J Ind Microbiol Biotechnol (2017) 44:773–784
DOI 10.1007/s10295-016-1863-2
BIOENERGY/BIOFUELS/BIOCHEMICALS - REVIEW
Biomanufacturing: history and perspective
Yi‑Heng Percival Zhang1,2   · Jibin Sun1 · Yanhe Ma1 
Received: 14 June 2016 / Accepted: 30 October 2016 / Published online: 11 November 2016
© Society for Industrial Microbiology and Biotechnology 2016
Abstract Biomanufacturing is a type of manufactur-
ing that utilizes biological systems (e.g., living microor-
ganisms, resting cells, animal cells, plant cells, tissues,
enzymes, or in vitro synthetic (enzymatic) systems) to pro-
duce commercially important biomolecules for use in the
agricultural, food, material, energy, and pharmaceutical
industries. History of biomanufacturing could be classi-
fied into the three revolutions in terms of respective product
types (mainly), production platforms, and research technol-
ogies. Biomanufacturing 1.0 focuses on the production of
primary metabolites (e.g., butanol, acetone, ethanol, citric
acid) by using mono-culture fermentation; biomanufactur-
ing 2.0 focuses on the production of secondary metabolites
(e.g., penicillin, streptomycin) by using a dedicated mutant
and aerobic submerged liquid fermentation; and biomanu-
facturing 3.0 focuses on the production of large-size bio-
molecules—proteins and enzymes (e.g., erythropoietin,
insulin, growth hormone, amylase, DNA polymerase) by
using recombinant DNA technology and advanced cell
culture. Biomanufacturing 4.0 could focus on new prod-
ucts, for example, human tissues or cells made by regen-
erative medicine, artificial starch made by in vitro synthetic
Tribute to Arny Demain, Industrial Microbiologist Extraordinaire
Celebration of the 90th birthday of Arnold Demain.
* Yi‑Heng Percival Zhang
zhang_yh@tib.cas.cn
* Yanhe Ma
ma_yh@tib.cas.cn
1 Although biomanufacturing has played a relatively
Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Science, 32 West 7th Avenue, Tianjin Airport
Economic Area, Tianjin 300308, China
2
Biological Systems Engineering Department, Virginia Tech,
304 Seitz Hall, Blacksburg, VA 24061, USA
biosystems, isobutanol fermented by metabolic engineer-
ing, and synthetic biology-driven microorganisms, as well
as exiting products produced by far better approaches. Bio-
manufacturing 4.0 would help address some of the most
important challenges of humankind, such as food security,
energy security and sustainability, water crisis, climate
change, health issues, and conflict related to the energy,
food, and water nexus.
Keywords  Advanced biomanufacturing ·
Biomanufacturing 4.0 · Bioeconomy · In vitro synthetic
biosystem · Metabolic engineering and synthetic biology ·
Sustainability revolution
Introduction
Biomanufacturing is a type of manufacturing that utilizes
biological systems (e.g., living microorganisms, resting
cells, plants, animals, tissues, enzymes, or in vitro synthetic
(enzymatic) systems) to produce commercially impor-
tant value-added biomolecules for use in the agricultural,
food, energy, material, and pharmaceutical industries [37].
Its products may also be isolated from natural sources,
such as blood, cultures of microbes, animal cells, or plant
cells grown in specialized equipment or dedicated cultiva-
tion environments. The cells/tissues or enzymes used may
be natural or modified by genetic engineering, metabolic
engineering, synthetic biology, and protein engineering
[62].
important role in the past three industrial revolutions, bio-
manufacturing 4.0 (or advanced biomanufacturing) will
become one of the most important cornerstones of the sus-
tainability revolution happening in the twenty-first century
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774 J Ind Microbiol Biotechnol (2017) 44:773–784

Table 1  Classification of biomanufacturing revolutions and representative products

Biomanufacturing Starting time Representative product Production platform Research tool
revolution

Pre-modern Several thousand Beer, cheese, soy sauce, wine, Mixed microorganism Solid state fermentation
Biomanufacturing years ago bread, steamed bun, pickles, cultures
vinegar
Biomanufacturing 1.0 1910s #1 metabolites Mono-culture bacteria or Anaerobic liquid
Acetone, fungi fermentation
n-Butanol, Aerobic submerged
Glycerol, fermentation
Amino acids,
Organic acids
Vitamin C
Biomanufacturing 2.0 1940s #2. metabolites Mutated fungi or bacteria Aerobic submerged
Penicillin, fermentation
Tetracycline,
Streptomycin
Biomanufacturing 3.0 1980s # protein drugs GM (animal) cell cultures Recombinant DNA
Erythropoietin, GM microorganisms Cell culture
Insulin, Enzymatic catalysis
Growth hormone
# Enzymes
Amylase,
Protease,
Cellulase
Biomanufacturing 4.0 2000s # Translational research Embryonic stem cells Tissue engineering & stem
Human cells, tissues, or organs Induced pluripotent stem cells
# Renewable energy cells (iPSC) Metabolic engineering and
Isobutanol Engineered microorgan- synthetic biology
Hydrogen isms Advanced protein
# New food In vitro synthetic (enzy- engineering
Artificial starch matic) biosystems

GM genetically modified

[70, 74]. The past three industrial (technology) revolutions
are (1) the first industrial revolution that began in Britain in
the late eighteenth century, with representative examples of
the mechanization of the textile industry powered by steam
engines and coal mining; (2) the second industrial revolu-
tion that came in the early twentieth century in USA, with
representative examples of wide use of internal combus-
tion engines, liquid (fossil) fuels, electrification, mass pro-
duction based on moving assembly lines; and (3) the third
industrial revolution, with representative examples of wide
use of computers and internet. In this century, manufactur-
ing is going digital, called industry 4.0 [53]. It is expected
that the fourth industrial revolution may focus on customi-
zation production by using clever software, artificial intel-
ligence, novel materials, more dexterous robots, new pro-
cesses (notably three-dimensional printing), and a whole
range of web-based services. As compared to Industry 4.0,
biomanufacturing 4.0 would become an enabling platform
to produce new products or existing products in far better
ways than current technologies.
