Branched-Chain Higher Alcohols - Biotechnology in China: Biofuels and Bioenergy - page 113

regular metabolite in almost all organisms, to one molecule of acetoacetyl-CoA.
Next, an acetoacetyl-CoA transferase transfers CoA from acetoacetyl-CoA to
acetate or to butyrate, forming acetoacetate, which is then converted to acetone
and carbon dioxide by an acetoacetate decarboxylase. Finally, a primary-
secondary alcohol dehydrogenase (such as the secondary alcohol dehydrogenase)
converts acetone to isopropanol in an NADPH-dependent reaction.
With renewed interest in alternative fuels, the production of isopropanol,
especially by a renewable route (fermentation or synthetic biosynthesis), has
become an important topic to study. In the traditional fermentative process, iso-
propanol can be produced naturally by various strains of Clostridium, with max-
imum production levels reaching 2 g/L [ 45 ]. In the synthetic biosynthesis
pathway, the production of isopropanol has been investigated in tractable heter-
ologous organisms. The first work using a synthetic pathway for isopropanol
production in E. coli was reported by Hanai et al. [ 37 ]. Their strategy utilized the
pathway models of Clostridium. Various combinations of genes from Clostridium
species were cloned and expressed in E. coli, and the engineered E. coli was able
to produce 81.6 mM isopropanol after 30.5 h in shake flasks with a mole yield of
43.5% in the production phase. Afterward, Jojima et al. [ 38 ] engineered E. coli
harboring the isopropanol-producing pathway consisting of thiolase, CoA trans-
ferase, actoacetate decarboxylase, and alcohol dehydrogenase from Clostridium,
and produced up to 227 mM isopropanol with a mole yield of 51% from glucose
under aerobic fed-batch conditions. Recently, Inokuma et al. [ 47 ] further improved
isopropanol production by optimization of the fermentation conditions and
removal of isopropanol using metabolically engineered E. coli strain TA76, pro-
ducing about 2,378 mM (143 g/L) isopropanol after 240 h with a mole yield of
67.4%, which indicates great potential for commercial fermatative isopropanol
production. However, the low production rate is still a significant hurdle for the
low-cost fermentative production of isopropanol, and improvement is still neces-
sary, which would be likely approached by additional metabolic engineering and
optimization of all aspects of the fermentation system. Unfortunately, no engi-
neering microorganisms for isopropanol production have been reported in China.
Isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol are major organoleptic
compounds in fermented foods, such as alcoholic beverages, soy sauce, and bread.
The synthetic pathway for the production of these BCHAs was proposed by
Ehrlich [ 48 ]. The Ehrlich pathway involves three enzymatic activities (Fig. 4 ).
Firstly, branched-chain amino acids are transaminated to the corresponding
oxoacids by branched-chain amino acid transaminases. The ammonia binds to
2-oxoglutarate and the glutamate so formed can subsequently serve as a nitrogen
donor for all other nitrogen-containing compounds in the cell [ 49 , 50 ]. Secondly,
branched-chain oxoacids are converted to branched-chain aldehydes via a decar-
boxylation reaction. Finally, the branched-chain aldehyde is reduced to the cor-
responding BCHA by an alcohol dehydrogenase. BCHAs can also be synthesized
from the corresponding intermediate a-keto acids in the branched-chain amino
acid ( L -valine, L -leucine, and L -isoleucine) metabolic pathway by decarboxylation
and reduction. Therefore, these a-keto acids are formed via two major pathways: