Available online at www.sciencedirect.com
Review
Precision fermentation for food proteins: ingredient
innovations, bioprocess considerations, and outlook —
a mini-review
J. Lucas Eastham and Adam R. Leman
The growing demand for dietary protein presents challenges for
environmentally sustainable food production within global limits.
Precision fermentation (PF) offers an innovative solution by
employing engineered micro-organisms to ecosustainably produce
animal or other proteins of interest for food applications. While
microbes have been used to modify and create foods for
thousands of years, microbial bulk protein production is relatively
new. Some of the most compelling PF products are food proteins
previously only derived from animal sources that can now be
expressed in micro-organisms. The alternative protein space is
flourishing with PF-derived protein research and development for
formulation in dairy, egg, meat, and sweetener applications. This
review discusses recent innovations in the space, popular target
ingredients, bioprocesses, production microbes, and the regulatory
landscape, all in a space poised for expansion this decade.
Address
Department of Science and Technology, The Good Food Institute,
Washington D.C., USA
Corresponding author: Leman, Adam R. (adaml@gfi.org)
Current Opinion in Food Science 2024, 58:101194
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cell factories, often programmed through strain en­
gineering, produce a target ingredient in a controlled
fermentation process. The ingredient is then separated
and purified from the micro-organism (Figure 1). This
mini-review explores protein ingredients produced by
genetically engineered micro-organisms to match pro­
teins traditionally produced by animals or plants.
The food industry has long produced molecules through
PF, such as citric acid, for preservation and processing
enzymes such as amylase, pectinase, and rennet (chy­
mosin-B). Chymosin has been used extensively in
cheese production since 1990 as one of the first animalfree recombinant PF proteins. PF-produced proteins for
direct ingredient use are being commercialized to enable
food functionality and nutritional characteristics while
addressing environmental concerns, enhancing food
safety by reducing animal-borne risks, and providing
health benefits. As PF technology evolves, a combina­
tion of bioengineering and food science innovations will
propel the industry. This review will highlight the most
common recombinant protein targets, their functional
properties, and microbial hosts, as well as the PF com­
mercial and regulatory landscape.
This review comes from a themed issue on Food Bioprocessing
Edited by Mattheos Koffas and Yong Su Jin
Precision fermentation–produced protein ingredients
The demand for dietary protein is expected to increase
through 2050, driven by industrialization and population
growth [1].
Producing animal-free PF proteins is similar to enzyme
biomanufacturing, which generates biocatalytic proces­
sing aids for the food industry. Briefly, upstream bio­
processing (USP) is the collection of steps where
microbes are provided nutrient feedstocks in an opti­
mized environment inside a bioreactor to generate the
protein macromolecules of interest. Temperature,
mixing, feeding rate, nutrient concentration, and cell
concentration are all potential optimization parameters
for USP. In downstream processing (DSP), the protein of
interest is isolated and purified from the rest of the
biomass and leftover media and then typically dried. A
diverse suite of technologies, such as centrifugation,
filtration, and spray drying, may be implemented to
produce a protein ingredient with a high purity
(Figure 1).
Precision fermentation (PF) offers one solution: lever­
aging engineered or evolved micro-organisms to eco­
sustainably produce food ingredients, such as proteins,
fats, flavors, pigments, and vitamins. These microbial
Precision protein targets, encompassing dairy, egg, meat
enhancing, and novel sweet proteins, are selected for
commercialization to mirror current ingredients and en­
hance flavor and nutritional profile. Some of these
For complete overview of the section, please refer to the article
collection, “Food Bioprocessing 2024”
Available online 1 July 2024
https://doi.org/10.1016/j.cofs.2024.101194
2214–7993/© 2024 The Authors. Published by Elsevier Ltd. This is an
open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
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Current Opinion in Food Science 2024, 58:101194
2
Food Bioprocessing
Figure 1
Current Opinion in Food Science
Common PF bioprocesses. A commercial fermentation process includes an array of steps from upstream to downstream production. USP includes all
the processing and preparation before harvest, such as substrate/medium preparation, medium sterilization, micro-organism growth for inoculation
(seed train), equipment sterilization, and finally, growth/production in the main bioreactor. DSP includes all processing after harvest, such as
dewatering, extraction, and purification of the fermentation product from the biomass (solid fraction) or supernatant (liquid fraction), along with
equipment sterilization and wastewater effluent treatment. SIP: sterilize-in-place; CIP: clean-in-place; TFF: tangential flow filtration. Created with
BioRender.com.
