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Leading Edge

Review
Biofuels for a sustainable future
Yuzhong Liu,1,2,3,8 Pablo Cruz-Morales,1,2,3,8 Amin Zargar,1,2,3 Michael S. Belcher,1,2,4 Bo Pang,1,2,3 Elias Englund,1,2,3
Qingyun Dan,1,2,3 Kevin Yin,1,2,4 and Jay D. Keasling1,2,3,5,6,7,*
1Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA
2Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
3California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA
4Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA
5Departments of Chemical and Biomolecular Engineering and of Bioengineering, University of California, Berkeley, Berkeley, CA, USA
6Novo Nordisk Foundation Center for Biosustainability, Technical University Denmark, Horsholm, Denmark
7Center for Synthetic Biochemistry, Shenzhen Institutes for Advanced Technologies, Shenzhen, China
8These authors contributed equally

*Correspondence: keasling@berkeley.edu
https://doi.org/10.1016/j.cell.2021.01.052

SUMMARY

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation
of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop,
test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well
developed, while other feedstock resources and processes have also shown high potential to provide effi-
cient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis,
as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further
engineered to not only increase the product yield but also expand the chemical space of biofuels through the
rational design and fine-tuning of biosynthetic pathways toward the realization of ‘‘designer fuels’’ and
diverse future applications.

INTRODUCTION and fermentation processes, bioethanol and biodiesel produc-

tion have once again become viable and sustainable surrogates
There is a clear need to transition energy dependence from fossil for petroleum-based fuels. Bioethanol is derived from corn and
fuels to renewable energy sources to address the unprecedented sugar cane in the United States and Brazil, respectively, which
pace of climate change due to the accumulation of greenhouse together account for 84% of the total global production. In the
gases (GHGs) in the atmosphere. Overwhelming evidence has United States, bioethanol production has reached a volume of
shown that human activity is the major driver of climate change 15.7 billion gallons in 2019 (US Department of Energy, 2020a),
and that its consequences are impacting food production, migra- thus meeting the mandatory 10% supplementation requirement
tion patterns, economic, and political stability on a global scale. In for gasoline (109th US Congress, 2005). In Europe, the lack of
the US alone, 6.677 gigatons of GHG were emitted in 2018 with cultivable land and the ban on genetically modified crops has
the largest fractions being attributed to transportation (28%), elec- largely limited bioethanol production. As such, 75% of the bio-
tricity generation (27%), industry (22%), commercial and residen- fuel market in the European Union is composed of biodiesels
tial applications (12%), and agriculture (10%) (US Environmental derived from rapeseed, palm oil, soybean, and used cooking
Protection Agency, 2020). As all these activities are largely depen- oil. As of 2015, biofuels have reduced carbon emissions by
dent on fossil fuels, technological advances and diversification of 589.3 million tons (Figure 1) and will continue to play an important
alternative energy sources hold promise to significantly reduce role in renewable energies (Biotechnology Innovation Organiza-
carbon emissions and alleviate climate change. tion, 2014).
Predating the use of petroleum itself, biofuels such as vege- Recent advances in battery technology have substantially
table oils, animal fats, and ethanol were used for heat and illumi- increased the power density of electrical energy storage de-
nation (Figure 1). This is exemplified by the first mass-produced vices, thus accelerating the development of electric vehicles.
car, the Ford model T, which ran on corn-derived ethanol. As However, to date, electricity in the US is still predominantly
automobile production became increasingly industrialized in derived from fossil fuels such as gas and coal (38% and 23%,
the early 20th century, it became evident that the ethanol produc- respectively) (US Department of Energy, 2020b). Furthermore,
tion scheme could no longer meet the ever-growing fuel demand limitations in the driving range, high capital cost, the lack of
for internal combustion engines. infrastructure, and power-to-weight ratios preclude the imple-
Today, with environmental policies pushing for a reduction of mentation of electric long-haul vehicles and aviation. To reach
GHG emission, aided by recent advances in crop engineering a carbon-neutral to -negative transportation scheme, a more

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Figure 1. A timeline of biofuel production
Non-fossil fuels have been used long before the
beginning of the oil era in the late 19th century.
Scientific advancements, legislation, and the
pressing need to lower CO2 emissions has led to
an increase in the production of biofuels, reaching
15.8 billion gallons and 1.7 billion gallons per year
of ethanol and biodiesel, respectively.

