Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli

Abstract

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), beta-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.

Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways

“…We found that either route led to production of trans-2-pentenoate, suggesting all enzymes examined here, Hbd, Crt, PhaB, and PhaJ1, are able to accept five-carbon substrates. The low titers compared with 3HV synthesis (∼55 mg/L versus ∼2 g/L) also identified dehydratase activity as a potential bottleneck in the full pathway, despite previous reports of high enzyme activities measured in vitro in E. coli extracts (19).…”

“…This result was corroborated by the appearance of improved protein expression for the codon-optimized variant by SDS/PAGE analysis. Interestingly, the original AdhE gene has been used previously to produce butanol (6,19,20). This result might point to a lower activity with the C5 substrates, such that improved expression is now more important.…”

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

“…For example, YqhD has a Km value of approximately 0.5 mM for butyraldehyde; this is lower than the reported 1.6 mM for Clostridium acetobuylicum's butyraldehyde dehydrogenase (Palosaari and Rogers 1988) and 4.7 mM for aldehyde dehydrogenase ALDH in Lactobaccilus brevis (Berezina et al 2010). Thus, it seems that the high background level of butyraldehdye reductase activity observed in E. coli by Inui et al could be attributed to YqhD (Inui et al 2008).…”

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

“…In another case, E. coli JM109 strain overexpressing the crt, bcd, etfAB, hbd, adhE and thiL genes of C. acetobutylicum was developed. This engineered E. coli strain was able to produce 16 mM butanol using 4% (w/v) glucose as a carbon source [58]. More recently, metabolic engineering has been used for the development of C. cellulolyticum H10 for isobutanol synthesis directly from cellulose [59] (Fig.…”