Most technological innovations are incremental in
nature, that is, improving existing technologies (extended
technologies) for meeting current market needs or extended
markets, but disruptive innovations can drive rapid and
adaptive change in terms of new market and value net-
work and eventually disrupt an existing market and dis-
place established market leaders and alliances (Table 1)
[20]. Such rare innovations are being driven by paradigm-
shifting concept or theory, novel research tools, and game-
changing production methods, as evidenced in Industrial
Revolutions. In this context, the introduction of disruptive
biomanufacturing technologies often provide some compa-
nies with a huge competitive edge, allowing early adaptors
to focus on process efficiency, flexibility, manufacturing
convenience, and drastic decreases in manufacturing costs.
Table 2 presents the classification of biomanufacturing his-
tory based on production platform and research tool/theory
as well as representative products. For example, mass pro-
duction of penicillin in the World War II led by Merck and
Pfizer helped build solid foundations to become two of the
largest pharmaceutical companies; the successful produc-
tion of erythropoietin (EPO) made Amgen to become one
of the most successful biotechnology companies for several
decades.

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 Table 2  Enzyme production and purification methods
Name Protein Size Representative protein expression Representative protein purification Cost Representative example

Academic Lab μg to 10 mg E. coli Ultra-sonification, adsorption of Ni– $1000 per batch Research enzymes
1-L flask (200 mL) NTA resin, PLC, more
J Ind Microbiol Biotechnol (2017) 44:773–784

Academic pilot plant
Industrial pilot plant (CRO)
Bulk enzyme (relatively costly) 10 kg to100 tonnes E. coli
Bulk enzyme less costly)
Ultra enzyme
LB media + IPTG
A600 = 3–4
100 mg to 10 g E. coli Homogenization, selective adsorp- $6000 per batch Research enzymes, special tool
~50-L bioreactor tion, PLC, more enzymes
Medium-density media + lactose
A600 = 30–100
10 g to 1 kg E. coli Homogenization, precipitation (e.g., $15,000 per batch Redox enzymes used for synthesis of
~1000-L bioreactor heat, (NH4)2SO4), centrifugation, fine chemicals (1–100 tonnes), tool
High-density media + lactose ultra-filtration, (freeze drying) enzymes
A600 = 50–300
Homogenization, precipitation (e.g., $50-500/kg Enzymes used to produce biocom-
~20 m3 bioreactor heat or NH4SO4), centrifugation, modities (>10,000 tonnes)
High-density media + lactose ultra-filtration, (freeze drying)
A600 = 50–300
100–10,000 tonnes Secretory protein hosts (e.g., Centrifugation, ultra-filtration, (spray $10–30/kg Protease ($~10/kg)
Trichoderma, Bacillus) drying) Amylase ($~20/kg)
50 + m3 bioreactor Cellulase ($~20/kg)
High-density media Phytase ($10/kg)
[E] = 50–200 g/L
>100,000 tonnes Best microbial fermentation Ibid $2–5/kg Single cell proteins
GM plants Plant protein extraction Soy bean proteins
Future bulk enzymes

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776
Fig. 1  The evolution of bio-
manufacturing history from pre-
modern biomanufacturing (solid
state fermentation) to Biomanu-
facturing 1.0 (anaerobic liquid
fermentation) to Biomanufac-
turing 2.0 (aerobic submerged
fermentation) to Biomanufac-
turing 3.0 (advanced cell cul-
ture) to Biomanufacturing 4.0
(new directions). iPSC induced
pluripotent stem cells, ME&SB
metabolic engineering and syn-
thetic biology, APE advanced
protein engineering, ABivSB
advanced biotransformation by
in vitro synthetic biosystem
In this perspective review, we attempt to classify the
history of biomanufacturing into the four revolutions like
industrial revolutions. New directions of Biomanufactur-
ing 4.0 could revolutionize biomanufacturing to produce
a number of products from new food, renewable energy,
drugs and medicines, and materials better than existing
biomanufacturing processes in terms of product yield,
titer, volumetric productivity, biomanufacturing costs, and
sustainability.