(e.g., corn and sugar cane), which require

fertilization, water, and soil, and thus
directly compete with food production.
Tight regulations on the use of pesticides
and genetically modified crops further
limit their utilization in sustainable trans-
portation. In order to mitigate these short-
comings, second-generation biofuels are
derived from non-edible lignocellulose
remnants of plants, which consist of up
to 70% polymerized sugars and consti-
tute the most abundant form of biomass
on Earth (Isikgor and Becer, 2015). These
biofuels are attractive because their
net carbon footprint (emitted carbon –
consumed carbon) can be neutral or
even negative (Field et al., 2020; Tilman
et al., 2006), and their generation from
agricultural and forest residues or white
wood chips provides economical advan-
tages compared to crops. However, us-
ing lignocellulose for biofuel production
requires energetically and financially
diversified approach therefore requires the use of both electric costly extraction of fermentable sugars such as thermal, chem-
vehicles and biofuels alike. Specifically, electric vehicles hold ical, and/or biochemical pre-treatment. As a result, despite the
promise in short-range and light-weight configurations, whereas fact that the Energy Independence and Security Act (EISA) of
the use of biofuels offers significant advantages for conventional 2007 set an annual blending target of 16 billion gallons of cellu-
long-distance ground transportation and aircraft. losic biofuels by 2022 for the US (110th US Congress, 2007),
To mitigate GHG emissions while meeting the global fuel de- by 2017 production had amounted to less than 2% of this
mand, biofuel technology advancements need to focus on (1) benchmark (US Department of Energy, 2020). Significant tech-
optimization of current biofuel-production technology for higher nological progress has since been made in the production of
productivity and efficiency of lignocellulosic biomass conver- lignocellulosic biofuel toward a clean and economically viable
sion, (2) diversification of feedstocks to ensure the viability of process, including advances in energy crop engineering strate-
biofuel production within existing ecological and economic con- gies, efficient degradation of lignocellulose, and simultaneous
straints (e.g., carbon fixation through photosynthetic and elec- manufacturing of higher-value products.
trochemical means as well as conversion of biowaste into The climate benefits of large-scale lignocellulosic biofuel pro-
value-added products), and (3) expansion of the chemical space duction were initially questioned due to its potential competition
toward designer molecules that improve fuel economy and per- with land use for reforestation; it is believed that energy crop culti-
formance while reducing carbon emissions. Major efforts need vation may result in less carbon capture efficiency than reforesta-
to be devoted not only to overcome technological barriers but tion, leading to a carbon debt that must be compensated by the
also to integrate social, economic, and environmental factors carbon negativity of the resulting biofuels. However, recent anal-
to provide long-term, cost-effective, and reliable production sys- ysis of switchgrass production on transitioning crop/pasture land
tems for the biofuel industry. showed that in fact, its GHG mitigation potential is comparable
with reforestation of this land and has several times more mitiga-
Advancing lignocellulosic feedstocks for biofuel tion potential than grassland restoration (Field et al., 2020). Addi-
production tionally, the ability of energy crops such as sorghum to grow on
Biofuel technology has evolved through several generations of marginal lands provides an avenue for biofuel production that
significant advancements. The predominant problem with first- minimizes the competition for necessary farmable land to sup-
generation biofuels is that they are derived from food crops port the growing population (Jiang et al., 2019; Diallo et al., 2019).

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Figure 2. Engineering strategies in planta
to improve CO2 fixation
Acceleration of NPQ relaxation, truncation of the
chlorophyll antenna, and introduction of a photo-
respiratory bypass are promising strategies to in-
crease CO2 fixation and biomass yield in plants.