proteins improve food products at low concentrations,
such as meat protein enhancers (< 2%), sweeteners
(< 0.1%), or bioactive proteins (< 1%), whereas egg and
dairy proteins are often present at significant levels in
the final product for functionality and organoleptic
properties (5–35%; Figure 2) [2].
globular aggregates called micelles, which play a crucial
role in cheese curd coagulation [5]. Caseins are highly
desired for their emulsification and gelation properties,
which are imperative for animal-free cheese production
[5]. Example start-ups targeting caseins include Formo,
New Culture, and Standing Ovation.
Dairy proteins are diverse proteins that contain the es­
sential amino acids (AAs) for growth and are critical for
cheese, yogurt, and milk products [3]. Major dairy pro­
teins include casein and whey, which drive flavor, gela­
tion, and texture in dairy products [4]. Caseins, including
alpha, beta, and kappa caseins, are associated with large
Whey proteins are globular proteins found in the liquid
fraction that separates from curds during cheese pro­
duction [3]. Whey proteins have excellent solubility,
emulsification, and foaming properties, enabling animalfree dairy, bakery, and confectionary formulations
(Figure 2) [4,6]. Beta-lactoglobulin has an immediate
Current Opinion in Food Science 2024, 58:101194
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Current Opinion in Food Science
PF protein targets, functional/nutritional properties, and commercial landscape. End-product properties and use concentration are based on GRAS notices and in-text references. The commercial
landscape represents commercialized companies or start-up examples. *GRAS notice inventory used for microbial host strain information. **GRAS inventory used for Novonesis (formerly
Novozymes) lysozyme; it is not derived from egg protein sequence (fungal based). Otherwise, microbial host strains are based on patents: New Culture US20230074278A1; Change Foods
W02023133417 (pending); ReMilk casein IL276823A (pending); Paleo EP4271200A1 (pending); Geltor US11174300B2; Amai Protein US20210139546A1 (pending); Novozymes US9273320B2
(Brazzein); Mycotechnology US20220183333A1 (pending). Created with BioRender.com.
Figure 2
Precision fermentation for animal-free proteins Eastham and Leman
3
Current Opinion in Food Science 2024, 58:101194
4
Food Bioprocessing
commercial interest as a bovine whey protein replace­
ment, while alpha-lactalbumin has potential infant for­
mula applications, as the most abundant whey protein in
human milk [3,7]. Several start-up companies have tar­
geted beta-lactoglobulin, such as ImaginDairy, ReMilk,
and Perfect Day, along with large corporate partnership
interest from DSM-Fermenich/Fonterra and Novonesis/
Arla Foods.
Lactoferrin is an iron-binding, whey glycoprotein valued
for immune protection and has antimicrobial, and antiinflammatory properties. Lactoferrin production is an
attractive PF target for its high value and low con­
centration in bovine milk, making the current animalderived source valuable and costly to isolate [3,8]. Lac­
toferrin is well suited for nutraceuticals and infant for­
mula, as targeted by start-ups TurtleTree and Helaina,
respectively. Although, it has been explored to enhance
color and flavor in meat alternatives [9].
Egg white proteins (EWPs) have diverse food applica­
tions, from baked goods, ice creams, and nutritional
supplements, for their complete AA profile and func­
tional properties. EWPs are often used as a binding
agent in vegetarian formulations of meat alternatives,
such as mycoprotein [10]. Common EWPs include
ovalbumin (54%), ovotransferrin (12%), ovomucoid
(11%), ovoglobulin (4%), ovomucin (3.5%), and lyso­
zyme (3.4%). Ovalbumin enhances foaming, gellation,
and emulsification for baking. Ovomucoid, valued for
heat stability, prevents enzymatic browning. Lysozyme
can extend product shelf-life with its natural anti­
microbial activity [11].
