Biomanufacturing has evolved into four successive generations underpinned by microbiology, genetic engineering and synthetic biology. Generation 1.0 utilised pure‑culture and anaerobic fermentation to produce primary metabolites, suffering from scale‑up barriers, food‑feedstock dependence and poor automation. Triggered by penicillin industrialisation, Generation 2.0 deployed mutagenesis and aerobic submerged fermentation for large‑scale secondary‑metabolite antibiotic production. Centred on recombinant‑DNA technology from the 1980s, Generation 3.0 achieved heterologous synthesis of complex therapeutic proteins and industrial enzymes through targeted host‑cell engineering. Generation 4.0 marks the AI‑integrated synthetic‑biology era. Leveraging the iterative Design‑Build‑Test‑Learn (DBTL) cycle, rationally redesigned biological systems convert renewable carbon feedstocks into pharmaceuticals, chemicals, biofuels and biomaterials, as demonstrated by yeast‑based artemisinin biosynthesis. This review outlines technical features, milestone cases and limitations of each stage, highlighting how technological innovation drives sustainable biomanufacturing to address global energy, environmental and public‑health challenges.
Biomanufacturing 1.0: Monoculture Technology and Conventional Anaerobic Fermentation
In 1854, German scientist Robert Koch proposed the concept of microbial pure culture, arguing that microorganisms must be isolated from complex mixed microbial communities for research. He subsequently developed the tissue‑section method and introduced agar into culture media to produce solidified media, enabling the isolation and cultivation of single colonies. This marked a major breakthrough in microbial pure‑culture technology, allowing microbiologists to cultivate individual colonies. These technological advances ushered conventional biomanufacturing into the Biomanufacturing 1.0 era.
Biomanufacturing 1.0 originated in the 1910s with Chaim Weizmann’s discovery of acetone‑butanol‑ethanol (ABE) fermentation. Two core features defined this phase: the replacement of mixed‑culture systems with purified monocultured microorganisms, and large‑scale anaerobic liquid‑phase fermentation. Microorganisms functioned as “micro‑factories”, converting simple carbon sources such as sugars and starch into target products including ethanol, acetone, butanol, amino acids and organic acids — primary metabolites.
During World War II, acetone served as a critical chemical feedstock for explosive production. Wartime chemical shortages prompted the British Government to establish the Solvent Committee to identify alternative manufacturing routes. Weizmann discovered that Clostridium acetobutylicum could produce acetone, butanol and ethanol under anaerobic conditions via ABE fermentation. Adopted by the British Government, this process supported the construction of multiple acetone‑production facilities to satisfy wartime demand. Post‑war continuous improvements boosted process yield and productivity, delivering key feedstocks for the chemical‑processing industry.
In 1956, Japanese scientist Shukuro Kinoshita isolated Corynebacterium glutamicum. One year later, he pioneered glutamate production via fermentation using this strain, opening a new chapter for microbial‑based amino‑acid manufacturing. Monosodium glutamate, the highest‑volume amino‑acid product, transitioned from extraction via acid hydrolysis of plant proteins and chemical synthesis to microbial fermentation. Featuring low cost and environmental friendliness, fermentation displaced competing technologies across glutamate manufacturers. Today the global amino‑acid market exceeds USD 20 billion annually; lysine is also produced by microbial fermentation. Major amino‑acid producers are located in China, Japan, the Republic of Korea, the United States and Europe.
Case Study: Biosynthesis of Acetone and Butanol
In 1912, Weizmann identified Clostridium acetobutylicum, a bacterium capable of fermenting starch and glucose into acetone, butanol and ethanol under oxygen‑free conditions. This bioprocess is known as ABE fermentation, or the Weizmann process.
The outbreak of the First World War created urgent demand for key chemical feedstocks. Weizmann’s findings attracted attention from the British Government, which rapidly deployed the technology and built multiple acetone‑production plants throughout the war. Facilities utilised carbohydrate‑rich biomass feedstocks such as sweet potato, corn, cassava and bagasse. Grains or cassava were milled and mixed with water to prepare fermentation broths, which were sterilised at high temperature and cooled before inoculation with purified Clostridium acetobutylicum. Anaerobic fermentation proceeded, followed by distillation to recover a product mixture typically comprising acetone, butanol and ethanol in a 3:6:1 ratio.
