Food synthetic biology-driven protein supply transition:From animal-derived production to microbial fermentation

Yanfeng Liu,Xiaomin Dong,Bin Wang,Rongzhen Tian,Jianghua Li,Long Liu,Guocheng Du,Jian Chen

1 Science Center for Future Foods,Jiangnan University,Wuxi 214122,China

2 Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China

3 Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China

4 National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China

ABSTRACT Animal-derived protein production is one of the major traditional protein supply methods,which continues to face increasing challenges to satisfy global needs due to population growth,augmented individual protein consumption,and aggravated environmental pollution.Thus,ensuring a sustainable protein source is a considerable challenge.The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins,which is an important way to solve the protein supply problem.This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis.Moreover,using artificial bioengineered milk and artificial bioengineered eggs as examples,the progress of food protein supply transition based on synthetic biology has been systematically summarized.Additionally,the future of food synthetic biology as a potential source of protein has been also discussed.By strengthening and innovating the application of food synthetic biology technologies,including genetic engineering and high-throughput screening methods,the current limitations of artificial foods for protein synthesis and production should be addressed.Therefore,the development and industrial production of new food resources should be explored to ensure safe,high-quality,and sustainable global protein supply.

Keywords:Artificial food Biotechnology Food processing Food synthetic biology Protein source Protein supply

1.Introduction

Protein is an essential nutrient for maintaining human life and growth,and is used as an important component of human physiology and biochemistry [1].Thus,the quality and quantity of the ingested protein directly affect human health [2,3].According to the recommendations outlined in the “Dietary Nutrient Intake of Chinese Residents”,the protein intake for adult men and women should be 65 g per day and 55 g per day,respectively.For certain populations,including athletes,elderly,and pregnant women,studies have shown that the optimal daily protein intake should be higher than the amount recommended for the general population,due to its positive effect on their bodies [4].According to the predictions made by the United Nations,the world’s population is expected to grow up to 9.7 billion by 2050,indicating that an additional 260 million tons of protein per year needs to be produced to meet the increasing demands.

To meet such a substantial demand for proteins,it is necessary to increase the supply of protein-rich foods.Plants and animals are the traditional sources of protein,of which animal-derived proteins account for 40% of the total human protein consumption,and this proportion is predicted to increase considerably [5,6].The United Nations Food and Agricultural Organization (FAO)predicts that from 2000 to 2050,global consumption of meat and dairy products will increase by 102% and 82%,respectively,which necessitates the production of an additional 233 million tons of meat and 466 million tons of milk [5].The growth of animals and plants and realization of such a high requirement indicates the input of limited earth resources,such as water and land.Animal husbandry alone accounts for usage of 1/4 of the world’s fresh water and 1/3 of the available land[7,8].Moreover,the production process of animals will have a significant impact on the climate,biodiversity,and other environments.Simultaneously,the process of food transformation,especially animal-derived protein transformation,also include loss of a considerable amount of resources.Considering beef production as an example,the resources consumed to produce 4 g of beef protein may produce 100 g of plant-derived protein [9].Limited natural resources increase difficulty for traditional protein supply methods to meet the population needs.Additionally,the intake of animal protein can cause safety issues,such as bovine spongiform encephalopathy,avian influenza,and residual antibiotics [10].Moreover,there has been an increase in the perception that eating animals is inhumane.All the above-mentioned problems pose challenges to the traditional protein supply methods.Therefore,it is necessary to explore sustainable protein sources to compensate for the traditional protein supply model.

Synthetic biology is a key technology in line with sustainable development that can provide support for global agriculture,medical care,and can alleviate the current environmental burden[11–14].However,the current research on synthetic biology is mainly focused on fields related to chemistry and medicine,while research on food is relatively less [15].Food synthetic biology aims to synthesize food components or nutritional chemicals with renewable biomass as raw materials[16].The use of synthetic food biology to produce proteins can(1)reduce air pollution,energy consumption,and land use area,and (2) reduce animal infectious diseases that may be caused by practices in animal husbandry [17].

In addition to meat,the supply of eggs and milk accounts for a large proportion of the total animal protein supply.The consumption ratio of meat,egg,and milk in China is about 15:5:6.Particularly,according to the Digestible Indispensable Amino Acid Score(DIAAS),proteins in eggs and milk have higher scores,and the amino acid composition/content is similar to that required by humans [18,19].Previous reviews focused mainly on the current status of artificial meat research technology [20–23].This review aimed to address how food synthetic biology could promote the transformation of protein source,as well as the research progress and challenges of artificial bioengineered milk and eggs.

