Protein engineering in designing tailored enzymes and microorganisms for biofuels production

Abstract

Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performance of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field.

Figure 1

A simplified overview of the traditional lignocellulose-to-biofuels process. This process involves multiple complex, costly, and energy-intensive steps, including pretreatment of plant biomass, enzyme production, enzymatic hydrolysis of pretreated biomass, and fermentation of the hydrolysate (monomeric sugars) to produce biofuels using engineered microorganisms. The most expensive processing steps, namely pretreatment, enzyme production, and enzymatic hydrolysis, are used to overcome the recalcitrance of biomass. Concerted effort from various fields is necessary to lower the production cost of lignocellulosic biofuels and the strategies covered in this review are underlined.

Figure 2

A schematic stereoview of the active site of ketoisovalerate dehydrogenase (KIVD) [30]. In order to produce long-chain alcohols (C₅–C₈) from amino acid biosynthetic precursors, the active site of KIVD was modeled and altered to fit larger substrates. The shaded areas are representations of altered binding pockets and those of double mutants M538A/V461A (green) and F381L/V461A (red), which offer less steric hindrance to larger substrates like 2-keto-4-methylhexanoate. Dotted side chains are wild-type and highlight the changes in the substrate binding pocket as a result of mutations. ThDP: thiamine diphosphate, a co-substrate.

Figure 3

A schematic representation of the global transcriptional machinery engineering gTME method developed by Stephanopoulos and coworkers [47••,49]. Complex phenotypic changes are possible via altered regulation of multiple genes. Such transformations can be achieved by concentrating mutagenesis on a single protein among the various components of yeast’s transcriptional machinery. Expression of the mutant TATA-binding transcriptional factor Spt15–300 (red) resulted in significant up-regulation of 14 genes. All these overexpressions act in a concerted manner to increase yeast’s tolerance to high concentrations of ethanol and glucose.

Figure 4

DNA assembler method developed by Zhao and coworkers [61•]. Multiple gene expression cassettes (encoding promoter-gene-terminator) are prepared by splicing PCR. Each fragment contains sequence homology to adjacent fragments at its termini enabling crossover events at both ends. All fragments are co-transformed and correctly assembled in S. cerevisiae with a linearized vector (or a helper fragment, not shown in the figure), yielding a plasmid encoding a functional pathway (or integration of a functional pathway into yeast chromosome). This method should significantly simplify the protein engineering of biochemical pathways and has various potential applications including biofuels production.