Biomanufacturing history
Here we arbitrarily attempt to divide the biomanufacturing
history in terms of product types, biocatalysts, and technol-
ogy tools (Fig. 1; Table 2), wherein new technologies and
new products are usually developed in parallel and syner-
gized to lead to the new biomanufacturing revolution. Most
times, needs and products (or money) represent the major
driving force while technologies help product commerciali-
zation and market needs simulate technology development.
Pre‑modern biomanufacturing
Human beings utilized spontaneous (mixed-culture) micro-
bial processes to convert a food source into another form
thousands of years ago, although they did not have a clue
as to the nature of fermentation. Such living microbes are
readily accessible (indeed, essentially unavoidable), often
easy to cultivate, and possessed “mysterious” capabili-
ties for efficiently generating desired products from sim-
ple feedstocks, such as sugar solutions, fruits, vegetable
mashes, or molasses. For example, ancient Chinese made
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J Ind Microbiol Biotechnol (2017) 44:773–784
wine from a mixture of rice, honey, and fruits as early as
9000 year ago [36], the Sumerians and Babylonians prac-
ticed the brewing of beer before 6000 BC, and Egyptians
used yeast for baking bread, as recorded in the Bible. Prior
to the Louis Pasteur’s discovery of the essential role of liv-
ing microorganisms in fermentation, numerous fermenta-
tions utilized diverse and disparate cultures for the produc-
tion of foodstuffs, dyes, and other items, for example, the
preservation of cabbage, cucumbers, and other crops by
pickling; the use of calf rennet for cheese manufacture; the
manufacturing of specific food condiments (e.g., soy sauce,
vinegar); the bating of hides using dung microbes; and the
production of indigo for dyeing wool and cotton. Such
spontaneous fermentations can be regarded as Pre-modern
Biomanufacturing, which features solid-state (anaerobic)
fermentation (most times) and natural mixed microorgan-
ism cultures. In 2015, the wine industry produced more
than 24 billion liters of wine, accounting for the largest
fraction of drink market [14]. To increase wine output and
quality, more developments in the wine industry are going
on pertaining to grain saccharification, mixed yeast ecol-
ogy, and yeast metabolism [14].
Biomanufacturing 1.0
Biomanufacturing 1.0 starts from the Chaim Weizmann’s
acetone, butanol, and ethanol (ABE) fermentation in 1910s.
This biomanufacturing platform has two new features: the
use of the purified mono-culture microorganism instead of
mixed cultures and large-scale anaerobic liquid fermenta-
tion (Table 2). The typical products of Biomanufacturing
1.0 are primary metabolites produced by microorganisms,
where primary metabolites are directly involved in normal
J Ind Microbiol Biotechnol (2017) 44:773–784 777
Fig. 2  Product focus switch for
the ABE fermentation, where
market determines product
selection
cell growth, development, and reproduction. They usually
perform physiological functions in the organism (i.e. an
intrinsic function). They include ethanol, acetone, butanol,
amino acids, organic acids (e.g. lactate, acetate, citrate),
and so on.
The history of ABE fermentation teaches us two lessons:
(1) a focus shift of products over time and (2) a rapid tech-
nology development was driven by market needs (Fig. 2).
At the start of the twentieth century, a shortage of natural
rubber produced by trees, along with soaring prices of rub-
ber, stimulated interest in alternative feedstock, and routes
to produce synthetic rubber. The chemical firm Strange and
Graham Ltd. (London and Manchester, UK) decided that
butadiene and isoprene could be best precursors, which can
be prepared by oxidation of n-butanol and isoamyl alcohol,
respectively (Killeffer 1927). In 1912, Chaim Weizmann
isolated an anaerobic bacterium Clostridium acetobutyli-
cum that can produce acetone, butanol, and ethanol from
starch and glucose. However, the world rubber market
collapsed at the same year so that making synthetic rub-
ber from n-butanol was not economically appealing at all.
The product goal of his pioneering study was refocused to
acetone in World War I due to Britain’s critical need for
acetone as a solvent for manufacturing smokeless explo-
sive cordite. During the war, this fermentation was rapidly
scaled up to produce 30,000 tonnes of acetone per year.
When acetone was produced, twice as much n-butanol was
co-produced, the second in volume as an industrial bioman-
ufacturing product only to ethanol at that time. Surplus of
n-butanol lead to its new markets: a substitution for amyl
acetate in lacquers, its use in manufacturing of solvents,
plasticizers, paints, and resins. Recently, the focus of ABE
fermentation was shifted back to n-butanol again because it
is a good drop-in biofuel, which can be blended with gaso-
line at any ratio [32]. Now Chaim Weizmann is widely rec-
ognized to be the father of industrial fermentation [62].