ergy conversion efficiency of the plant

(Shih et al., 2014; South et al., 2019).
Eventually, many of these traits can be
stacked into individual biofuel crops to in-
crease plant productivity and maximize
biomass production as technologies for
advanced plant engineering and gene
regulation are developed (Belcher
et al., 2020).
Metabolic pathways to synthesize
products with higher values can also be
incorporated in the meantime to increase
the economic feasibility of lignocellulosic
biofuels as fossil fuels are currently
produced at a far lower price than bio-
fuels, and simultaneous manufacturing
of higher-value products with bio-
fuels would increase their economic
viability. ‘‘Molecular farming,’’ which cou-
ples agriculture with the production of
high-value small molecules and proteins
including therapeutics and antibodies, is
In order to maximize the land use for lignocellulosic biofuel a promising strategy for increasing the value of lignocellulosic
production, crops have been engineered to be more productive biomass (Buyel, 2019; Yang et al., 2020). Accumulating these
in accumulating biomass by increasing their photosynthetic ca- molecules after integrating their biosynthetic pathways in a bio-
pacity and carbon fixation efficiency (Figure 2). Biological pro- fuel crop background and introducing efficient extraction
cesses like non-photochemical quenching (NPQ) and photores- schemes into the processing pipeline can drastically decrease
piration are suboptimal for the bioconversion of photon energy production costs, thus increasing profits. This has additional im-
into fixed carbon. This is because the NPQ process dissipates plications for medicine, as cancer biologics and viral antibodies
excess photon energy as heat (unproductive), and the transition can be produced in planta at high levels in the field without the
from an NPQ state to a carbon fixation state (productive) is need for sterile manufacturing systems (Capell et al., 2020;
generally slow, leading to mass energy loss in field conditions. Dent and Matoba, 2020; Donini and Marusic, 2019; Mortimer,
It has been shown that overexpressing the genes responsible 2019). This is one of the most promising strategies to achieve
for NPQ relaxation in the model crop Nicotiana tabacum can economic viability for biofuel production, although transgene
accelerate the switching process, resulting in 15% increases biocontainment strategies will need to be implemented to pre-
in plant height, leaf area, and total biomass accumulation (Krom- vent unwanted transgene flow from engineered crops (Clark
dijk et al., 2016). Additionally, plants have evolved to maximize and Maselko, 2020). Because of the mismatch in the volumes
light capture with much of the energy wasted. While counterintu- of fuel needed versus the volume of each individual therapeutic
itive, diminishing a plant’s light harvesting capacity in dense field needed, it will be necessary to have a large number of crops,
conditions has a drastic and beneficial effect on biomass accu- each producing the same biofuel precursor and different high-
mulation. In fact, truncation of light-harvesting complex antenna value products, which will be agronomically challenging.
components decreased the capacity for light capture in engi- Biofuel production from lignocellulosic biomass relies on the
neered lines resulting in a 20% increase in total biomass accu- microbial bioconversion of cell-wall sugars and components
mulation under these conditions (Kirst et al., 2017). Photorespi- into fuels and products (Figure 3A) (Baral et al., 2019; Perez-Pi-
ration is another process that limits productivity due to mienta et al., 2019). A major hurdle to efficient bioconversion is
Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase’s (RuBis- the recalcitrance of the feedstock material and the inhibitory ef-
CO’s) capacity to react with molecular oxygen in place of CO2 fect that lignin has on this process (Dos Santos et al., 2019).
thus leading to a net loss of carbon sequestration efficiency. Cell-wall engineering has shown promise for decreasing overall
Therefore, introducing an engineered photorespiratory bypass recalcitrance by increasing the ratio of C6/C5 sugars, reducing
pathway into biofuel crops has the potential to increase the en- lignin content, and reducing the acetylation of cell-wall polymers

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Figure 3. Conversion of diverse feedstock sources to make biofuels

(A and C) Upon pretreatment, sugars and aromatic molecules can be extracted from lignocellulosic biomass; together with carbohydrates, lipids, proteins, and
plastic monomers from waste, these molecules can be used as carbon sources for the microbial production through glycolysis and beta oxidation pathways to
make biofuels and biogas.
(B) Instead of feeding biomass to microbes, photosynthesis and direct synthesis of biofuels can be achieved in a single cell in cyanobacteria and microalgae.
(D) Lithotrophs can be coupled to the cathode of an electrochemical cell, with delivery of electrons from the electrode driving CO2 reduction and carbon fixation.

that limit the conversion efficiency of the feedstock material (Az- Diversifying feedstocks for biofuel production
nar et al., 2018; Eudes et al., 2015; Yan et al., 2018). While lignin is Photosynthetic carbon fixation
a major contributor of feedstock recalcitrance, it is also a prom- While the first and second generations of biofuels use light and
ising substrate for specialized microbes that convert these aro- CO2 to produce biomass in crops that is later fed to microbes,
matic polymers into usable products (Fang et al., 2020; Incha third-generation or algal biofuels combine energy capture
et al., 2019). The introduction of specialized microbial hosts into and fuel production within a single cell of photosynthetic
various processing systems has the potential to optimize the con- cyanobacteria and algae (Figure 3B). Having the entire fuel-pro-
version of all lignocellulosic feedstock components into products duction process take place in one organism makes the process
with economic value, limiting the waste streams for biofuel pro- more direct and efficient with no energy invested in non-
duction and increasing the viability for their use on a global scale. fermentable parts such as plant stems, roots, and leaves. The
The synergistic application of these various strategies has the solar energy conversion in cyanobacteria and algae is higher
potential to make lignocellulosic biofuels economically viable than that in plants, reaching an efficiency of 3% in microalgae
while shifting the current paradigm of what an effective biofuel/ compared to less than 1% in most crops (Wijffels and Barbosa,
bioproduct production system achieves. Through a multidisci- 2010). Furthermore, many species can grow in wastewater or
plinary approach across all sectors, we have the potential to revo- marine environments with simple nutritional requirements and
lutionize the manufacturing of biofuels/bioproducts from lignocel- therefore do not compete for land use with agriculture. It is esti-
lulosic biomass ushering in a new era of green technologies. mated that microalgae can produce oil at a yield of 100,000