Wartime scale‑up enabled an annual acetone output of 30 000 tonnes. Concurrent n‑butanol generation reached twice the acetone titre, second only to ethanol among major industrial bioproducts of that era. Surplus n‑butanol created new market opportunities as a substitute for amyl acetate, finding broad application in solvents, plasticisers, paints and resin manufacturing.
Modern‑day ABE‑fermentation R&D refocuses on n‑butanol, a viable drop‑in biofuel miscible with petroleum‑derived fuels at any blending ratio. This historic fermentation technology has regained industrial relevance.
Case Study: Industrialisation Journey of Citric‑Acid Production
Citric acid is a ubiquitous organic acid widely deployed across food‑beverage, pharmaceutical and other sectors. Its industrial production dates back to the late 19th century.
Early explorations In 1893, German scientist Wehmer first observed citric‑acid formation by Penicillium species using sugar‑based substrates. In 1913, B. Zahorski documented citric‑acid biosynthesis by Aspergillus niger. In 1916, Thom and Currie experimentally confirmed citric‑acid production across multiple Aspergillus strains, with Aspergillus niger exhibiting the highest acid‑synthesis capacity.
Impact of World War I Wartime shortages of imported calcium citrate from Italy drove Pfizer to implement fungal fermentation for citric‑acid synthesis from sugars in 1919, resolving supply constraints. In 1923, Pfizer constructed the world’s first plant for citric‑acid manufacture via Aspergillus niger surface‑culture fermentation. Subsequent production capacity was built in Belgium, the United Kingdom, Germany and the Soviet Union. Fermentation progressively displaced traditional citrus‑fruit‑based extraction of natural citric acid.
Evolution of fermentation technologies Prior to the 1950s, citric acid was predominantly manufactured by surface‑culture fermentation, which suffered from long cycle times and low yields. In 1952, Miles Laboratories in the United States commercialised large‑scale citric‑acid production via submerged fermentation, superseding surface‑culture workflows. Submerged fermentation delivers shorter cycle times, elevated yields, reduced labour input, smaller footprint and improved process controllability for continuous manufacturing. Aspergillus niger is cultivated in sucrose‑ or glucose‑rich media. Post‑fermentation, calcium hydroxide is added to precipitate calcium citrate. Sulphuric‑acid treatment of isolated calcium citrate yields purified citric acid.
Citric‑acid production is now a mature industrial discipline. Its progression from laboratory discovery to full‑scale manufacturing exemplifies continuous innovation in microbial‑fermentation engineering.
Biomanufacturing 1.0 represents an experimental era centred on conventional fermentation and pure‑culture microorganisms for biobased fuels and chemical feedstocks. Nevertheless, multiple bottlenecks emerged.
First, substantial technical barriers existed during scale‑up from lab‑scale trials through pilot‑scale testing to full‑scale production. Even with promising laboratory performance, reactor‑scale‑up impairs heat‑ and mass‑transfer efficiency within large bioreactors. Precise control of fermentation parameters becomes difficult, generating significant performance gaps between laboratory and manufacturing scales.
Second, Biomanufacturing 1.0 relied heavily on food‑based feedstocks. Rising market demand created feed‑supply constraints that limited production capacity.
Furthermore, low‑level automation and digitalisation constrained data processing and process regulation, compromising product quality and batch‑to‑batch consistency. These limitations hindered commercial deployment and necessitated technical advancement toward subsequent biomanufacturing generations.
Biomanufacturing 2.0: Specialised Mutant Strains and Aerobic Fermentation
Biomanufacturing 2.0 was inaugurated by penicillin fermentation during World War II. A defining shift occurred: biomanufacturing expanded beyond primary‑metabolite synthesis toward complex secondary metabolites. These secondary metabolites evolved as microbial defence molecules, repurposed by humans for pharmaceuticals, flavourings and aromatics.
Two technologies, conceived before this era but widely adopted in Biomanufacturing 2.0, underpinned progress: specialised mutant‑strain development and aerobic submerged‑fermentation.
The emergence of antibiotics, epitomised by penicillin, marked a pivotal turning point. Prior to World War II, sulphonamides provided limited antibacterial efficacy. In 1928, Alexander Fleming discovered penicillin, a potent antibacterial agent active against diverse bacterial pathogens.
To enable large‑scale penicillin production, Merck & Co. deployed aerobic submerged‑fermentation technology in 1942. This technology dramatically boosted productivity. Extensive R&D optimised culture media, bioreactor design, high‑yield strain screening, oxygen supply and growth‑regulation strategies. These advances reduced penicillin production costs and laid foundational infrastructure for Biomanufacturing 2.0.