2.Protein Supply Transition

Dietary guidelines recommend a mixed protein diet,including animal-,dairy-,and plant-based foods.Plant proteins usually lack one or more essential amino acids and are not easy to digest,whereas animal proteins are more in line with the needs of the human body,which usually provides all essential amino acids[4].For example,beef is rich in lysine,leucine,and valine;pork contains threonine,phenylalanine,and lysine;while eggs are rich in methionine,phenylalanine,tryptophan,and histidine [24–26].However,the production of animal proteins is generally considered to be highly polluting and unsustainable.First,animal husbandry accounts for 18%of the global greenhouse gas emissions,including carbon dioxide,nitrous oxide,and methane [23,27].Second,compared with the production of 1 g of protein from plants,production of 1 g of animal protein requires more resources and leads to increased emissions;36%of the calories produced by crops worldwide are consumed by livestock,but only 12% of the calories in these feeds ultimately contribute to the human diet(such as meat,eggs,milk,and other animal products)[6,27,28].Since 7 kg of grain is necessary to produce 1 kg of beef,it can be inferred that the conversion process from feed to animal food is not efficient.It is estimated that livestock consume 77 million tons of protein from feed every year,while livestock products only provide 58 million tons of protein for human consumption[27].Third,there are safety issues associated with the use of animal proteins because more than 65%of the human infectious diseases originate from animals and livestock [29].Pathogens such asSalmonellacontained in feces,carcasses,and other wastes pose risks to public health in the process of farming.When humans ingest animal-derived proteins,the food consumed may also have residual antibiotics,which may pose health risks.Therefore,there is an urgent need for new alternative proteins to solve these problems.

In recent years,new protein resources,including algae,insects,and some new plant foods,have emerged (Table 1).Algae from fresh water or salt water are a new source of high-quantity and high-quality proteins [36].The protein content of some chlorella and spirulina can reach 40%–60% of their dry weight,and proteins from these organisms comprise all eight essential amino acids necessary for the human body[37].Insects,such as crickets,scorpions,and tarantulas,which usually use protein waste produced in the food industry as feed,are also a source of high-quality proteins[38].Compared with other animal-based proteins,insect-based proteins consume less energy and utilize less area,which can contribute for reduced burden on the environment and energy loss[39].More recently,researchers have discovered a few plant species that,until now,were not being used as protein sources,such as chickpeas,coconuts,lupin,quinoa,and hemp seeds [35].Compared with the traditional animal protein sources,these foods have high resource benefits and low environmental costs.Nonetheless,there are also difficulties in using them to supply proteins,such as complex protein isolation processes and low acceptance [40].To date,compared with the traditional protein supply sources,algae and insect proteins account for only a small proportion of the total human protein intake,and the pressure on the environment from food production remains a considerable issue.

Additionally,artificial bioengineered foods produced using food synthetic biology technology have also appeared in the market,including artificial bioengineered meat,eggs,and milk[41].Unlike the above-mentioned three protein sources,artificial bioengineered foods are more nutritious and environmentally friendly;they contain precisely designed ingredients and are produced by microorganisms using renewable biomass as raw materials.Moreover,survey data from the United States,India,and China have indicated that the consumers of different ages and genders show preference towards consumption of artificial meat products.Notably,the acceptance of clean meat (also known as lab-grown meat,which is produced by culturing animal cells in a suitable medium)in China and India is significantly higher than that in the United States [42].Particularly,93.2% of the respondents,especially the Chinese respondents said that they might procure clean meat,which also indicated that most consumers were not accustomed to the consumption of artificial foods.Recently,progress has been observed in the production of artificial bioengineered food;hence,timely and systematic discussion and reporting of data is crucial.To date,most reviews have focused on artificial meat,mainly including plant meat analogues and cultured meat [20].Additionally,artificial bioengineered eggs and milk are also viable alternatives to traditional animal-derived proteins,which have not been systematically discussed.Therefore,it is important to discuss the available information on artificial bioengineered eggs and artificial bioengineered milk.

3.Artificial Bioengineered Milk

Milk is an important source of high-quality proteins.Casein and whey protein are the major proteins in milk,accounting for about 76%–88% and 16%–22% of the total protein content,respectively(Table 2) [43–45,49],and their functions and coding genes have been well studied [54].In recent years,the global consumptionof dairy products has increased steadily.According to a survey from the United States Department of Agriculture(USDA),the global milk output was 523 million tons by 2019,which has increased by 0.96% compared to that reported in 2018.Concurrently,global milk consumption was 188 million tons in 2019,which represented an increase of 0.56% from that reported in 2018.However,milk may also cause health issues such as lactose intolerance,cow milk allergy,and hypercholesterolemia [55,56].Further,the concerns of milk hormones and residual antibiotics,different lifestyles of the consumers,the destruction of the environment by animal husbandry,and ethical issues,must be considered and urgently explored [56–61].

