Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis

Abstract

To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H(2)O(2)) stress, on isoprene production were further examined. Heat, salt, and H(2)O(2) induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

FIG. 1.

Pathways of isoprenoid biosynthesis in E. coli, B. subtilis, and Saccharomyces cerevisiae (14, 38, 39). G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methylerythritol; CDP-MEP, CDP-ME 2-phosphate; MEC, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate; AcCoA, acetyl coenzyme A (acetyl-CoA); AACoA, acetoacetyl-CoA; HMG-CoA, hydroxymethylglutaryl-CoA; MVAP, mevalonate-5-phosphate; MVAPP, mevalonate-5-diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; Dxs, 1-deoxy-d-xylulose-5-phosphate synthase; IspC, 1-deoxy-d-xylulose-5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase; IspE, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; IspF, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase; IspA, farnesyl diphosphate synthase; IspS, isoprene synthase; AtoB, acetoacetyl-CoA thiolase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MPD, mevalonate pyrophosphate decarboxylase; Idi, isopentenyl pyrophosphate isomerase.

FIG. 2.

Real-time RT-PCR analysis of the relative levels of dxs and dxr mRNAs in DSM 10 and three overexpression strains. The averages of results obtained from three independent RNA preparations are shown. All transcript levels were measured in triplicate for each RNA preparation. Error bars represent standard deviations from the means.

FIG. 3.

SDS-PAGE analysis of B. subtilis DSM 10, DSM 10/pHTdxs, DSM 10/pHTdxr, and DSM 10/pHTdxsr. Lanes: 1 and 6, protein markers; 2, wild type; 3, DSM 10/pHTdxs; 4, DSM 10/pHTdxr; 5, DSM 10/pHTdxsr. The arrow indicates the expected size for Dxr (43 kDa). The expected band for Dxs (70 kDa) could not be identified from SDS-PAGE.

FIG. 4.

Growth curve analysis of the wild-type strain DSM 10 and overexpression strains in LB medium at 30°C. The curves indicate the average OD₅₉₅ of each culture over time. Each sample was measured in triplicate.

FIG. 5.

Isoprene production is induced by heat (48°C), salt (0.3 M NaCl), and oxidative stress (0.005% H₂O₂) and is repressed by 1% ethanol in the B. subtilis strain DSM 10 and the sigB in-frame deletion mutant. The average concentrations (ng/ml/OD₆₀₀) obtained from three independent cultures are shown. Error bars represent standard deviations from the means.