In the twentieth century, biomanufacturing 1.0 had been
developed to produce a number of primary metabolites,
such as organic acids and amino acids. For example, World
War I also caused a shortage of calcium citrate imported
from Italy, which forced Pfizer to search for an alternative
supply. In 1919, Pfizer commercialized the production of
citric acid from sugars by using fermentation of a fungus.
Monosodium glutamate is the largest amino acid pro-
duced, being approximately 2 million tonnes [50]. Its prod-
uct approaches were changed from extraction from acidic
hydrolysate of vegetable proteins to chemical synthesis to
microbial fermentation. The advantages of the fermentation
method, such as reduction of production costs, environ-
mental load, and food safety concerns, were large enough
to cause all glutamate manufacturers to shift to fermenta-
tion [50]. Currently, the amino acid industry has annual
market size of more than 20 billion US dollars. The second
largest amino acid—lysine is also produced by microbial
fermentation [8]. The major producers of amino acids are
based in China, Japan, South Korea, the US, and Europe.
The great potentials of biodegradable plastics (e.g., biopol-
yesters) is opening new markets for a lot of organic acids,
such as d-lactic acid, l-lactic acid, succinic acid, furmaric
acid, glucaric acid, and so on.
Biomanufacturing 2.0
Biomanufacturing 2.0 started from penicillin fermentation
in World War II. The most distinguishing feature of Bio-
manufacturing 2.0 is the production of a secondary metabo-
lite instead of a primary metabolite. Secondary metabolites
are often restricted to a narrow set of species within a phy-
logenetic group and often play an important role in micro-
organism defense against other organisms. Humans usually
use secondary metabolites as medicines, flavorings, and
fragrances. Biomanufacturing 2.0 also adopted two new
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technologies: the use of the dedicated mutants and aerobic
submerged fermentation (Table 2). But it is worth mention-
ing that these two technologies had been developed in a
few cases of Biomanufacturing 1.0 (e.g., citrate) but were
not widely adopted until Biomanufacturing 2.0.
The production of antibiotics drove the rapid develop-
ment of Biomanufacturing 2.0. Before World War II, anti-
microbials, most notably sulfonamides discovered by Ger-
hard Domagk (Nobel Prize in Medicine 1939), were widely
used, but they had many limitations relating to spectrum,
efficacy, tolerability, and emergence of resistance [22]. In
1928, penicillin was discovered from Penicillium fungi by
Alexander Fleming (Nobel Prize Medicine, 1945). Penicil-
lin antibiotics are among the first medicines to be effective
against many bacterial infections caused by staphylococci
and streptococci. To decrease penicillin manufacturing
costs, aerobic submerged fermentation was introduced by
Merck in 1942. Also, intensive efforts were made, includ-
ing fermentation media, aerobic fermenter design, identi-
fication of a super-producer penicillin strain, oxygen sup-
ply, growth physiology control, regulation, etc. In 1950, Dr.
Elmer Gaden presented his groundbreaking Ph.D. disserta-
tion that provides the optimal amount of oxygen to allow
greater fermentation energy for penicillin mold to grow and
multiply more rapidly. He is widely regarded as “Father of
Biochemical Engineering” [24].
Dr. Arnold Demain is one of the world’s leading indus-
trial microbiologists for his pioneering research on the
elucidation and regulation of the biosynthetic pathways
of penicillins and cephalosporins and being instrumen-
tal in the development of beta-lactam industry. He joined
Merck as a research microbiologist in 1954 for studying
the synthesis of penicillin. Later, he moved on to Merck’s
penicillin research laboratories and worked on fermenta-
tion microbiology, β-lactam antibiotics, flavor nucleotides,
and microbial nutrition. In 1961, he started working on
the biosynthesis of the very important β-lactam antibiotic
cephalosporin C [7]. To make a number of semisynthetic
penicillins, 6-aminopenicillanic acid (6-APA), core of
penicllins, was prepared from penicillin by using penicillin
amidase. In 1965, he founded the Fermentation Microbiol-
ogy Department at Merck and directed research and devel-
opment on processes for monosodium glutamate, vitamin
B12, streptomycin, riboflavin, cephamycin, fosfomycin,
and interferon inducers. In 1969, he joined MIT, where he
set up the Fermentation Microbiology Laboratory. Along
with several professors (for example, Elmer Gaden, Dan-
iel IC Wang), a sub-discipline of chemical engineering—
biochemical engineering was established to address key
problems related to aerobic microbial fermentations in sub-
merged liquid cultures.
Later, a number of microorganism-derived antibiotics,
such as tetracycline, streptomycin, were discovered and
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introduced as medicines. For example, streptomycin, dis-
covered by Selman Waksman in 1943 (Nobel Prize Medi-
cine, 1952) became the first effective medicine to treat
tuberculosis. Currently, annual market size of anti-infective
antibiotics and semisynthetic antibiotics is approximately
60 billion US dollars [8].