Table 1 Sources of protein supply substitutes

Table 2 Concentration and biological functions of major bovine milk proteins

To address the above-mentioned problems,plant-based milk alternatives have been investigated [62,63].They are obtained by extraction of the soluble components of the degraded plant materials via water-based extraction methods,followed by filtration,centrifugation,homogenization,and heating processes [64].Oat,peanut,and almond milk,as well as other plant-based milk are the most recent products on the market [65–67].However,the unbalanced nutritional profile and undesirable sensory flavor of these products limit their consumption [62,68].Nonetheless,the plant-based milk substitutes currently on the market differ considerably in terms of nutritional content,especially for insufficient protein or vitamin content,which may limit their applications.

In recent years,with the rapid development of food synthetic biology,numerous cell factories have been constructed to efficiently synthesize important food components and functional food additives[16,69,70].Synthetic biology has been used to synthesize certain proteins and oligosaccharides additives for milk,such as lactoferrin,human milk oligosaccharide 2′-fucosyllactose and lacto-N-neotetraose [71–78].Compared with the production processes of traditional milk and plant-based milk alternatives,there are many advantages involved in the application of synthetic biology to produce animal-free milk.First,the microbial synthesis of milk components can be performed in a bioreactor to avoid environmental pollution,and antibiotic and hormone contamination caused by traditional methods.Second,the fermentation of cell factories producing milk components can be conducted using a simple medium with raw materials available,such as glucose,soy peptone,corn syrup,urea,and inorganic salts,with a relatively low cost [79].Third,the merit of microbial fermentation is the short cycle,and fermentation is not affected by the environment and weather.Fourth,cell factories can avoid several problems such as inefficient extraction of plant materials,loss of target products,and complicated post-processing protocols.

Currently,the research on milk proteins is mainly focused on the biosynthesis of lactoferrin.Bovine lactoferrin is an antimicrobial agent and immunomodulator that is present in low concentrations in bovine milk[80–82].Thus,construction of a cell factory for its biosynthesis may represent a promising strategy.Kimet al.and Kooet al.successfully expressed bovine lactoferrin C-lobe and N-lobe inRhodococcus erythropolis,and a green algae ofChlorella vulgaris,respectively[83].Isuiet al.expressed and purified a recombinant bovine lactoferrin inEscherichia coliusing a coexpression strategy in which thioredoxin (Trx) and lactoferrin were expressed as a transcriptional fusion protein.The accumulation of recombinant bovine lactoferrin(rbLf)was verified by western blot analysis,and the purified protein was obtained with a concentration of 15.3 mg·L-1and a purity of 90.3%.Moreover,rbLf inhibited the growth ofE.coliBL21(DE3)and Mach1-T1 strains by 87.7% and 79.8%,respectively [84].Bovine lactoferrin was also highly expressed inPichia pastorisby optimization of the codon usage and with selection of a strong promoter,AOX1.The final rbLF expression yield was 3.5 g·L-1by batch fermentation after induction,cell lysis,and purification.In food synthetic biology,the GARS strain,Bacillus subtilis,is an ideal host for protein expression.Jinet al.expressed bovine lactoferrin N-Lobe inB.subtilis168 through promoter optimization and codon engineering [85].The yield of lactoferrin,which was purified by three steps including ammonia sulfate precipitation,Ni-NTA affinity chromatography,and Superdex 200 chromatography,was 16.5 mg·L-1with a purity of 93.6%.Eventually,the researchers verified the desired antibacterial activity againstE.coliJM109,Pseudomonas aeruginosa,andStaphylococcus aureus.

In addition to lactoferrin,there are few studies on the production of other major milk proteins by synthetic biology.In 2014,Perfect Day conducted a research on the production of artificial bioengineered milk (https://www.perfectdayfoods.com/learnmore/#background).The core technology involved the introduction of the DNA sequence of milk protein into yeast cells and production of casein and whey protein through fermentation.Thereafter,the milk proteins were mixed with water and other ingredients to produce dairy substitutesi.e.,artificial bioengineered milk.The development of artificial bioengineered milk prompts the production of milk based on the technology of food synthetic biology.However,the above-mentioned studies were focused on laboratory-scale research of a single component in milk protein.There are many problems and challenges which should be overcome to realize the industrial production of artificial bioengineered milk.Thus,further research is warranted.