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L/hectare/year, while palm and sunflower oil can only reach cle (Claassens et al., 2016). However, due to the pathway nor-
1,000–6,000 L/hectare/year. Algal fermentation could also mally operating under anaerobic conditions, it might have limited
lead to 9,000 L/hectare/year of bioethanol production, transferability to organisms with oxygenic photosynthesis.
compared to 600 L/hectare/year derived from corn (Alalwan Nevertheless, an in vitro study has demonstrated that enoyl-
et al., 2019). CoA carboxylases/reductases with better properties than Ru-
Despite these favorable comparisons, attempts at large-scale BisCO, along with 16 other enzymes from all three domains of
cultivations have struggled with high production costs. Unlike life can be incorporated in a circular pathway where cofactors
agriculture, which has been optimized over millennia by humans, can be regenerated (Schwander et al., 2016).
the technology for mass scale cultivation of photosynthetic mi- One key parameter that dictates the viability of algal biofuels is
croorganisms is still in its early developmental stage. The cultiva- the productivity of the host strain. Certain microalgae can store
tion can be done in either an open system like a raceway pond, or up to 80% of their dry weight as lipids, making them an attractive
in a closed system such as a photobioreactor. Open ponds have target for biodiesel production, while other strains accumulate
lower operating costs but their use is limited due to the risk of carbohydrates that can be fermented to make ethanol (Alalwan
contamination, as well as strict regulation against genetically et al., 2019). Further engineering strategies have demonstrated
modified organisms in an uncontained system (Abdullah et al., the feasibility to upregulate lipid production (e.g., triacylglycerol,
2019). Closed systems, on the other hand, can have more tightly De Bhowmick et al., 2018) and convert residual proteins and car-
controlled cultivation conditions and have a low risk of contam- bohydrates toward further fermentation (Rashid et al., 2013). The
ination but the operating costs are high: $2,743 per ton of lipid length and level of saturation can also be altered through
biomass versus $1,227 per ton of biomass in open ponds (algae metabolic engineering, which can yield biodiesel for direct use
farm cost model) (Zhu et al., 2018). These costs can be lowered if in vehicles without extensive engine modification (Kings
the following limitations are addressed: (1) light dissipation in the et al., 2017).
NPQ process, (2) narrow usable light spectrum, and (3) poor car- To produce a specific biofuel directly by photosynthetic micro-
bon fixation efficiency of microbes. organisms, metabolic engineering is often required, and cyano-
Light dissipation in NPQ in algae is akin to that of energy crops, bacteria are typically used due to their genetic tractability.
which can also be addressed by a similar engineering strategy: A range of different fuel molecules have been produced,
truncation of the chlorophyll A in green algae (Melis et al., including alcohols, free fatty acids, molecular hydrogen, and al-
1999) and modification of the light-harvesting complex antenna kanes (Knoot et al., 2018). Standout examples include 5.5 g/L
size of the cyanobacterial photosystems have been shown to (212 mg/L/day) of ethanol and 4.8 g/L (302 mg/L/day) of butanol,
improve the solar-to-product conversion efficiency by up to both in the cyanobacterial model strain Synechocystis sp.
3-fold (Melis, 2009). The second issue is due to the fact that cy- PCC6803 (Gao et al., 2012; Liu et al., 2019). In these cases,
anobacteria and microalgae only capture the visible light range the most efficient strategy to reach high titers was to use the
(400–700 nm), which makes 50% of incident solar energy inac- strongest promoters available to drive overexpression of
cessible (Blankenship et al., 2011). However, terrestrial cyano- pathway enzymes. However, a better understanding of the
bacteria, when grown in far-red light environments, express a native metabolism and the development of more sophisticated
novel chlorophyll f (Chen et al., 2010). Heterologous expression genetic tools will likely lead to more efficient approaches.
of this pigment in Synechococcus sp. PCC7002 successfully Indeed, transcriptomics studies have revealed the importance
extended light absorption up to 750 nm, thereby broadening of small RNAs for cellular control (Kopf et al., 2014), and genome
the usable solar spectrum for fuel production (Tros et al., 2020). scale models have allowed coupling production with growth
The issue with poor carbon fixation is more complex in nature (Shabestary and Hudson, 2016). Still, the field has a long road
since RuBisCO, the main CO2 fixing enzyme, suffers from poor ahead before cyanobacteria can reach the productivity of model
catalytic activity and its promiscuity toward O2. In fact, attempts heterotrophic hosts.
to improve the activity of RuBisCO have had limited success due Electrochemical carbon fixation
to a trade-off between CO2 affinity and carboxylation rate (Savir Man-made devices, such as photovoltaics, are more efficient
et al., 2010). As an alternative, efforts to engineer the Calvin cycle than photosynthesis at capturing sunlight, partly due to their abil-
for increased regeneration of RuBisCO’s substrate, ribulose-1,5- ity to capture most of the solar spectrum (Blankenship et al.,
biphosphate (RuBP), resulted in a 69% increase in ethanol yield 2011). The problem with photovoltaics is energy storage, which
(Liang et al., 2018). In addition, CO2 fixation can be improved by in photosynthesis can be easily achieved through the formation
engineering a carbon sink that pulls fixed carbon away from the of chemical bonds. Microbial bioelectrocatalysis is a hybrid be-
Calvin cycle. By introducing the 2,3-butanediol pathway into tween the two approaches (Figure 3D). This is a novel research
Synechococcus elongatus PCC 7942, as well as enzymes gener- area that uses electricity as a source of electrons, and CO2 fixa-
ating pyruvate from 3-phosphoglycerate, the total carbon yield tion plus energy storage from autotrophic microbes to make
was increased by 1.8-fold (Oliver and Atsumi, 2015). Unnatural fuels and other valuable products. The energy transfer can be
carbon fixation pathways have also been introduced to circum- achieved either through direct contact with the electrode or by
vent the poor properties of RuBisCO and its complicated regula- using an electron mediator. It has been known for decades
tion. Certain archaea use a reductive acetyl-CoA pathway to fix that oxidoreductase enzymes can use electrodes as either a
carbon with formate dehydrogenase and CO dehydrogenase/ source or a sink of electrons (Cooney et al., 2008). While the
acetyl-CoA synthase. This pathway is ATP efficient, requiring 2 membrane of living cells has low conductivity, a number of mi-
ATPs to synthesize acetyl-CoA, compared to 7 in the Calvin cy- croorganisms have the ability to shuttle electrons across the