Biomanufacturing 3.0
Biomanufacturing 3.0 started from 1980s for the produc-
tion of large-size proteins (that is, polypeptide/protein-
based drugs and enzyme-based biocatalysis) instead of pri-
mary or secondary metabolites (Table 2). The development
of this biomanufacturing platform was driven by two break-
throughs: the introduction of recombinant DNA technology
and advanced cell cultures. In 1973, Stanley Cohen and
Herbert Boyer created the first in vitro recombinant DNA
plasmid. Commercial ventures quickly started up with the
objective of capitalizing on Boyer and Cohen’s recombi-
nant DNA technology. Initial efforts focused on high-value
biopharmaceutical proteins because proteins are large com-
plex molecules that cannot be economically synthesized
by chemical synthesis and are too costly to be isolated
from natural organisms. In 1976, Genentech was founded
by venture capitalist Robert Swanson and Herbert Boyer
for the production of human insulin and growth hormone.
Among hundreds of recombinant proteins as biopharma-
ceuticals, non-glycosylated proteins are usually made in E.
coli or yeasts, accounting for 40% of the therapeutic protein
market [9]. In 1980, Amgen was founded for the produc-
tion of recombinant human EPO, a protein hormone that
increases the rate of production of red blood cells. EPO,
a heavily glycosylated protein that cannot be produced by
microbial fermentation, was engineered for expression in
mammalian cell cultures. Carbohydrates attached to the
EPO peptide are responsible for the different biological
activities of these proteins. It is one of the most success-
ful protein drugs and its market size rose to 13 billion dol-
lars in 2007 [8]. The biological activities of recombinant
glycoproteins produced in heterologous systems may vary
depending on the host cells in which the proteins are modi-
fied [19]. To address challenges of low-cost production of
constant-quality glycosylated proteins in mammalian cell
cultures, Dr. Daniel IC Wang, called the son of biochemi-
cal engineering, made the most significant contribution to
this area [1]. Another large market of glycosylated proteins
is antibody–drug conjugates (ADCs) for cancer treatment,
such as Adcetris® (brentuximab vedotin) and Kadcyla®
(ado-trastuzumab emtansine). Unlike conventional treat-
ments that damage healthy tissues upon dose escalation,
ADCs utilize monoclonal antibodies (mAbs) to specifi-
cally bind tumor-associated target antigens and deliver a
highly potent cytotoxic agent. The synergistic combination
J Ind Microbiol Biotechnol (2017) 44:773–784 779
of mAbs conjugated to small-molecule chemotherapeutics,
via a stable linker, has given rise to an extremely effica-
cious class of anti-cancer drugs with an already large and
rapidly growing clinical pipeline [23, 57].
Besides protein drugs, (high-cell density) fermentation
of microbial cells can produce a number of recombinant
enzymes, which are used for biocatalysts in life sciences
and industrial biocatalysis. In 1974, New England Biolabs
was founded for the commercial production of restric-
tion enzymes, DNA polymerases, and so on, for academic
researchers working on recombinant DNA technology.
Now a number of companies, such as Bio-Rad, Takana,
Sigma, Thermo Fischer, and so on, are producing numer-
ous research tool enzymes for researchers.
At the same time, enzyme-based biocatalysis has been a
practical and environmentally friendly alternative to chem-
ical synthesis on an industrial scale [4]. In the first wave
of biocatalysis based on natural enzymes, the main chal-
lenge for these applications is the limited stability of the
biocatalyst, and such shortcomings were primarily over-
come by enzyme immobilization, which also facilitated the
reuse of the enzyme [4, 34, 40]. Novozymes and Genencor
were founded in 1980s to focus on the production of bulk
enzymes for the food industry (e.g., amylase, glucoamyl-
ase, glucose isomerase, lipase), the textile industry (e.g.,
cellulase, hemicellulase), the detergent industry (e.g., amyl-
ase, lipase, protease), and the fine chemical industry (e.g.,
racimases, ketoreducases, dehydrogenases). In the second
wave of biocatalysis (1980s to 1990s), protein engineering
technologies, typically structure based, extended the sub-
strate range of enzymes to allow the synthesis of unusual
synthetic intermediates. This change expanded biocataly-
sis to the manufacture of pharmaceutical intermediates and
fine chemicals [4]. The third, and present, wave of bioca-
talysis started with the work of Frances Arnold and Pim
Stemmer in the mid and late 1990s. They pioneered molec-
ular biology methods that rapidly and extensively modify
biocatalysts via an in vitro version of Darwinian evolution.
Now rational design and directed evolution are not mutu-
ally exclusive; researchers often apply both to achieve the
tailored enzymes [55, 66].