Ovalbumin and ovomucoid have been primary PF tar­
gets. Researchers at VTT and spinout company OnegoBio have demonstrated the production of ovalbumin in
Trichoderma reesei with excellent end-product function­
ality [12]. While the EVERY Company has developed
PF-ovomucoid (GRN967) and ovalbumin (GRN1107)
using a yeast, Komagataella phaffii. The PF-ovomucoid is
highly soluble and forms optically clear, high-protein
liquids for beverage applications. Novonesis has devel­
oped a PF-derived lysozyme (fungal based) for food
processing applications [2] (Figure 2).
While fermentation-derived ingredients such as AAs and
yeast extracts are already widely used as food ingredients
to improve taste, PF can enable meat alternatives by
producing proteins that replicate meat sensory char­
acteristics. For instance, heme proteins, such as myo­
globin, hemoglobin, and leghemoglobin, can mimic the
red ‘blood-like’ color and meaty flavor of conventional
meat and have been shown to enhance the aromatic
profile of meat alternatives [13]. Leghemoglobin, a heme
protein from legume root nodules, is produced by Im­
possible Foods [2]. Myoglobin, muscle heme protein,
Current Opinion in Food Science 2024, 58:101194
enhances the aroma profile of plant-based meats and is
commercialized by Motif Foodworks and Paleo [13].
Collagen, derived from animal connective tissue, is widely
used in food and nutraceuticals due to its neutral sensory
properties (Figure 2). Gelatin, a collagen derivative, is es­
sential in products such as gummies and gel-based des­
serts, improving gelation and moisture retention. Animalsourced collagen faces safety, reproducibility, and sustain­
ability challenges, driving PF applications. Currently,
Geltor offers PF collagen for nutraceuticals and personal
care, with plans to scale dietary collagen [14].
Finally, sweet proteins, originating from plants and fungi,
such as brazzein, monellin, thaumatin, and mycodulcein,
are another PF-derived ingredient area of interest [15,16].
These PF-produced proteins are significantly sweeter
than sugar, enabling sugar reduction in food and beverage
while catering to the evolving preferences for healthier,
ecosustainable, and great-tasting foods [15]. Oobli is
commercializing brazzein (GRN1142), while Myco­
technology is exploring truffle sweet protein [2,16].
Food and bioprocess considerations for
precision fermentation proteins
These diverse protein targets have various functional­
ities and techno-economics. The expression of a target
protein is not always a drop-in replacement for their
animal-derived sources due to impacts on technofunc­
tional properties during processing or formulation and
from target protein sequence and microbial protein posttranslational modification (PTMs).
Each protein product’s unique attributes, along with the
bioprocessing medium, impact its conformation, equili­
brium state, and functional behavior in food applications.
For instance, globin proteins that bind heme must
maintain iron-binding functionality, requiring proper ion
concentrations, pH, and solvents during DSP. For dairy
proteins, while foaming is a desirable ingredient attri­
bute, foaming interferes with bioreactor operation during
fermentation. Mitigation requires food-grade antifoam
dosing during USP to prevent filter fouling, poor oxygen
delivery, or overflow. Furthermore, antifoam addition
can affect production efficiency [17].
While the production of PF-derived protein ingredients
is often compared to enzyme production, there are un­
ique considerations. Many food-grade enzymes undergo
chromatography purification for concentration and
functionality. Avoiding chromatography reduces costs,
but lower purity could impact functionality (like iron
binding) or organoleptic properties.
Microbial PTMs also significantly impact protein struc­
ture and function. Collagen’s triple helix structure can
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Precision fermentation for animal-free proteins Eastham and Leman
be difficult to produce, requiring proper PTMs.
However, gelatin (denatured collagen) does not require
full PTMs for its functionality and dietary applications
[14,18]. In dairy, beta-lactoglobulin (18.4 kDa protein)
has shown native functionality through microbial ex­
pression [12,18], whereas lactoferrin (80 kDa) may be
impacted by varying microbial PTMs affecting ironbinding and folding. Lactoferrin evaluations have de­
monstrated proper functionality albeit with higher purity
DSP, which increases costs [18,19].