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membrane for anaerobic respiration using conductive pili or (RNG) matching the quality of fossil natural gas while effectively
outer membrane cytochromes (Logan et al., 2019). In one study, lowering carbon emissions ranging from 17 to 70 kg CO2/ton of
multiple acetogenic bacteria, including species from the genera waste (Slorach et al., 2019). AD is achieved through bacterial hy-
Geobacter and Clostridium, were shown to reduce CO2 to ace- drolysis of materials with high molecular weight or high granular
tate as well as a small amount of 2-oxobutyrate with a faradaic content into smaller soluble fragments (e.g., fatty acids, glucose,
efficiency of more than 85% (Nevin et al., 2011). This electrosyn- and amino acids), which are degraded into volatile fatty acids to
thetic system mimics the natural process of photosynthesis with be further digested into biogas acetate, CO2, and H2. This pool of
oxygen evolution at the anode and CO2 reduction to value- molecules can then be utilized by methanogens to produce
added molecules at the cathode. It should be noted that in methane. Besides methane, biohydrogen is a non-carbon and
contrast to photosynthesis, carbon and electron flux is directed pollution-free fuel with the highest energy yield of known
primarily to the synthesis of reduced carbon products instead of fuels—122 kJ/g (compared to 50.1 kJ/g of methane) (Han
biomass (Nevin et al., 2010). A drawback of this method is the et al., 2016). By adding a dark fermentation stage to the existing
limited available surface area on the cathode where a biofilm AD, a mixture of methane and hydrogen (biohythane), can also
can be formed, thereby making large-scale productions difficult. be converted from waste. Several key parameters have been
An alternative approach is to use an electron mediator that demonstrated to increase methane and hydrogen yield as well
shuttles electrons from the electrode to the cells. Immobilized as system stability, including mesophilic temperature (30 C–
Desulfovibrio vulgaris have been used to produce H2 with methyl 40 C) to increase metabolic rates as well as destruction of path-
viologen as the redox mediator between the cathode and hy- ogens, pH 6.5–7.2 for favorable growth of methanogens, a
drogenases (Tatsumi et al., 1999). In another study, Ralstonia carbon to nitrogen ratio of 20–30, and an optimal lipid concentra-
eutropha (also known as Cupriavidus necator) was engineered tion (Zhang et al., 2016). Co-digestion of food waste with other
to fix CO2 using electrochemically generated formate to synthe- organic substrates has also been shown to improve the perfor-
size isobutanol and 3-methyl-1-butanol (Lu et al., 2012). H2 mance of anaerobic fermentation by 383% due to buffering
generated by electrochemical water splitting was used as an capacity and nutrient balance (Misi and Forster, 2001), high-
electron mediator to fuel production of methane in Methanosar- lighting the need to engineer bacteria toward higher tolerance
cina barkeri (Nichols et al., 2015). Although methane can be for pH and a wider spectrum of nutrient supplements.
generated directly by nonorganic electrocatalysis, higher effi- Plastic waste degradation and conversion to biofuels
ciency and lower overpotential was achieved with this hybrid More than 350 million tons of plastic are produced in the world
bioinorganic approach, demonstrating a fully solar-driven every year (Danso et al., 2019). Synthetic plastics have high resis-
methane production system. In another study, a cobalt-phos- tance to many physical and chemical factors, and, as such, the
phorus water-splitting catalyst, more compatible with bacterial degradation in a natural setting is extremely slow. A few microbes
growth, was used to evolve Ralstonia eutropha to grow on H2 have shown great potential toward the degradation and eventu-
(Liu et al., 2016). This setup captured 180 g of CO2 with 1 kWh ally, conversion of plastics (Figure 3C). For example, Ideonella sa-
of electricity. An issue with using H2 as electron mediator is its kaiensis is capable of metabolizing polyethylene terephthalate
low solubility, which hinders the maximum throughput of (PET) as a primary carbon and energy source, and two enzymes,
reducing equivalents being transferred to the cells. To overcome PETase and MHETase, are responsible for degrading PET into its
that problem, a biocompatible nanoemulsion has been used to two monomers, terephthalic acid and ethylene glycol (Yoshida
increase the kinetics of H2 transfer and subsequent oxidation et al., 2016). Other bacteria and fungi have also been reported
by more than 3-fold (Rodrigues et al., 2019). to degrade ester-linked polyurethane, polyethylene, polystyrene,
Conversion of food waste into biogas and polyamide plastics. Despite the lack of well-studied depoly-
Turning waste into fuel is a solution to two pressing problems merizing enzymes, polystyrene, polypropylene, and polyethylene
faced by biofuels: (1) the cost of food waste is low, especially if can be non-enzymatically treated, e.g., via pyrolysis. The result-
collection schemes are routinely practiced, and it is a more sus- ing long-chain hydrocarbons can then be readily metabolized by
tainable alternative than burying it in landfills; (2) food waste has microbes such as Yarrowia lipolytica (Mihreteab et al., 2019). The
high content for carbohydrates, proteins, and lipids that can be partial or complete plastic degradation pathways from existing
readily consumed by microbes, forgoing the need of extensive microorganisms could potentially be incorporated into recombi-
pretreatment. According to the Food and Agriculture Organiza- nant microbial hosts to not only break down polymers but also
tion of the United Nations, a third of all food produced for human convert the degradation products into fuels, valuable chemicals,
consumption is lost or wasted worldwide, i.e., 1.3 billion wet tons or biodegradable plastics.
per year, which is equivalent to $161 billion USD (FAO, 2015). Biofuel chemical space and applications
Conversion of food waste is not only favorable for economic Petroleum-derived fuels are made of hundreds of hydrocarbons
feasibility of biofuels, but also has a positive impact on land di- that are obtained by distillation. The differences in the average
versity as well as in the environment, as nearly 400 kg of CO2 number of carbon atoms and content of aromatic, branched,
emission per ton of organic waste in landfilling can be avoided and aliphatic hydrocarbons define their boiling point, freezing
(Smith et al., 2001). point, and combustion energy. This in turn determines their per-
Bacterial anaerobic digestion (AD) (Figure 3C) is a common formance and application space: lighter fractions and gasoline
practice to convert food waste to biogas that is 50%–75% are used in heating as well as light and medium-duty transporta-
methane and 25%–50% CO2 (Atelge et al., 2018). This process tion; kerosene is used in the aerospace industry; diesel is used
can be further upgraded to produce renewable natural gas for medium and heavy-duty transportation.