Case Study: Discovery of Penicillin
The discovery of penicillin constitutes a landmark serendipitous scientific achievement. In 1921, Alexander Fleming (1881‑1955) identified lysozyme, a bactericidal substance, although this compound was not penicillin.
In 1928, returning from vacation, Fleming observed green‑coloured mould growing on a Staphylococcus aureus agar plate, with complete bacterial inhibition surrounding the mould colony. Filtrates from the mould culture killed Staphylococcus aureus within hours even at 800‑fold dilution. Repeated experimentation confirmed that metabolites secreted by Penicillium exerted antibacterial activity; Fleming named this substance penicillin.
Fleming’s original report initially attracted limited medical attention. In 1938, Howard Florey and Ernst Chain commenced research at the University of Oxford to characterise penicillin and develop scalable production routes, representing the “rediscovery” of penicillin. In 1942, United‑States‑based pharmaceutical manufacturers launched large‑scale penicillin production. Penicillin substantially reduced wartime mortality from bacterial infections and contributed to Allied wartime victory. Fleming, Florey and Chain were jointly awarded the 1945 Nobel Prize in Physiology or Medicine for the discovery and therapeutic realisation of penicillin.
Penicillin discovery catalysed Biomanufacturing 2.0 and accelerated the maturation of industrial submerged‑liquid‑fermentation platforms. Adequate aeration and agitation delivered sufficient dissolved oxygen to sustain robust microbial metabolism.
Mutagenesis technology emerged to introduce targeted alterations to microbial DNA, either spontaneously or induced by ultraviolet irradiation, chemical mutagens such as nitrosoguanidine, or cobalt‑60 gamma‑ray exposure. Mutagenesis enabled strain engineering for enhanced production performance.
Mutagenic screening delivered multiple antibiotics including tetracycline and streptomycin. For instance, Streptomyces aureofaciens yielded low native tetracycline titres. Ultraviolet‑mutagenesis programmes isolated high‑producing mutant strains that drastically elevated titres and lowered costs, establishing tetracycline as a mainstream antibiotic. Iterative X‑ray‑ and UV‑mutagenesis campaigns substantially improved penicillin yields in production strains.
Biomanufacturing 2.0 transitioned from Biomanufacturing 1.0’s anaerobic monoculture workflows to aerobic fermentation. Rational strain breeding improved product titres, enhanced quality, diversified product portfolios and simplified manufacturing workflows. Submerged‑fermentation and continuous‑fermentation technologies advanced alongside bioreactor hardware. Production evolved from manual small‑batch operations to full‑scale industrial manufacturing. Optimised microbial growth and metabolic pathways drove higher yields and efficiencies for multiple antibiotic classes, realising genuine large‑scale biomanufacturing.
Biomanufacturing 3.0: Gene‑technology‑empowered Cell Culture
Emerging in the 1980s, Biomanufacturing 3.0 focused on large‑protein production, predominantly protein therapeutics and industrial enzymes. Two foundational breakthroughs drove this era: recombinant‑DNA technology and advanced cell‑culture systems. Recombinant‑DNA technology represents the core of Biomanufacturing 3.0: defined gene sequences are transferred across species to express target proteins and biomolecules in heterologous host cells. Microorganisms serve as expression hosts for diverse heterologous genes to generate vaccines, antibodies, interferons, insulin, hormones and other complex compounds. Metabolic‑regulation theory accelerated industrial translation by redirecting microbial carbon flux toward accumulation of target metabolites.
Biomanufacturing 3.0 enabled rational reprogramming of organismal genetics, overcoming limitations of natural screening to support customised cellular design and construction.
In 1972, Stanford University scientist Paul Berg (1926‑2023) deployed restriction endonucleases (“molecular scissors”) to cleave and re‑ligate DNA fragments from disparate organisms, generating chimeric recombinant‑DNA molecules. This procedure established the fundamentals of modern genetic engineering. Recombinant‑DNA technology ranks among the most transformative achievements in modern molecular biotechnology.
In 1973, Stanley Cohen and Herbert Boyer constructed the first self‑replicating chimeric plasmid assembled from heterologous DNA fragments. Recombinant‑DNA technology enabled biosynthesis of complex proteins inaccessible or prohibitively expensive via chemical synthesis. In 1976, venture capitalist Robert Swanson co‑founded Genentech with Herbert Boyer to produce recombinant insulin and human growth hormone.