For industrial enzyme biocatalysis, decreasing bulk
enzyme production costs are essential besides enzyme
immobilization. To accomplish this goal, intensive studies
have been made for high-cell density fermentation, selec-
tion, and modification of protein-producing hosts, etc. [9].
The easiest and quickest expression of heterologous pro-
teins can be carried out in Escherichia coli. In academic
labs, the use of flasks plus lysogeny broth media results in
very high protein production costs (e.g., $1000 per mg of
the purified enzyme, that is, $1 billion per kg). This infor-
mation often gives most academic researchers a misleading
impression that industrial recombinant proteins are very
Fig. 3  The relationship between the production costs of enzymes
and enzyme market sizes. The ultimate production costs of proteins
(enzymes) could be as low as 2–5/kg dry weight protein because soy
bean protein produced by dedicated soy bean crops costs $~1.00/kg
costly. Indeed, high-cell density fermentations of E. coli
have dry cell contents of approximately 20 to more than
100 g/L [33]. It is estimated that industrial production of
recombinant proteins by E. coli in fermenters could cost
in a range of $50 to $500 per kg, depending on protein
expression level, fermentation technology, protein puri-
fication cost, and fermentation scale (Table 2; Fig. 3). To
further decrease recombinant protein production costs by
decreasing protein purification costs and increasing protein
yields based on substrates, the ability to secrete recombi-
nant proteins across the cell membrane is important for
microbial production strains, such as Bacillus subtilis,
Aspergillus niger, Trichoderma reesei, and so on. Two of
the most important industrial enzymes (i.e., subtilisin for
detergent and alpha-amylase for starch hydrolysis and bak-
ing) are produced by B. subtilis, and their production costs
are as low as $10/kg of protein [37]. Up to 50 g/L secretory
glucoamylase is produced by Aspergillus niger and up to
200 g/L secretory cellulase including endoglucanases and
cellobiohydrolase is produced by T. reesei [9, 73]. After
intensive efforts in host mutagenesis and fermentation opti-
mization, the production costs of glucoamylase and cellu-
lase also have been decreased to a level of $~10/kg of dry
protein [68]. The use of transgenic plants such as Arabi-
dopsis thaliana, tobacco, and others may be the most prom-
ising means to further decrease biomanufacturing costs of
recombinant bulk enzymes because of cost-effective plant
cultivation, high level accumulation of proteins in plant tis-
sues, easy and cheap scale-up, and simple protein purifica-
tion [6, 70, 71]. In comparison of the lowest production of
plant protein—soy bean protein (i.e., $~1/kg), it may be
possible to decrease recombinant protein production costs
as low as $2–5/kg by using transgenic plants in the future
[6, 70, 71] (Table 2). Figure 3 presents an exponential
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relationship between the enzyme production costs and
enzyme market size. In a word, enzyme market needs moti-
vate drastic decreases in enzyme production costs.
Emerging biomanufacturing 4.0
In the beginning of this century, new product needs (e.g.,
renewable energy, artificial food, and regenerative medi-
cines) and new research tools (such as, induced pluripotent
stem cells (iPSC), metabolic engineering, synthetic biol-
ogy, systems biology, cascade biocatalysis, etc.) would lead
to a new revolution of biomanufacturing, which will pro-
duce new products (i.e., new functional tissues, new drugs)
or produce existing products by far more effective means as
compared to existing platforms in terms of product yield,
volumetric productivity, product titer, scale-up feasibility,
and sustainability.
Regenerative medicines
Regenerative medicines deal with the process of replac-
ing, engineering, or generating damaged human cells, tis-
sues, or organs to restore normal functions [35]. The ini-
tial focus of studies was engineering damaged tissues and
organisms via stimulating the human body’s own repair
mechanisms to heal previously irreparable tissues or organ-
isms. Later, most studies focus on growing cells, tissues
and organs in vitro in the laboratory and safely implanting
them in patients. When regenerated cells, tissues or organs
are derived from the patients themselves, such transplanta-
tion may potentially solve the problems of the shortage of
donated organisms and of transplant rejection.
In practice, regenerative medicine often refers to bio-
medical approaches related to stem cells, which have two
properties: self-renewal and potency. One of the most
promising stem cell solutions is induced pluripotent stem
cells (iPSCs), where various types of pluripotent cells
can be regenerated from adult cells without embryos. Dr.
Shinya Yamanaka (Nobel Prize Medicine, 2012) first dem-
onstrated conversion of adult cells to pluripotent stem cells
by the introduction of four specific genes encoding tran-
scription factors [59, 60]. Pluripotent stem cells hold great
promise in the field of regenerative medicine because they
can propagate indefinitely, as well as give rise to every
other cell type in the body, such as, heart, neuron, bone,
skin, kidney, pancreatic, and liver cells.