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While bioethanol and biodiesel will likely remain the front run- sized from acetyl-CoA through the mevalonate (MVA) pathway,
ners in the near future of biofuels due to their existing market and or from acetyl-CoA and glyceraldehyde 3-phosphate (G3P)
technological advancement, the lack of chemical diversity through methylerythritol 4-phosphate (MEP) pathway (Figure 4).
compared to that of gasoline and diesel limits their use in a These C5 units are condensed in linear pyrophosphate interme-
wide range of applications. In addition, new generations of bio- diates such as dimethylallyl pyrophosphate (GPP, C10) and far-
fuel molecules must meet the following criteria: (1) higher energy nesyl pyrophosphate (FPP, C15) (Peralta-Yahya et al., 2012).
content—pure ethanol energy content is only 70% of gasoline These pyrophosphate intermediates can be transformed by
and pure biodiesel 90% of D2 diesel (US Department of energy, terpene synthases or chemical semi-synthesis to produce fuel
2014); (2) low freezing temperatures—soybean biodiesel has a molecules. Examples are isoprenol/prenol (C5) (Zheng et al.,
cloud point of 1 C, while D2 diesel has a cloud point of between 2013) and farnesol (C15) (Wang et al., 2016) produced by phos-
7 and 28 C (US Department of agriculture, 2019), which phatases or pyrophosphatases, and pinene (C10) (Sarria et al.,
makes biodiesel incompatible with the current distribution infra- 2014), limonene (C10) (Chuck and Donnelly, 2014), farnesene
structure, thus leading to geographical and seasonal con- (C15) (Brennan et al., 2015), and bisabolane (C15) (Peralta-Yahya
straints; and (3) tailored molecular functionalities for highly et al., 2011) produced by terpene synthases and followed by
specialized applications, e.g., high isomeric carbon number optional chemical hydrogenation. These pathways can generate
and strained bond angles for aerospace applications (Holladay products with isomeric carbons and strained angles yielding
et al., 2020). To synthesize such molecules, novel bioproduction better fuel properties for aviation. In fact, farnesane, a jet fuel
approaches that incorporate complex, unnatural biosynthetic derived from farnesene has a freezing point of 70 C and was
pathways are needed. As synthetic biology has demonstrated approved for use as a 10% blend in jet fuel.
the capability of delivering target molecules with precise control Reverse b-oxidation (Dellomonaco et al., 2011), fatty acid syn-
of their molecular structures and functionalities, such versatility thases (FASs) (Steen et al., 2010), and polyketide synthases
can be translated into future biofuel-production schemes. (PKSs) (Yuzawa et al., 2018) can be used to produce long-chain
Biosynthetic pathways to produce alcohol, fatty acid, biofuels (Figure 4). These pathways have in common thiol carrier-
and terpene-based biofuels bound intermediates derived from acyl-Coenzyme A (Acyl-CoA).
Extant biofuels are usually simple molecules such as short-chain In reverse b-oxidation, CoA acts directly as the thiol carrier, while
alcohols, fatty acid esters, and terpenes. Naturally occurring in FASs and PKSs an acyl carrier protein (ACP) is activated with a
metabolic pathway that converts carbohydrates, pyruvate, and phosphopanteteinyl group, which is in turn derived from CoA.
other glycolytic intermediates can be engineered to make bio- Another common feature of these pathways is that the chain
fuels, which requires additional reducing equivalents to convert extension occurs via a Claisen condensation mechanism. After