Approximately 40 % of therapeutic proteins are non‑glycosylated and expressed in hosts such as Escherichia coli or baker’s yeast.
Amgen was founded in 1980 to develop recombinant human erythropoietin (EPO), a hormone that stimulates erythrocyte biosynthesis. EPO requires site‑specific glycosylation for biological activity; bacterial or yeast hosts cannot recapitulate authentic human glycosylation patterns, necessitating mammalian‑cell expression systems. Glycosylation patterns directly determine EPO bioactivity. In 1988, published studies demonstrated variable biological activity for EPO produced in different mammalian‑cell lines. Daniel I. C. Wang developed key methodologies for low‑cost, consistent‑quality glycoprotein manufacturing using mammalian cell culture, advancing the field substantially.
Glycoproteins constitute a major product class for oncology antibody therapeutics, including antibody‑drug conjugates such as Adcetris and Kadcyla indicated for selected malignancies.
Beyond protein therapeutics, microorganisms produce diverse industrial enzymes that catalyse biotransformations. High‑cell‑density fermentation supports high‑level recombinant‑enzyme expression.
New England Biolabs was founded in 1974 to supply recombinant enzymes including DNA polymerases for molecular‑biology research. Today suppliers including Bio‑Rad, Takara, Sigma‑Aldrich and Thermo Fisher Scientific provide comprehensive enzyme portfolios for research workflows.
Enzyme‑based biocatalysis emerged as an industrially‑scalable, environmentally‑friendly alternative to chemical synthesis. Natural enzymes frequently exhibit insufficient stability for process conditions. Immobilisation technology mitigates this limitation: enzymes are physically or chemically anchored to solid supports to enhance stability, enable catalyst recycling and improve volumetric productivity.
Novozymes and Genencor, established in the 1980s, manufactured bulk industrial enzymes for food processing (amylases, glucoamylases, glucose isomerase, lipases), textiles (cellulases, hemicellulases), detergents (amylases, lipases, proteases) and fine‑chemical synthesis (racemases, ketoreductases, dehydrogenases).
Protein‑engineering technology enables rational modification of protein structure‑function relationships. Targeted amino‑acid alterations re‑engineer enzyme properties to produce rare chemical species inaccessible via conventional synthetic routes.
Case Study: Recombinant Human Insulin
In 1974, Genentech inserted synthetic genes encoding insulin A‑ and B‑chains into separate Escherichia coli plasmids and transformed the constructs into bacterial hosts. Recombinant plasmids replicated and expressed the respective insulin chains. Isolated A‑ and B‑chains were combined in‑vitro via disulphide‑bond formation to yield biologically‑active human insulin.
Recombinant‑DNA technology launched the biosynthetic‑insulin era in the 1980s. Novo Nordisk engineered biosynthetic insulin with amino‑acid sequences identical to native human insulin. Recombinant human insulin delivers high purity, potent hypoglycaemic activity, low immunogenicity, minimal drug‑resistance risk and reduced adverse‑event profiles, establishing a new paradigm for industrial‑scale insulin production.
These technological advances expanded biomanufacturing capabilities beyond simple metabolites toward high‑value pharmaceuticals and fine chemicals.
Biomanufacturing 4.0: Advances in Synthetic Biology and Artificial Intelligence
Biomanufacturing 4.0 applies engineering principles to biology for precise design and reprogramming of biological systems. In 2000, synthetic biology was formally defined as a discipline with landmark publications. Analogous to assembling building blocks, synthetic biology enables rational design and construction of novel biological systems. Renewable carbon feedstocks including starch, cellulose and carbon dioxide are converted into chemicals, pharmaceuticals, foods, biofuels and biomaterials via synthetic‑biology workflows, delivering clean, efficient and sustainable manufacturing.
Genomics revolutions, coupled with advances in computational, quantitative and systems biology, underpinned the emergence of synthetic biology. High‑throughput sequencing and analytical technologies automated the characterisation and functional prediction of biological parts and genetic circuits, accelerating development cycles. Synthetic biology integrates biology, engineering, physics, chemistry and computer science. Its core objective is the engineering‑driven reprogramming or de‑novo synthesis of biological systems. Custom‑engineered cells are deployed for large‑scale production of target compounds, therapeutics and functional materials.
