Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels

https://doi.org/10.1016/j.cbpa.2016.08.003Get rights and content

Highlights

  • Advanced biofuels beyond bioethanol are attracting increasing attention.

  • Microbes are metabolically engineered for advanced biofuels production.

  • A few advanced biofuels are under commercialization, but most are not.

  • More efficient strains can be developed through systems metabolic engineering.

  • Enzyme screening and engineering are important for novel pathway construction.

As climate change has become one of the major global risks, our heavy dependence on petroleum-derived fuels has received much public attention. To solve such problems, production of sustainable fuels has been intensively studied over the past years. Thanks to recent advances in synthetic biology and metabolic engineering technologies, bio-based platforms for advanced biofuels production have been developed using various microorganisms. The strategies for production of advanced biofuels have converged upon four major metabolic routes: the 2-ketoacid pathway, the fatty acid synthesis (FAS) pathway, the isoprenoid pathway, and the reverse β-oxidation pathway. Additionally, the polyketide synthesis pathway has recently been attracting interest as a promising alternative biofuel production route. In this article, recent trends in advanced biofuels production are reviewed by categorizing them into three types of advanced biofuels: alcohols, biodiesel and jet fuel, and gasoline. Focus is given on the strategies of employing synthetic biology and metabolic engineering for the development of microbial strains producing advanced fuels. Finally, the prospects for future advances needed to achieve much more efficient bio-based production of advanced biofuels are discussed, focusing on designing advanced biofuel production pathways coupled with screening, modifying, and creating novel enzymes.

Introduction

Future energy security and the ongoing climate change are urging us to develop more sustainable energy alternatives including biofuels produced from renewable biomass. Today's most representative biofuel is bioethanol fermented from corn or sugarcane [1]. However, bioethanol possesses several undesirable characteristics, including low energy content, hygroscopy, and high vapor pressure [2], leading to the development of advanced biofuels having much better fuel properties (Figure 1). Also, much effort has been exerted to use non-food feedstocks such as oleaginous microalgae, greenhouse gases, or wastes derived from human activities, instead of food crop-derived feedstocks [3, 4, 5]. Speaking of waste-derived feedstocks particularly, European countries have given attention to agricultural and forest industry wastes for the production of renewable biofuels [6]. Furthermore, a large amount of food waste generated in Asian and Asia-pacific countries has the potential of becoming a versatile feedstock for production of advanced biofuels [7, 8•]. Recent advances in metabolic engineering and synthetic biology have accelerated the capability to engineer various microorganisms allowing the engineered microbial strains to efficiently convert such feedstocks into various value-added products, including advanced biofuels.

So far, most metabolic engineering strategies for advanced biofuels production have utilized routes that fall into four major metabolic pathways: the 2-ketoacid pathway (Figure 2a), the fatty acid synthesis (FAS) pathway (Figure 2b), the isoprenoid pathway (Figure 2c), and the reverse β-oxidation pathway (Figure 2b). More recently, the polyketide biosynthetic pathway mediated by polyketide synthases (PKSs) has been tweaked for advanced biofuel production, with results showing good potential (Figure 3).

There have been several review papers on metabolic engineering and synthetic biology strategies employed for designing and optimizing pathways for microbial production of biofuels [1, 2, 3, 4, 5]. These recent review articles provide an overview and also a detailed idea for engineering the major metabolic pathways for the production of biofuels including higher alcohols [2] and hydrocarbons [4]. Instead of repeating the information reported in these review papers, we focus on the characteristics and the significance of engineering strategies employed for the production of the three major classes of advanced biofuels: higher alcohols, biodiesel and jet fuel, and biogasoline (Figure 1). Higher alcohols contain longer-chain alcohols with or without branched chains, with lower hygroscopy and higher energy content than ethanol, making them suitable as diesel or gasoline substitutes. Biodiesel contains both long-chain fatty acid esters and long-chain alkanes with similar properties as petroleum diesel, while bio-based jet-fuels consist of terpenoid-derived branched-chain or cyclic alkanes. Biogasoline consists of short-chain alkanes identical to those found in petroleum gasoline. Finally, key enzymatic reactions involved in the production pathways are revisited with focuses given on selection and/or evolution of enzymes (Table 1).

Section snippets

Higher alcohols

Despite the drawbacks discussed above, bioethanol has already established a large market size worldwide (Renewable Fuels Association; http://www.ethanolrfa.org/). However, it is expected the bioethanol market will be replaced with more advanced biofuels with better fuel properties such as higher alcohols [2]. Microbial production of primary higher alcohols such as 1-propanol [9, 10], 1-butanol [11], and other linear-chain fatty alcohols [2] has been widely studied (Figure 2a and b). Several

Diesel and jet fuel

So far, chemical trans-esterification of various lipid feedstocks using methanol or ethanol has been the typical method for producing biodiesel, but additional processes for trans-esterification reduce the cost-effectiveness and overall production efficiencies [4]. To circumvent this drawback, there have been several recent studies on bio-based production of diesel and jet fuel by direct microbial fermentation. Accumulation of knowledge on microbial lipid metabolism together with advanced

Gasoline

According to the U.S. Energy Information Administration (EIA), gasoline is responsible for 55% of the total transportation fuels used in the U.S.A. in 2014 (U.S. Energy Information Administration; http://www.eia.gov/), clearly showing the need for renewable production of this highly demanded fuel. Consequently, the current trend in microbial production of advanced biofuels has moved toward production of short-chain hydrocarbons (four to twelve carbons) that are very similar to commercial

Importance of enzyme engineering

It is obvious that the efficiency of microbial advanced biofuel production needs to be maximized to enable successful industrial-scale production. Yet, except for a few, most of the reported advanced biofuels production cases have been rather proof-of-concept type works and show rather low productivities and titers. For successful commercialization of cheap products such as fuels, productivities and titers should typically be in the order of several grams per liter and hour, and tens to

Perspectives and conclusions

In this paper, we reviewed the recent studies performed on the production of advanced biofuels, which are classified into three different types, higher alcohols, diesel and jet fuel, and gasoline, based on their properties. Among these, isobutanol and some long-chain hydrocarbons are reaching full-scale commercialization. Numerous examples of producing advanced biofuels are still mostly in proof-of-concept stages, but rapid progress on strain development by systems metabolic engineering

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556). Martin Gustavsson was additionally supported by the Swedish Research Council Formas (2014-1620).

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