4.Artificial Bioengineered Egg

According to the FAO data,eggs are one of the main sources of human protein intake,with an output of more than 82.86 million tons worldwide in 2018.The protein content of egg white varies from 9.93%to 10.71%,and the protein content of yolk ranges from 16.28% to 17.85% [86,87].Overall,80% of the egg white is composed of four proteins,namely ovalbumin (54%),ovotransferrin(12%),ovomucin (11%),and lysozyme (3.4%),while the egg yolk is a complex homogeneous system composed of water,lipids,and lipoproteins(Table 3)[88,89].However,use of eggs as a traditional protein source poses multiple challenges.First,from a nutritional perspective,eggs have a considerable amount of cholesterol that can contribute towards the risk of developing cardiovascular diseases.Although the cholesterol limit was removed from the 2015–2020 Dietary Guidelines for Americans,the current nutritional viewpoint continues to recommend that the elderly or patients with heart disease should reduce their cholesterol intake[90–93].Second,eggs contain a variety of allergenic ingredients that can cause IgE-mediated food hypersensitivity,which is one of the most common food allergies in both adults and children[94].Third,there are many unstable factors in the egg production process.Through the analysis of the total chemical composition of eggs,it has been found that the quality of eggs is affected by many factors such as the chicken genotype (breed,line,hybrid),age,housing systems,and feeding,among others[86,87].Although this situation is substantially alleviated in the modern egg production industrial system,environmental pollution also plagues the egg production industry.The poultry farming industry is also an industry with high energy consumption and pollutant emissions.According to FAO,an average of 2.3 kg of dry matter feed is necessary to produce 1 kg of eggs.Moreover,the protein efficiency of eggs is 25%,which implies that 25% of the protein in the chicken feed inputs is effectively converted to egg products and the remaining 75%would be lost during conversion.This is an evident waste of matter and energy when the global food supply faces scarcity.Additionally,the egg production industry also faces constraints due to other factors.For example,animal welfare is regulated by the “European Convention for the Protection of Animals kept for Farming Purposes”which stipulates the basic conditions of poultry breeding,such as minimal space,maximal number of hens,and furniture [86,87].

Subject to the various constraints of the egg industry,and dietary restrictions for special consumers with religious issues,health issues,and personal lifestyle choices,a variety of egg substitutes from other protein sources,especially vegetable proteins,have been tested and developed.In the earliest research,the eggs in yellow cakes were completely replaced with white lupine protein,but it was necessary to add emulsifiers and xanthan gum to improve the hardness,moisture content,volume,and shape characteristics of the cake [95].Subsequently,gluten,soy milk,and soybean protein isolates were also tested to replace egg protein [96,97].However,the use of alternative proteins is often unsuccessful,and additional ingredients,such as polysaccharide hydrocolloids or emulsifiers,are used to produce egg products of good quality.This indicates that the substituted vegetable protein possesses a different function compared to egg white protein.Egg white protein has a variety of properties that vegetable proteins usually do not have,such as good foaming capacity,emulsification,stabilization,and elasticity.A recent study found that Aquafaba(AQ),a viscous water produced during cooking of chickpea or other legumes,has theability to form foams and emulsions,which enables it to be used as an egg protein substitute [98].However,the emulsifying ability and stability of AQ produced by different chickpea cultivars are significantly different,and the cooking and storage processes also affect its physicochemical properties and hydration characteristics[99].Furthermore,considering the various advantages of plant proteins,some plant-based artificial eggs made from legume proteins,such as plant protein liquid products made from mung bean protein,have also appeared on the market.However,the price of vegetable eggs is usually more than 10 times that of ordinary eggs,which is not well accepted by most consumers.These studies show that the use of vegetable protein egg substitutes also has problems such as poor product stability and complex technology,and plantderived proteins may also have unreported allergens.Thus,a new and safer egg substitute production method is warranted.

Table 3 Concentration and biological functions of major egg white protein components

In the exploration of egg substitutes,whey protein concentrate proved to be a good egg substitute[100].In the baking substitution experiments of biscuits and cakes,parameters such as physicochemical,color,textural,microbial,and sensory attributes were all within acceptable ranges,indicating that the compound ovalbumin was a potential egg substitute [101].Simultaneously,consumers continue to choose egg white products with relatively more protein and less carbohydrates.Therefore,the production and compounding of egg white proteins may be an effective solution.Food synthetic biology provides the most potential solution for the artificial synthesis of egg white proteins,such as ovalbumin,the most abundant protein in egg white.In previous studies,various microorganisms have been used to produce ovalbumin [102].As early as 1978,the synthesis and extracellular secretion of 43,000 daltons of chicken ovalbumin byE.coliwas achieved through genetic engineering [103].In recent studies,correctly folded ovalbumin has also been artificially synthesized byE.coliandB.subtilis[104–106].Although ovalbumin synthesized by microorganisms is essentially free from post-translational modifications,it continues to exhibit similar antigenicity and biological activity to ovalbumin from natural eggs.Additionally,there have been companies dedicated to the microbial production of egg whites.For example,Clara Foods has attempted to produce a combination of two or more egg white proteins in a variety of microorganisms,such asSaccharomyces cerevisiaeandB.subtilis.These attempts show that food synthetic biology can pave the way for the synthesis of the main egg white protein by microorganisms and through compounding,and for artificial production of egg substitutes with similar functional properties (including solubility,water binding/absorption,viscosity,gelation,cohesion,adhesion elasticity,emulsification,and foaming).