For cheese, casein micelle formation and PTM phos­
phorylation present challenges for PF-derived casein
production. Research has shown that the degree of
phosphorylation impacts artificial casein micelle and
curd formation [5]. However, novel formulations can
develop cheese products with comparable properties, as
demonstrated in New Culture’s PF-enabled mozzar­
ella [20].
PF can also produce familiar food proteins with modified
structures that alter functionality. For instance, betalactoglobulin functionality can be modified through AA
substitutions or lack of native PTMs. Researchers have
demonstrated modified aggregation of recombinant betalactoglobulin through cysteine substitution, which pro­
vides valuable insights into protein structure modulation
for particular functionalities [21]. And, Perfect Day has
demonstrated that beta-lactoglobulin with altered phos­
phorylation has improved solubility, enabling beverage
formulation [22].
Other opportunities include targets from non­
domesticated animals with improved functionalities or
targeted native protein expression of nonallergenic pro­
teins, such as beta-casein variants. Similarly, genetic
modification can reduce allergenicity of common food
products, as demonstrated by Perfect Day for a betalactoglobulin [23].
5
different PTM profiles than their animal-derived coun­
terparts, which can impact food functionality and for­
mulations. Enzymatic postprocessing of bacterial
proteins can catalyze proper PTMs, which may improve
ingredient functionality but adds cost and complexity
(Figure 3).
Escherichia coli, the most common industrial bacteria, has
been used for PF-derived collagen and Chymosin-B
production. While E. coli titers up to 14 g l−1 have been
reported, as a gram-negative bacteria, endotoxins can
challenge food-grade production [14,25]. Thus, grampositive bacteria such as Bacillus subtilis, Corynebacterium
glutamicum, and Lactococcus lactis are popular industrial
strains due to the lack of endotoxins, availability of en­
gineering tools, protein secretion pathways, and low
endogenous extracellular protein levels [25]. B. subtilis
and L. lactis have been used to produce casein proteins
[20]. C. glutamicum can express recombination heme and
leghemoglobin up to 3 g l−1 titer [26–28]. With the
shorter production time of bacterial strains, commercial
production operational costs could be competitive with
mid-to-high-value food proteins.
Budding yeasts are well suited for fermentation, espe­
cially with complex protein targets. Advantages include
scalability, fast growth, high protein-folding fidelity, and
PTM signatures analogous to animals. Furthermore,
proteins can be trafficked intracellularly or extra­
cellularly, increasing bioprocess production and pur­
ification options (Figure 3) [29].
Saccharomyces cerevisiae, Komagataella phaffii, and
Kluyveromyces sp. are common food-safe industrial yeasts.
S. cerevisiae has long been a mainstay of food and bio­
technology, with many genetic tools for modulating
protein levels. Still, it is not a common PF-derived food
protein production microbe, likely due to its preference
for anaerobic metabolism and its hyperglycosylating
PTM behavior [18,30].
Production micro-organisms
Microbial host selection can strongly impact PF protein
ingredient technofunctionality and techno-economics.
Substantial R&D is devoted to increasing strain pro­
duction (yield, productivity, and titer). However, as
mentioned above, protein structure/function can also
impact micro-organism selection due to varying PTMs.
Thus, micro-organism choice depends on various factors,
including the desired protein complexity, scalability, and
production efficiency (Figure 3).
Bacteria are often chosen for PF due to rapid growth,
scalability, genetic tractability, and breadth of en­
gineering tools. However, many production systems
suffer from relatively low protein titers or protein traf­
ficking to intracellular inclusion bodies, complicating
DSP [24]. Many bacterial PF-produced proteins possess
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K. phaffii, a methanol-metabolizing yeast, is a preferred
microbe with established genetic engineering tools and
high secretory efficiency [18,31] (Figure 3). Myoglobin
(0.3 g l−1), lactoferrin (3.5 g l−1), and leghemoglobin
(3.5 g l−1) expression have been demonstrated in K.
phaffii [32–34]. Kluyveromyces sp. are promising bio-in­
dustrial yeast for their high protein secretion levels with
demonstrated commercial enzyme applications in K.
lactis [18,35] and inulinase/chymosin-B production in K.
marxianus [18,36].