Metabolic engineering‑ and synthetic biology‑driven
whole cell fermentation
Metabolic engineering, which emerged 20 years ago,
is directed modulation of metabolic pathways for
13
J Ind Microbiol Biotechnol (2017) 44:773–784
over-production of desired metabolites or the improvement
of cellular properties, whereas synthetic biology mainly
focuses on fundamental research facilitated by the use of
synthetic DNA and genetic circuits [58]. Synthetic biology
applies engineering principles (e.g., design, extraction, and
standardization) and combines science (biology and chem-
istry) in order to design and build novel biological func-
tions and biosystems that function unnaturally or function
much better than natural counterparts [3, 12]. The concept
of synthetic biology was promoted greatly by engineers
so that most synthetic biology projects are goal-oriented,
decreasing biomanufacturing costs for existing products or
producing new products. Although synthetic biology can be
divided into two directions: in vivo and in vitro [15, 68],
most synthetic biology projects are based on living micro-
organisms because they have high surface-to-volume ratio
(facilitating rapid uptake of nutrients, fast biosynthesis
rates), ability of self-duplication, diversity of metabolic
reactions to make specific compounds, and ability to adapt
to different environmental conditions.
Several well-known synthetic biology examples are the
production of antimalarial drug artemisinin by engineered
E. coli and yeast [43, 47], of isobutanol from engineered E.
coli [2], and of opiods by engineered yeast [16]. Microbial
production of artemisinin and opiods represents a promis-
ing biomanufacturing platform to replace cultivating spe-
cial dedicated plants for meeting existing needed (Table 1)
but their biomanufacturing advantages over natural or engi-
neered plants need more justification on large scales.
It is worth noting that five hard truths for synthetic biol-
ogy were highlighted recently [31]. First, many of the parts
are neither defined nor standardized. Although they are
often defined in one special host under a special condition,
their performance could be different when in another host
or in other reaction conditions. Second, the very compli-
cated circuitry of living cells is unpredictable. Third, the
complexity is unwieldy. Fourth, many parts are incompat-
ible. Fifth, variability crashes the system. Synthetic biology
holds great promises in biomanufacturing of something
new. However, new microbial cell factories improved by
synthetic biology approaches do not undoubtedly exhibit
greater biomanufacturing advantages in biomanufacturing
of existing products than existing cell factories because
complexity of living microorganisms is still beyond our
limited knowledge and the primary goal of living microor-
ganism (that is, self-duplication) is a mismatch from bio-
manufacturing of desired products [71].
Advanced biotransformation by in vitro synthetic
(enzymatic) biosystems
To address major biomanufacturing problems of living
microorganisms [71] and high technical challenges of
J Ind Microbiol Biotechnol (2017) 44:773–784 781
in vivo synthetic biology [31], a few scientists and engi-
neers proposed advanced biotransformation catalyzed by
in vitro synthetic (enzymatic) biosystems (ivSBs) that usu-
ally contain more than four enzymatic components or even
tens of ones in one vessel for implementing complicated
biological reactions. These cell-free (in vitro) biosystems
are proposed to become an emerging biomanufacturing
platform. Assembling an in vitro biosystem is much easier
than modifying a living organism [68]. Engineering flex-
ibility in vitro is much greater, i.e., it is unshackled from
cellular viability, complexity, physiology, and the pres-
ence of membranes and/or cell walls [68]. A number of
enzymes isolated from different organisms (e.g., yeasts,
bacteria, archaea, animals and plants) are highly exchange-
able [72]. In the literature, this platform has a number of
different names with different emphasis, such as, systems
biocatalysis as compared to systems biology or biocatalysis
[13, 52, 61], in vitro synthetic biology [63, 75], synthetic
biochemistry as compared to synthetic biology [30, 41, 42],
synthetic pathway biotransformation (SyPaB) as compared
to whole-cell fermentation [68, 69], in vitro or cell-free
metabolic engineering as compared to metabolic engineer-
ing [11, 17, 21, 25, 48], and so on.
Compared to fermentation catalyzed by living whole
cells, biotransformation catalyzed by ivSBs has numerous
advantages: (1) high product yields mainly due to neither
of the production of side-products nor the synthesis of cell
mass, such as high H2 production from sugars [39, 48], bio-
electricity generation powered by sugars [77], high-yield
biopolymer synthesis from glucose [42], 1,3-propandiol
production from glycerol [46], lactate production from
glucose [65], and so on; (2) high volumetric productivity
due to no cell membrane and/or high volumetric enzyme
loading without dilution effects of other cellular proteins,
such as, enzymatic fuel cells [49, 77], high-speed biohydro-
gen generation [28, 48]; (3) high product titer due to high
enzyme tolerance to toxic compounds, such as biohydro-
genation in biomass hydrolysate [63], isobutanol biotrans-
formation [21]; (4) easy product separation mainly due
to no cell membrane, such as, xylulose 5-phosphate [29],
poly-3-hydroxybutyrate [51], synthetic amylose [44]; (5)
high product quality, such as synthetic amylose [44]; (6)
easy process control and optimization, such as cell-free
protein synthesis [18, 26, 64], rapid pathway test & optimi-
zation and building block validation [27, 48, 76], synthetic
or biomimetic pathways [28, 38]. Although this platform
seems to be advantageous, it cannot produce some prod-
ucts more economically better than microbial fermentation,
such as bulk enzymes (complex biomolecules) and antibi-
otics (secondary metabolites). For example, cell-free pro-
tein synthesis could produce high-value protein drugs but
not bulk enzymes ($10–30/kg) because we cannot make
less value products from high-value substrates.
