Engineering Corynebacterium glutamicum for isobutanol production

Abstract

The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2522-6) contains supplementary material, which is available to authorized users.

Involvement of Regulatory Interactions among Global Regulators GlxR, SugR, and RamA in Expression of ramA in Corynebacterium glutamicum

“…C orynebacterium glutamicum is a high-GϩC-content Grampositive soil bacterium which is used in biotechnological production of amino acids, organic acids, and alcohols (1)(2)(3)(4)(5)(6). Since the complete genome sequence of C. glutamicum became available (7)(8)(9), a number of transcriptional regulators controlling genes involved in central carbon metabolism have been characterized (10,11).…”

Harnessing novel chromosomal integration loci to utilize an organosolv‐derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum

“…About 15.5 ± 0.6 mM (46 ± 1%) and 1.7 ± 0.0 mM (43 ± 1%) of d ‐xylose and l ‐arabinose were metabolized respectively, and CIsArXy produced 7.2 ± 0.2 mM of isobutanol within 28 h of cultivation. With respect to the analysed pentoses, a carbon molar product/substrate yield (Y P/S ) of 0.31 ± 0.02 C‐mol isobutanol per C‐mol substrate ( d ‐xylose + l ‐arabinose) was achieved, which is already in the range of d ‐glucose‐based processes with engineered C. glutamicum strains (0.15–0.52 C‐mol C‐mol −1 ; Blombach et al ., 2011; Smith et al ., 2010; Yamamoto et al ., 2013). Isobutanol production based on the pentoses d ‐xylose and l ‐arabinose has so far not been demonstrated and therefore represents a promising example for the valorization of HF within a novel value chain.…”

Current knowledge on isobutanol production withEscherichia coli,Bacillus subtilisandCorynebacterium glutamicum

“…However, the overexpression of malE in our producer strains might be a possibility to further improve isobutanol production with C. glutamcum. 5 Smith et al 4 also engineered C. glutamicum for the production of isobutanol and tried to increase NADPH + H + availability by inactivation of the glucose 6-phosphate isomerase gene to redirect the carbon flux into the NADPH + H + generating pentose phosphate pathway. Unfortunately, this attempt to improve isobutanol production failed, probably due to an imbalance in the redox state of the cell.…”