Food-safe yeasts are ideal hosts for food proteins, with
published titers reported as high as 17–22 g l−1
[3,18,29,32,34,37,38]. Additionally, production batches
typically run for 3–6 days. The lower range productiv­
ities could be sufficient for mid-high value ingredients
Current Opinion in Food Science 2024, 58:101194
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Food Bioprocessing
Figure 3
Current Opinion in Food Science
Common PF organism types. General commercial attributes of bacterial, morphological yeast fungi, and morphological filamentous fungi. Positive
attributes for PF are denoted by a green plus (+) sign. Negative attributes are denoted by a red minus (−) sign. See text for additional titer ranges and
references. Created with BioRender.com.
(e.g. lactoferrin, $750–$1500/kg) with lower end-use
concentrations, but higher productivities (e.g.
25–50 g l−1) are needed for high volume, lower marketpriced proteins (e.g. whey protein ∼$12/kg) [3].
commercially relevant titers [12]. While achievable,
higher titers are crucial for techno-economic and sus­
tainability goals [42,43].
Regulatory frameworks
Filamentous fungi are efficient protein factories adapted
for scavenging carbohydrates. Species such as Aspergillus
sp. and Trichoderma reesei produce abundant starch-de­
grading amylase and cellulase enzymes at industrial scales
[12]. They can express high titer, extracellular re­
combinant proteins with eukaryotic PTMs, though cul­
ture viscosity can challenge production performance [39].
Food regulations vary worldwide, but two leading and
contrasting frameworks are the United States Food and
Drug Administration (FDA) generally regarded as safe
(GRAS) process and the European Food Safety
Authority (EFSA) novel food program. However, as al­
ternative proteins have gained market traction, world­
wide policies have been updated [44].
High-titer production is necessary for commodity-level
scales, and endogenous proteins secreted by T. reesei
have reached > 100 g l−1 with strain optimization [12,39].
However, longer batches (5–10 days) can reduce annual
production and increase batch failure risk.
The U.S. FDA regulatory GRAS framework focuses on
the safety of the finished product and process, including
toxicity, genotoxicity, and carcinogenicity [45]. Compa­
nies typically publicly or privately ‘self-affirm’ their
product as GRAS before submitting a safety dossier.
FDA does not approve or reject applications for food
additive proteins; rather, they review the safety dossier
for the ingredient. If GRAS criteria are satisfied, the
FDA responds with a ‘No Questions’ letter, which in­
dicates the FDA reviewers are satisfied with the safety
assays performed and do not question the conclusion for
Several companies currently use filamentous fungi for
recombinant protein production due to their high-titer
production [40,41] (Figures 2 and 3). However, pub­
lished titers of PF targets such as ovalbumin (2 g l−1) and
beta-lactoglobulin (1 g l−1) in T. reesei lag behind
Current Opinion in Food Science 2024, 58:101194
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Precision fermentation for animal-free proteins Eastham and Leman
the intended end use. Many PF-derived food enzymes
and dietary proteins have been concluded to be GRAS
with a ‘No Questions’ letter from the FDA [2]. Coun­
tries such as Singapore and Canada follow a similar fra­
mework requiring detailed scientific safety data
submission for review [45].
In contrast to the FDA, EFSA has a different purview
for regulating PF-derived proteins. Rather than a sub­
mitter-prepared GRAS conclusion, EFSA performs
safety evaluations of novel foods [45]. PF-derived pro­
ducts are mostly classified as genetically modified mi­
crobes, and EFSA mechanisms require additional
regulatory oversight. EFSA closely scrutinizes the or­
ganism that produces fermentation-derived proteins and
their genetic material [46]. Thus, EFSA safety dossiers
include genetic sequences, genome modifications, and
the safety of the process and product. To date, no PFderived ingredient proteins have been approved
by EFSA.