these oxidized substrates into highly saturated products. Two each condensation, the resulting b-ketone is fully reduced to
structural changes are often needed: (1) transformation of the form a methylene moiety, and the final products, fatty acids,
natural precursor into burnable products by elimination of func- are released from the carriers by a thioesterase (Figure 4). Ester-
tional groups, e.g., carboxyl and phosphate groups in lipids, or ification of these carboxylic acids yields fatty acid methyl ester
addition of simple functionalities such as alcohols, (m)ethyl-es- (FAME) and ethyl ester (FAEE) biofuels such as ethyl-octanoate
ters in fatty acids or methylene in unsaturated terpenes; and (2) and ethyl-decanoate (Saerens et al., 2006). Hydride substitution,
modification of the carbon number and density, which can be from NADPH or NADH, releases the chain to fatty aldehydes
achieved by extending the length of the molecule or introducing (Schirmer et al., 2010), further reduced to fatty alcohols (Steen
modifications, such as branches and rings. et al., 2010), or decarbonylated to burnable hydrocarbons
Short-chain alcohol production pathways directly convert pri- (Schirmer et al., 2010). Alternatively, thioesterases release the in-
mary metabolites to the fuel molecules without additional chain termediates to carboxylic acids. These free fatty acids can be
extension. For example, ethanol production pathways reduce reduced to alcohols by the carboxylic acid reductase-aldehyde
pyruvate to ethanol, in the presence or absence of acetyl-CoA reductase pathway (Akhtar et al., 2013) or decarboxylated to hy-
(Liao et al., 2016). Due to their simplicity and natural occurrence drocarbons by enzymes like nonheme iron oxidases (Rui et al.,
in microorganisms, these pathways can be very efficient and are 2014), ferulic acid decarboxylases (Aleku et al., 2018), and
compatible with many microbial hosts, leading to ethanol’s prac- light-driven fatty acid decarboxylases (Sorigué et al., 2017),
tical usage in gasoline blends. However, the short chain length which has been used for the production of pentadecane from
and high oxygen content of ethanol results in low-energy density palmitic acid.
and high hygroscopicity (Liu and Khosla, 2010). Alcohols pro- Engineering of polyketide synthases for novel biofuels
duced by keto acid pathways, such as butanol and propanol, Among these thiol carrier-dependent pathways, modular type I
have longer chains and better fuel properties over ethanol (higher PKSs are the most versatile synthetic platform due to their
energy content and lower hygroscopicity). In these pathways modular biosynthetic logic. PKSs are very large proteins which
(Figure 4), 2-keto acids, intermediates from amino acid biosyn- consist of multiple domains that work together to extend the
thesis, are converted to aldehydes by 2-keto-acid decarboxy- chain length while adding functionalities to the product: the acyl-
lases (KDCs) and then to alcohols by alcohol dehydrogenases transferase (AT) domain loads acyl-coA precursors, such as ma-
(ADHs) (Atsumi et al., 2008; Liu and Khosla, 2010). lonyl-CoA and its a-substituted analogs, the ketosynthase (KS)
Iterative assembly of building blocks using a common mecha- domain catalyzes the Claisen condensation, while optional ke-
nism such as fatty acid and isoprenoid biosynthesis is ideal for toreductase (KR), dehydratase (DH), and enoylreductase (ER)
long-chain fuel molecules. For isoprenoids, C5 units are synthe- domains convert the resulting b-keto moiety to a b-hydroxyl