5.Challenges and Perspectives

Food synthetic biology,an important research direction in the field of food production,provides important technical support to solve the challenges of food manufacturing.Biomanufacturing research,represented by the protein components in artificial bioengineered milk and eggs,has made progress.However,there are two persistent common challenges in the artificial bioengineered food production process (Fig.1).First,the expression of a single protein by a microbial host component is insufficient,and low productivity increases the cost of protein acquisition.Second,obtaining artificial bioengineered foods with a certain nutritional ratio requires complex protein purification and compounding processes,which increase production costs and limit the industrialized protein production strategy.

Fig.1.Challenges and potential solutions for production of microbial-derived proteins.

To address the first above-mentioned challenge,microbial chassis cells with high specific growth rates and high productivity can be constructed to improve protein expression.For example,protein synthesis efficiency can be enhanced by optimizing gene expression regulatory elements such as promoters,ribosome-binding sites,N-terminal coding sequences,and signal peptide elements to achieve efficient secretory expression [107].Additionally,a high-throughput and high-sensitivity screening method is also a powerful tool for screening high-efficiency chassis cell factories.To address the second described challenge,two types of potential protein-producing strategies may be developed,namely “singlehost multi-protein”and “multi-host multi-protein”(Fig.1).For example,to synthesize the protein components in artificial bioengineered milk,the“single-host multi-protein”strategy indicates that a single cell factory can synthesize four major types of proteins including αS2-casein,β-casein,κ-casein,and α-lactalbumin at the same time without producing the major allergens αS1-casein and β-lactoglobulin[108,109].Meanwhile,a certain proportion of the target product can be finally obtained by regulating the expression of different genes.The “multi-bacteria multi-protein”strategy indicates that different types of proteins can be obtained simultaneously through co-cultivation and fermentation of several cell factories.For the synthesis of milk protein,one cell factory can express one type or several types of milk proteins simultaneously,and then different cell factories can be used as co-culture and coferment systems to express multiple protein components.This approach can also directly express certain protein ratios by regulating the growth rate and by optimizing the gene expression elements of different strains,and artificial bioengineered products can be obtained without compounding.Microbial fermented proteins have higher production efficiency compared with plant-derived proteins and require less land than that required for the production of animal-derived proteins.Moreover,microbial fermented proteins require less raw materials due to their high conversion efficiency,and the equipment costs of the production process are inexpensive.Based on this,microbial fermented proteins have the potential to achieve lower production costs.Thus,the proposal of microbial chassis cells with high-efficiency protein expression and microflora with different protein production strategies provides considerable potential for realizing whole-cell utilization and industrial-scale production of food proteins.

Additionally,considering artificial bioengineered food,safety issues should also be noted.In general,the microorganisms used in food production are recognized as safe(GRAS)strains,including includeB.subtilis,Corynebacterium glutamicum,and Lactobacillus,thereby implying that these strains have been commonly consumed by a large number of people since a long time [110,111].The nutraceuticals produced by them have been widely used in food,medicine,and other fields.Moreover,purification of the protein produced by the engineered strain is necessary before it can be used as a food component,which further ensures the safety of the raw material.Therefore,the microbial fermented protein that has passed the food safety test,with approval from the respective regulatory bodies,can be mass-produced and safely consumed.

In summary,food synthetic biology is one of the main methods for boosting the future protein supply transition.Therefore,it is necessary to encourage and increase the development and application of food biotechnology,such as food synthetic biology,to lead industrialization,and to seize the forefront of the world’s technological frontiers and industrial highlands,ultimately benefiting mankind and the environment.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFA0900300),National Natural Science Foundation of China (31972854,21676119),Natural Science Foundation of Jiangsu Province (BK20200085),Key Research and Development Program of Jiangsu Province(BE2019628),Fundamental Research Funds for the Central Universities (JUSRP22036,JUSRP52020A),and the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-16).

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.