Engineering Microbes to Produce Biofuels

The Need for Transition to Renewable Energy Sources

Fossil fuels, such as coal, natural gas, and petroleum are important in our lives; much of the energy being used to power comes from them. However, they are rapidly depleted due to their non-renewable nature. Their extraction, transportation, refining, and use lead to air, water, and soil pollution. According to the Intergovernmental Panel on Climate Change (IPCC), fossil fuels contribute significantly to global warming, as evidenced by 89% of global CO2 emissions (ClientEarth Communications, 2022). Therefore, there is a need to transition to cleaner, renewable energy like biofuels for a more sustainable energy source and a healthier environment. Without relying too much on fossil fuels, energy security, global politics, and economies could be stabilized by diversifying the energy market, thereby reducing carbon emissions and mitigating climate change and air pollution (Ramos et al., 2022). 

Generation of Biofuels

Biofuels are produced when biomass is converted into gas or liquid through natural or thermal processes. There are two types of biofuels—primary and secondary. Primary biofuels, also known as natural biofuels, are generated directly from wood chips, plants, animal waste, forest crop deposits, and firewood. On the other hand, secondary biofuels can be classified into three generations based on feedstock type and processing methods. First-generation biofuels are derived from food crops that have high levels of starch, sugars, and oils. Mainly used for bioethanol and biodiesel production, first-generation biofuels can be extracted from sugarcane waste, sugar beet, wheat, sunflower seeds, and rapeseeds. Unlike first-generation, second-generation biofuels do not compete with food crops which does not pose challenges to food security as they are produced from lignocellulosic biomass, which are non-food crops, and agricultural waste. They are high in lignin, cellulose, and hemicellulose, which form the plant cell wall. Finally, third-generation biofuels are extracted from algal biomass with high lipid content. Compared to other feedstocks, algae yield higher biofuel output and has higher efficiency and sustainability, thus it receives high attention as a promising future biofuel source (Bhaskar and Pandey, 2015) (Antony et al., 2024).

Genetic Engineering Transforming the Biofuel Industry

Microorganisms are unicellular organisms used in biofuel production. Beyond their role as simple catalysts, microorganisms can utilize fatty acid substrates and alcohols to synthesize biofuel. Various microbes are used in biofuel production due to their unique metabolic capabilities. For instance, propionic acid bacteria are cultured in simple sugar substrates to produce propionic acid, acetic acid, CO₂, and H₂O. In addition, Bacillus megaterium can break down plant biomass to produce lactic acid and acetic acid. The most widely used in bioethanol production is Saccharomyces cerevisiae, or yeast, which ferments sugars and organic acids to produce ethanol and CO2. Microorganisms accumulate intracellular lipids like triacylglycerol (TAG), which are raw materials for biofuel production, to undergo transesterification. Their lipid content and composition can be influenced by types of metabolic pathways and environmental factors like temperature, pH, and nutrient availability. Some fast-growing microbes use feedstocks other than traditional energy crops, including sugarcane, to produce higher lipid content thus increasing more biofuel per hectare (Antony et al., 2024). 

Since energy demands are rising, scientists are working on improving biofuel yield to match the demand by genetically modifying microbes such as bacteria, yeast, and algae. Genetic engineering refers to the modification of genes to improve desired traits in an organism. Genetically microbes are transforming the biofuel industry by improving the efficiency to break down biomass more effectively and convert it into biofuels at a faster rate. By optimizing metabolic pathways, scientists can fine-tune microbial processes into desired fuel production with minimum resources used. Not only making biofuel synthesis more economically viable, but genetic modifications also allow microbes to use a wide range of feedstocks, including agricultural waste, to make biofuel synthesis more sustainable, reducing dependence on traditional raw materials (Antony et al., 2024).

Figure 1. Genetic Engineering Strategies for Improving Lipid Biosynthesis and Targeted Product Formation in Microbes (Antony et al., 2024)

Genetically modified Escherichia coli for biofuel production

Although some microbes isolated from the environment have natural biofuel-producing pathways, the limitations lie in insufficient quantities for economic viability. The most commonly targeted microorganism for metabolic engineering is Escherichia coli, due to its fast growth rate, metabolic adaptability, and user-friendly genetic tractability with public genomic databases providing gene variants to construct efficient pathways. It uses a wide range of carbon sources in a defined media and can grow aerobically and anaerobically (Adamczyk and Reed, 2017). Early biofuel metabolic engineering started with importing the ethanologenic pathway from Zymomonas mobilis into E. coli to drive ethanol production. However, ethanol has lower energy density and is not suitable for infrastructure due to moisture accumulation and corrosive properties (Lee et al., 2008). To overcome this, E. coli has been engineered to produce other fuels, which are divided into three types: alcohol-based (butanol and isobutanol), fatty-acid-based (fatty alcohol, alkanes, and esters), and terpenoid-based (isopentenols and farnesol). In butanol production, E. coli is engineered to produce it through a pathway by converting acetyl-CoA into butyryl-CoA and then to butanol. Additionally, fatty acids in E. coli are converted into alcohols, alkanes, and esters via engineered thioesterases and β-oxidation pathways. Terpenoid-based biofuels are derived from isoprenyl diphosphates, and engineered enzymes are used to convert these into isopentenols and farnesol (Wang, Pfleger, and Kim, 2017).