Also, it is worth mentioning that ivSB may have a syn-
ergy with living microorganisms. For example, simultane-
ous enzymatic biotransformation and microbial fermenta-
tion can convert cellulose to artificial starch and ethanol
in one pot [67]. In this bioprocess, the membrane of the
yeast separates the charged product—glucose 1-phos-
phate with glucose to produce artificial starch outside the
cell and ethanol inside the cell. This process has no sugar
losses and requires neither energy nor chemical inputs.
When enzymes used in the system are least costly and sta-
ble enough, the whole bioprocess would convert nonfood
biomass to starch at nearly no costs. It is envisioned that
next generation biorefineries plus dedicated cultivation
perennial plants would meet increasing needs in the food–
energy–water nexus, where perennial plant communities
have higher biomass yield per hectare, have easy resource
management, store more carbon, maintain better water
quality, utilize nutrients more efficiently, tolerate more
extreme weather events, and resist pests better than annual
crops [5, 70]. Also, properties of artificial starch, such as
chain length and branch frequency, could be tailored by
adjusting reaction conditions and enzyme use in vitro (data
not published). Beyond potential new food source, tailored
artificial starch could have some new applications, such as
drug-releasing carrier and bioplastic [5]. Further improve-
ments in this technology would be essentially important to
humankind in this century to address the food and water
security issues.
Although there are no products on market manufactured
by ivSB, great potentials of ivSB should not be under-
estimated because of its largest advantages in biomanu-
facturing—high or theoretical product yields or energy
efficiencies. It is highly likely that biocommodities with
huge-market sizes, most of which are primary metabolites,
could be preferentially produced by ivSBs. Due to a series
of breakthroughs in bulk enzyme production (Table 2), pro-
tein engineering [45, 66], and enzyme immobilization [34,
54] in past decades, several products with annual volume
of more than 10,000 tonnes (e.g., myo-inositol, artificial
starch) are under development for large-scale commerciali-
zation by ivSB.
Conclusions
The most important challenges of humankind, such as sus-
tainable development, food security, renewable energy, new
biomaterials, better health, clean water, climate change,
and so on, motivate rapid developments in new biomanu-
facturing platforms in this century. A lot of organizations,
magazines, and researchers have suggested a few grand
challenges or great questions. For example, the National
Academy of Engineer of the USA lists 14 grand challenges
13

782
for engineers in the twenty-first century (http://engineer-
ingchallenges.org/challenges.aspx). Several of them may
be partially addressed by developing new biomanufactur-
ing technologies, for example, engineering better medi-
cines (by regenerative medicines [59, 60] and new micro-
bial, plant or animal platforms [2, 16]), management of
10. Department of Economic and Social Affairs, United Nations
the nitrogen cycle (by planting low nitrogen-requiring
perennial plants and new biorefineries for making artificial
food [5] or artificial photosynthesis [69, 70]), and provid-
ing clean water by decreasing water consumption via less
water-consuming artificial photosynthesis [70, 74]. Sci-
ence magazine lists 125 hard questions [56]. Among the
top 25 questions, several questions are closely related to
biomanufacturing, for example, “what can replace cheap
oil—and when?” and “will Malthus continue to be wrong?”
The United Nations list six challenges for the sustainable
development; two of them may be addressed by advanced
biomanufacturing, such as hunger and malnourishment as
well as energy needs (Department of Economic and Social
Affairs and United Nations [10].
In a word, grand challenges and great questions mean a
lot of new opportunities for biomanufacturing-related sci-
entists, engineers, businessmen, policy-makers, administra-
tive as well as companies, governments, and organizations.
Acknowledgements  This paper could not have been written without
the support of the Tianjin Institute of Industrial Biotechnology, Chi-
nese Academy of Sciences, China and the Biological System Engi-
neering Department, Virginia Polytechnic Institute and State Univer-
sity, Virginia, USA. Also, it is partially supported by the Department
of Energy, Office of Energy Efficiency and Renewable Energy, Fuel
Cell Technologies Office under Award Number DE-EE0006968 to
YPZ. Also, authors JBS and YHM were partially supported by Tian-
jin Municipal Science and Technology Commission for the financial
supports of 13ZCZDSY04900 and 11ZCZDSY08400. Funding to
YPZ for this work was partially supported by the Virginia Agricultural
Experiment Station and the Hatch Program of the National Institute of
Food and Agriculture, U.S. Department of Agriculture.
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