Synthetic‑biology platforms act as a powerful engine for Biomanufacturing 4.0, enabling novel organisms and innovative products including functional tissues and new medicines, transforming pharmaceutical, food‑supply, energy‑material and agricultural industries.
Biomanufacturing 4.0 unfolded in four developmental phases:
1.Foundational Phase (2000‑2003) Scientists applied engineering paradigms to biological‑system design. Genetic‑circuit engineering emerged as a key discipline. Genetic circuits consist of biological parts (promoters, transcription factors, regulatory elements) forming regulatory networks analogous to electronic circuits, implementing genetic switches and control modules inside microbial hosts. These circuits are deployed in metabolic engineering to construct efficient intracellular biosynthetic pathways.
2.Expansion Phase (2004‑2007) The inaugural Synthetic Biology 1.0 conference convened at the Massachusetts Institute of Technology in 2004, marking a major milestone. Synthetic biology was formally recognised as an independent discipline with defined research priorities. The conference fostered international collaboration and gave rise to the iGEM competition, advancing synthetic‑biology education and outreach.
3.Innovation‑and‑Translation Phase (2008‑2013) Novel engineering tools emerged. CRISPR‑Cas9 genome‑editing technology delivered precise, programmable modification of genomic DNA sequences.
4.Comprehensive‑Advancement Phase (2014‑present) Synthetic‑biology research expanded rapidly. In 2014, researchers demonstrated stable replication of unnatural DNA base pairs inside bacterial cells. In 2015, groups at Harvard and Yale Universities achieved artificial amino‑acid biosynthesis. In 2014, multinational teams computationally redesigned and synthesised Saccharomyces cerevisiae chromosome III, pioneering whole‑eukaryotic‑genome re‑engineering to support microbial production of rare pharmaceuticals and biofuels.
Core Concepts of Biomanufacturing 4.0
DBTL Cycle: The Design‑Build‑Test‑Learn iterative workflow constitutes a core operating paradigm. Biological systems are computationally designed, physically assembled, experimentally characterised, and datasets are analysed to inform iterative redesign, continuously optimising biosynthetic performance.
Artificial‑Intelligence Augmentation: Advanced computing and artificial‑intelligence tools constitute a powerful analytical backbone. AI interprets massive biological datasets to accelerate target‑molecule discovery and streamline biomanufacturing workflows.
Technological Breakthroughs: Biomanufacturing 4.0 transcends limits of traditional fermentation. Instead of screening naturally‑occurring strains, researchers rationally design custom‑built biological systems, drastically expanding the product scope of biomanufacturing.
Strategic Mission: Biomanufacturing 4.0 addresses global challenges including energy scarcity, environmental degradation, food‑security risks and disease burden. Pursuing high‑quality, high‑efficiency and sustainable manufacturing, it drives transformation across the bioeconomy.
Case Study: Biosynthesis of Artemisinin
Artemisinin serves as a frontline therapeutic agent against malaria caused by Plasmodium parasites. Chinese scientists isolated artemisinin from Artemisia annua in the 1970s. Tu Youyou was awarded the 2015 Nobel Prize in Physiology or Medicine for this discovery.
Scientists characterised the artemisinin biosynthetic pathway. Transgenic approaches enhanced native artemisinin yields in Artemisia annua. More significantly, Jay D. Keasling’s research team at the University of California, Berkeley reconstituted the artemisinin biosynthetic pathway in Saccharomyces cerevisiae, publishing their landmark work in Nature in 2013. Plant‑derived artemisinin‑biosynthesis genes were heterologously expressed in yeast to construct microbial cell factories. Amyris subsequently industrialised semi‑synthetic artemisinin production, representing one of the most successful examples of heterologous biosynthesis of high‑value compounds.
Multiple optimisation rounds improved strain performance. In 2006, Royster’s group amplified farnesyl pyrophosphate (FPP) precursor supply to enhance pathway flux. In 2012, Westfall’s team deployed more robust yeast chassis strains and identified amorpha‑4,11‑diene‑to‑artemisinic‑acid conversion as the principal metabolic bottleneck. In 2013, Keasling’s laboratory introduced three plant‑origin reductases and implemented two‑phase fermentation, elevating artemisinic‑acid titres to 25 g/L. Optimised yeast‑cell‑factory performance strengthened global malaria‑treatment supply chains.