1642 Cell 184, March 18, 2021

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Figure 4. Engineering of microbial pathways to produce diverse biofuels and bioproducts with varying chain-length and structural func-
tionalities
Short- and medium-chain alcohols and fatty acids can be made from 2-keto acid pathways, as well as fatty acid and reverse b-oxidation pathways. Long-
chain and strained hydrocarbons, such as farnesene and a-pinene, can be converted from sugar via the terpene biosynthesis pathway. Engineering of multi-
modular PKS systems have also been successfully adjusted to incorporate natural and unnatural units to produce pre-designed compounds as biofuels and
bioproducts.

Cell 184, March 18, 2021 1643

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group, a-b alkene, or a saturated b carbon (Figure 4). PKS syn- and the low cost of petroleum-based fuels. Indeed, the advent
thetic pipelines often have a strong correspondence between of fracking and other methods to improve extraction of fossil
module order, domain composition, and the chemical structure fuels from the ground has made the situation even worse. Fortu-
of the final products, which enables the prediction of gene clus- nately, many of the technologies originally created for producing
ters from chemical structures, and vice versa, allowing for retro- advanced biofuels can be used to produce other products that
synthetic design for new molecules of various chain lengths and might have otherwise been synthesized from petroleum and
functionalities (Eng et al., 2018) and the potential production of which have a favorable profit margin or products that would be
‘‘designer’’ fuels. difficult or impossible to synthesize from petroleum. As the
The PKS biological design space is enormous considering (1) methods for producing these products become more efficient,
a large number of starter and extender units with different it will be easy to translate these gains to biofuel production
a-groups in combination (e.g., methyl and ethyl) with (2) the vary- when the world’s governments decide to prioritize climate
ing degrees of b-carbon reduction, and (3) the variations in the change over the petroleum industry.
number of modules (Pang et al., 2019). Engineering efforts to
controllably manipulate these characteristics has led to the ACKNOWLEDGMENTS
production of novel biomolecules, including new biofuels. Acyl-
This work was funded by the DOE Joint BioEnergy Institute (https://www.jbei.
transferase engineering through point mutations and AT domain
org/), supported by the U.S. Department of Energy, Office of Science, Office of
swapping has enabled the incorporation of many non-natural
Biological and Environmental Research, and by the Co-Optimization of Fuels &
extender units (Kalkreuter, 2019). For example, the AT domain Engines project sponsored by the U.S. DOE Office of Energy Efficiency and
of borrelidin PKS module 1 has been demonstrated to take not Renewable Energy, Bioenergy Technologies and Vehicle Technologies
only malonyl-CoA but also its a-branched derivatives, such as Offices, under contract DEAC02-05CH11231 between DOE and Lawrence
methylmalonyl-CoA into its assembly line, leading to highly Berkeley National Laboratory.
branched C3-C15 molecules (Curran et al., 2018). AT domain
swapping and further engineering have also led to the production DECLARATION OF INTERESTS
of C6 and C7 ethyl ketones, as well as C5 and C6 methyl ketones
J.D.K. has a financial interest in Amyris, Lygos, Demetrix, Maple Bio, Napigen,
at high titers of >1 g/L and several hundred mg/L, respectively Apertor Pharma, Ansa Biotechnologies, Berkeley Yeast, and Zero Acre Farms.
(Yuzawa et al., 2018). These short-chain ketones can be added
to gasoline as oxygenates to increase their octane number,
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