Navigating precision fermentation’s future
PF offers an opportunity to produce proteins sustainably
— mitigating environmental, animal welfare, and anti­
microbial resistance concerns. With environmental pro­
mise comes the potential for PF-derived ingredients to
provide functional and organoleptically favorable in­
gredients at a competitive price.
Life cycle assessments (LCAs) provide a model to
measure the environmental impacts of production pro­
cesses. Recent LCAs for beta-lactoglobulin and oval­
bumin help to measure the current potential for dairy
and EWP proteins to make meaningful progress toward
more sustainable food manufacturing. For beta-lacto­
globulin, LCA results indicated environmental impacts
of the same magnitude as pasture-based dairy proteins
[42]. While LCA results for ovalbumin production found
decreased emissions and land use compared with con­
ventional EWP [40]. Both LCAs demonstrated improved
sustainability for PF-derived protein production over
some established protein production systems but also
highlighted that increased sustainability could come
from improved feedstock sourcing and the use of re­
newable energy for electricity and thermal requirements
in fermentation. Additionally, reducing animal agri­
culture shrinks a significant source of antimicrobial re­
sistance risks [47].
Furthermore, PF LCA studies highlight the need for
better waste valorization. Carbon dioxide from fermen­
tation could be captured and used in biomanufacturing,
greenhouse crop production, or consumer goods. The
microbial biomass produced during PF bioprocessing is
substantial and valorization may be required to reach
their environmental and economic potential. This
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7
process could be akin to sidestreams industries built
around animal farming and biofuels. A suite of side­
streams industries need to be developed to maximize
the operational value of fermentation but also to prevent
environmental damage, such as freshwater eutrophica­
tion from biomass processing. Valorization could include
food, feed, and fertilizer applications but comes with
new market, technical, and regulatory development.
While these PF proteins demonstrate improved ecosustainability, the industry faces several challenges, such
as costs, regulatory/food safety studies, formulations, and
consumer perception. To compete in the food industry,
PF-derived products need to be delivered in high vo­
lumes and at competitive prices. New bioprocessing
technologies and strain development advances are
needed to increase the amount of protein produced for a
given amount of input energy, feedstocks, and labor.
Currently, many of these proteins are truly novel for
their intended use cases, with unique AA sequence
modifications, PTMs, and co-purifying molecules. Their
host organisms have demonstrated safety in a small, but
growing number of studies [2,48,49]. The industry must
balance the expansion of protein targets, intended uses,
and manufacturing approaches with the need for thor­
ough and clear demonstrations of food safety. Safety
studies are necessary for regulatory and consumer con­
fidence but represent a significant resource commitment
for start-ups and even established fermentation biopro­
cessors. Working with regulators on the relevant in­
formation required for effective safety analysis and
streamlined, standardized safety and purity expectations
should be a priority for producers.
Finally, PF-derived ingredients are often more precise
than the animal-derived ingredients they seek to re­
place. Egg whites and whey are both complex mixtures
of several proteins. These proteins often each contribute
particular functionalities that formulators wish to utilize.
Single protein purifications may provide some or all
those functionalities, but the optimal ingredient use pH,
temperature, salting, concentration, etc. must all be
characterized before effectively ‘dropping-in’ novel in­
gredients. In the future, as the repertoire of PF-derived
targets increases, the industry may see more mixtures of
proteins implemented, albeit at well-defined and char­
acterized ratios that improve upon the ratios originally
determined by their content in animal-derived isolates
and concentrates.
Finally, the industry must garner acceptance among the
consumer population to gain success. Many consumers,
driven by environmental and health concerns, have
made it clear that they are interested in novel foods such
as PF-derived ingredients [50]. However, consumers are
often also motivated by costs, organoleptic qualities, and
Current Opinion in Food Science 2024, 58:101194
8
Food Bioprocessing
ease of purchase. For PF-derived ingredients to continue
making inroads into ingredient markets and product
formulations; taste, cost, and availability will be the
principal goals of PF-derived ingredient producers.
Data Availability
No data were used for the research described in the ar­
ticle.
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
Acknowledgements
We thank our generous donors to The Good Food Institute. We also thank
Simone Costa for her critical manuscript review.
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