PH reached up to 95.0% in contrast to that of the raw PH (19.1%). Only three kinds of inhibitors including formic acid, levulinic acid, and a little furfural were formed during the pretreatment process, whose concentrations were too low to inhibit the isoprene yield for Escherichia coli fermentation. Moreover, compared with the isoprene yield of pure glucose fermentation (298 ± 9 mg/L), 249 ± 6.7 and 294 ± 8.3 mg/L of isoprene were produced using the pretreated PH as the carbon source by the engineered strain via separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) methods, respectively. The isoprene production via SSF had a 9.8% glucose–isoprene conversion which was equivalent to 98.8% of isoprene production via the pure glucose fermentation. Conclusions The optimized phosphoric acid/hydrogen peroxide combination pretreatment approach was proved effective to remove lignin and hemicellulose from lignocellulosic materials. Meanwhile, the pretreated PH could be converted into isoprene efficiently in the engineered Escherichia coli. It is concluded that this novel strategy of isoprene production using lignocellulosic materials pretreated by phosphoric acid/hydrogen peroxide is a promising alternative to isoprene production using traditional way which can fully utilize non-renewable fossil sources

Additional file 1: of A novel MVA-mediated pathway for isoprene production in engineered E. coli

Optimization of Fermentation Process and three Figs. (DOCX 216 kb

Comparison of isoprene production from different recombinant strains.

<p>The experiment was conducted under flask conditions in triplicate.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

The time course of isoprene production by YJM25.

<p>Isoprene accumulation (▪) and cell growth (Δ) in YJM25, Induction was carried out at an OD₆₀₀ of 12. Other experiment conditions were described in ‘Fed-batch fermentation’.</p

Strains, plasmids and Oligonucleotide primers used in this study.

<p>Strains, plasmids and Oligonucleotide primers used in this study.</p

Production of isoprene via the MVA pathways used in this study.

<p>Gene symbols and the enzymes they encode (all genes marked with white arrows were isolated from <i>S. cerevisiae</i>, the gene marked with light gray arrows derived from <i>P. alba</i> and all genes marked with gray arrows were from <i>Enterococcus faecalis</i>). MVA pathway: ERG10, acetoacetyl-CoA thiolase; ERG13, HMG-CoA synthase; tHMGR, truncated HMG-CoA reductase; MvaE, acetyl-CoA acetyltransferase/HMG-CoA reductase; MvaS, HMG-CoA synthase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, mevalonate pyrophosphate decarboxylase; IDI1, IPP isomerase; ispSPa, <i>P. alba</i> isoprene synthase was optimized to the preferred codon usage of <i>E. coli</i>. Pathway intermediates. MVA pathway: A-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, hydroxymethylglutaryl-CoA; Mev-P, mevalonate 5-phosphate; Mev-PP, mevalonate pyrophosphate. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate.</p