Next generation biofuel engineering in prokaryotes

Abstract

Next-generation biofuels must be compatible with current transportation infrastructure and be derived from environmentally sustainable resources that do not compete with food crops. Many bacterial species have unique properties advantageous to the production of such next-generation fuels. However, no single species possesses all characteristics necessary to make high quantities of fuels from plant waste or CO2. Species containing a subset of the desired characteristics are used as starting points for engineering organisms with all desired attributes. Metabolic engineering of model organisms has yielded high titer production of advanced fuels, including alcohols, isoprenoids, and fatty acid derivatives. Technical developments now allow engineering of native fuel producers, as well as lignocellulolytic and autotrophic bacteria, for the production of biofuels. Continued research on multiple fronts is required to engineer organisms for truly sustainable and economical biofuel production.

Figure 1. Four approaches to engineering next generation biofuel producers

Ideal biofuel producers should grow on cheap, renewable feedstock and, at the same time, produce high titers of advanced fuel. (A) One approach is to engineer both traits into model organisms, which have the major advantage of genetic tractability. A number of fuel pathways have been expressed in these organisms; less progress has been made in engineering them to utilize sustainable feedstocks. (B) A second approach involves using native fuel producers and re-engineering feedstock preferences. Solventogenic strains of Clostridium have long been used for industrial fuel production via the acetone–butanol–ethanol (ABE) fermentation process. However, fuel tolerance is low and genetic manipulations still remain challenging. (C and D) A third approach is to engineer biofuel production into organisms that are naturally capable of growth on renewable feedstocks. (C) Lignocellulolytic and some thermophilic organisms have the ability to use such feedstocks and grow at elevated temperatures, making biomass degradation and fuel extraction more efficient; however, genetic are sparse tools for these organisms and introduction of biofuel pathways is challenging. (D) Autotrophic organisms can naturally use CO₂ as a carbon source, but genetic tools are again limiting for the introduction of biofuel pathways.

Figure 2. Biosynthesis of alcohol biofuels

Alcohols can be synthesized via the CoA-dependent pathway (purple) or the keto-acid pathway (blue). The latter takes advantage of native amino acid biosynthesis by decarboxylation and reduction (via the Ehrlich pathway) of the keto-acid intermediates of the Val, Leu or Ile pathways. 2-Ketobutyrate can be produced via the native threonine or the heterologous citramalate pathway. Elongation using engineered enzymes can recursively increase the product length by one carbon each round. The CoA-dependent mechanism is similar to ABE fermentation in Clostridium. Replacing the key enzyme butyryl-CoA dehydrogenase (Bcd) with an irreversible trans-enoyl-coA reductase (Ter) increases flux to butanol. Successive cycles of this pathway elongate the product by two carbons per cycle, producing hexanol from one turn, and octanol from two turns of the pathway. Double-headed arrows represent multiple steps.

Figure 3. Biosynthesis of isoprenoid and fatty acid biofuels

Isoprenoids (green) are produced by successive condensation of the 5-carbon precursors isopentenyl-pyrophosphate (IPP) and dimethyl-allyl pyrophosphate (DMAP), which are isomers of each other. IPP and DMAP are synthesized by either the mevalonate pathway (found in the cytosol of plants) or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (also known as the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate (DXP) pathway), which is native to E. coli and cyanobacteria. Fatty acids (FAs, red) are synthesized from malonyl-CoA by multi-enzyme fatty acid synthases. Malonyl-CoA is made by acetyl-CoA carboxylase (ACC), the rate-limiting and committed first step in FA biosynthesis. The growing FA chains are attached to acyl-carrier proteins (ACP). Thioesterases cleave fatty acids off the ACP. Reverse β-oxidation offers an alternative pathway that uses CoA as a carrier molecule and acetyl-CoA to elongate the growing chain instead of malonyl-CoA. Double-headed arrows represent multiple steps.

Figure 4. Strategies for Consolidated Bioprocessing

There are two strategies for engineering functionality for both lignocellulose degradation and fuel production into one strain. Natively cellulolytic organism can be engineered to express fuel pathways, or cellulolytic enzymes can be expressed recombinantly in model organisms (or native fuel producers, which is more difficult and not diagrammed in this figure). Key features of each organism are highlighted in the figure and advantages and challenges are mentioned.