Since E. coli may naturally lack the necessary enzymes for the desired pathway, genes from different organisms are collected to artificially design a synthetic metabolic pathway and introduce the sequence of biochemical reactions into E. coli. For instance, CRISPR-Cas9 is a powerful and precise gene-editing tool that came from the bacterial immune systems, and it is used to insert a long DNA fragment encoding an isobutanol synthesis pathway into E. coli in a one-step approach (Bassalo et al., 2016). Other than that, CRISPR interference (CRISPRi) technology using Cas9 mutant without endonuclease activity is used to repress the transcription factor FadR to increase fatty acid synthesis and reduce β-oxidation in E. coli. E. coli has a single operon that can control the transcription of multiple genes. However, to control the expression level of each gene, the ribosomal binding site (RBS) can be designed in silico to have a desired translational initiation rate (TIR). Similarly, multivariate modular metabolic engineering (MMME) is employed to break down a synthetic metabolic pathway into smaller functional parts, each designed to perform a specific function within the pathway (Wang, Pfleger, and Kim, 2017). 

Figure 2. CRISPR technology for gene editing and silencing in metabolic engineering (Wang, Pfleger, and Kim, 2017)

E. coli does not have the mechanism to tolerate accumulated toxic biofuels in the biofuel production pathway. Therefore, scientists have developed efflux pumps to improve their tolerance to biofuel toxicity through genetic engineering by pumping out toxic biofuels from the cell. Similarly, transcription factors are modified to help E. coli control global gene expression in biofuel tolerance (Wang, Pfleger, and Kim, 2017). Looking at how other organisms naturally protect themselves from toxic organic solvents provides clues that can be applied in E. coli, such as changing the composition of their cell membranes and activating genes that help them survive stress. For instance, Geobacillus sp. Thioesterase that targets unsaturated medium chain length acyl-ACPs is used to change E. coli’s membrane lipid composition to improve its tolerance to high levels of fatty acid biofuels (Lennen and Pfleger, 2013).

Figure 3. Engineered E. coli with efflux pumps and transcription factor to improve tolerance for accumulation of biofuel products that are toxic to E. coli (Wang, Pfleger, and Kim, 2017)

Genetically modified Saccharomyces cerevisiae for biofuel production

Lignocellulosic biomass (LCB) is a plant-based material used in biofuel production. It is made up of cellulose, hemicellulose, and lignin, which are then pre-treated using cellulases and hemicellulases to break down into simpler sugars–hexoses, pentoses, and disaccharides. Saccharomyces cerevisiae efficiently ferments glucose into ethanol. But, it struggles to ferment other sugars like pentoses and disaccharides. This incomplete sugar utilization leads to low bioethanol production efficiency, not to mention the high cost of pre-treatment and the complexity of multiple steps in hydrolysis and fermentation, which reduces economic viability (Rezania et al., 2020). Therefore, yeast strains are engineered to produce the enzymes needed for LCB hydrolysis and eliminate the high-cost external enzyme addition while performing enzymatic hydrolysis and fermentation at the same time. This would make it more cost-effective for large-scale bioethanol production. A few aspects are looked at: engineering yeast strains to utilize more sugar types efficiently to ferment all sugars at the same time, not just glucose first, and survive toxic conditions in the process (Das, Sahoo, and Dasu Veeranki, 2023). 

When analyzing the gene expression of yeast strains that can ferment xylose, it was discovered that ergosterol biosynthesis genes, which are responsible for keeping cell membranes stable, were more active in yeast that ferment glucose. When yeast are fermenting xylose, genes related to starvation and stress response become more active, meaning that yeast does not view xylose as a good energy source, slowing down ethanol production. The reason behind this is yeast does not naturally have transport proteins to take in pentose sugars (xylose) efficiently (Matsushika, Goshima, and Hoshino, 2014). 

To fix this, scientists genetically modify yeast to add pentose transport proteins that are independent of the presence of glucose, allowing yeast to use both hexose (glucose) and pentose (xylose) sugars at the same time. Xylose transporters–H+ symporter (AraE) from Bacillus subtilis and xylose reductase from S. stipitis are introduced into the S. cerevisiae strain, showing a 4.1-fold increase in xylose utilization rate compared to strain without AraE (Kim et al., 2017). Evolutionary strategies are also used to optimize xylose transport by culturing S. cerevisiae with xylose as the only carbon source. Consequently, this selective pressure leads to a mutation in the Hxt7 transporter, improving its ability to transport xylose. Similarly, the mutation in S. cerevisiae GAL2 (N376F) was constructed to not only lose the transporting ability of hexoses but also possess the highest affinity for xylose. Apart from that, two types of xylose reductase are co-expressed to allow the utilization of both NADPH and NADH as cofactors to increase the efficiency of xylose reduction into xylitol (Sahoo et al., 2025).

Figure 4. Engineered xylose transport and metabolism in recombinant S. cerevisiae for xylitol production (Sahoo et al., 2025)

Microbial Biodiesel Production Process: From Cultivation to Transesterification

After culturing microbes, they are pre-treated to extract lipids to undergo transesterification into biofuel. Pre-treatment of microbial biomass is done before extraction to prepare the microbial cells for better lipid release. It includes using physical, chemical, and enzymatic methods that disrupt cellular structure, allowing lipids to be accessible for extraction. Subsequently, Bligh-Dyer and Folch extraction methods are used to extract lipids from lysed cells by treating biomass with methanol and chloroform (Antony et al., 2024). The last stage is transesterification, which converts lipids into biofuels using alcohol and catalysts. There are four types of catalytic transesterification—homogeneous acid-base catalysis, heterogeneous acid-base catalysis, enzyme catalysis, and newer catalytic methods. The traditional acid-base process faces several disadvantages, such as sensitivity to free fatty acids (FFA) and water content, high energy and capital consumption, slow reaction rates, and the corrosive nature of acid catalysts. Lipase, on the other hand, offers a more eco-friendly approach due to its operation at mild conditions at <60°C and atmospheric pressure, avoiding side reactions like saponification that might interfere with the downstream purification process, and no waste production (Wang et al., 2021). 

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This article was prepared by Tong Jing Ying (University of Bristol).

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