Li et al.

Biotechnol Biofuels (2018) 11:256

https://doi.org/10.1186/s13068-018-1234-5 Biotechnology for Biofuels

REVIEW Open Access

Biorefining of protein waste

for production of sustainable fuels
and chemicals
Si‑Yu Li1, I‑Son Ng2, Po Ting Chen3, Chung‑Jen Chiang4* and Yun‑Peng Chao5,6,7* 

Abstract 
To mitigate the climate change caused by ­CO2 emission, the global incentive to the low-carbon alternatives as
replacement of fossil fuel-derived products continuously expands the need for renewable feedstock. There will be
accompanied by the generation of enormous protein waste as a result. The economical viability of the biorefinery
platform can be realized once the surplus protein waste is recycled in a circular economy scenario. In this context, the
present review focuses on the current development of biotechnology with the emphasis on biotransformation and
metabolic engineering to refine protein-derived amino acids for production of fuels and chemicals. Its scope starts
with the explosion of potential feedstock sources rich in protein waste. The availability of techniques is applied for
purification and hydrolysis of various feedstock proteins to amino acids. Useful lessons are leaned from the microbial
catabolism of amino acids and lay a foundation for the development of the protein-based biotechnology. At last, the
future perspective of the biorefinery scheme based on protein waste is discussed associated with remarks on possible
solutions to overcome the technical bottlenecks.
Keywords:  Biorefinery, Protein waste, Biomass, Bio-based chemicals, Metabolic engineering

Background of technological bottlenecks and ecological issues associ-

Our daily life currently dependent on fossil feedstock has ated with land use [7].
been overshadowed by the global warming effect. This Replacement of the fossil fuels with biofuels used in
issue pressingly calls on the joined efforts of the inter- the heavy transport sector is necessary to meet the C ­ O2
national community towards climate change mitigation reduction requirement committed at Paris Climate Con-
by reducing ­CO2 emission. One emergent and promis- ference in 2015 (COP21) [7]. A sustainable production
ing field arises and devotes to exploration of plant bio- of biofuels relies on lignocellulosic biomass. This renew-
mass-based fuels and chemicals for replacement of fossil able biomass can produce the waste residues exceeding
resource-derived counterparts. Development of novel 2 × 1011 tons per year worldwide [8]. Biomass stemming
technology platforms for renewable production of energy from dedicated energy crops and the waste streams of
and chemicals still remains a focus of research efforts agro-food industries will be in a great demand because of
[1–6]. However, the progress of the second-generation the global incentives to the low-carbon alternatives. The
biofuel industry is fallen short of expectations because expanding need for renewable feedstock is accompanied
by the generation of a large volume of protein waste. As
estimated, protein waste with 100 million tons per year
could be generated if the use of biofuels (e.g., bioethanol
*Correspondence: [email protected]; [email protected] and biodiesel) accounts for 10% of the global fuel demand
4
Department of Medical Laboratory Science and Biotechnology, China [9]. The surplus protein waste necessitates recycling in a
Medical University, No. 91, Hsueh‑Shih Road, Taichung 40402, Taiwan
5
Department of Chemical Engineering, Feng Chia University, 100
circular economy scenario. Valorization of biomass waste
Wenhwa Road, Taichung 40724, Taiwan for various uses was estimated to rate bulk chemicals
Full list of author information is available at the end of the article

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Li et al. Biotechnol Biofuels (2018) 11:256 Page 2 of 15

with the highest value of $1000 per ton of biomass, as stove contain very few proteins. Interestingly, cassava
compared to the value of transportation fuel ($200–400), leafs have a high protein content reaching around 40%
cattle feed ($70–200), and electricity ($60–150) [8]. w/w and provide a promising source of proteins. Figure 1
Therefore, it appears incentive to produce chemicals shows the crude protein content of selected biorefinery
from protein waste [10]. feedstock. The global production volume of soybean
This article provides a concise overview of the advances meal, rapeseed meal, and sunflower meal amounts to
in refining protein waste for production of fuels and 200.8, 39.2, and 16 million metric tons (MMTs), respec-
chemicals with special emphasis on the biotechnology tively [13]. The production of maize DDGS and canola
platforms. The scope includes the available sources of seed meal in US is around 23.1 (Renewable Fuels Asso-
feedstock, protein recovery and pretreatment, amino- ciation) and 1.07 MMTs [13], respectively.
acid catabolism, and the enzyme- and microbe-based
production schemes of chemicals. As released from Alternative protein source
proteins, amino acids containing carbon skeletons with The volume of crops is largely limited by land availabil-
amino groups have functional similarities to the many ity. In contrast, microalgae which display a fast growth
petroleum-derived chemicals. The approach by enzy- produce a high protein level and can be grown in open
matic transformation (biotransformation) is straight- ponds. Microalgae approximately contribute to 40%
forward and enables conversion of amino acids to bulk of global photosynthesis by fixing C­ O2 [14]. There are
chemicals, particularly favorable for those with opti- more than 200,000 microalgae species on the planet,
cal purity. On the other hand, microbes naturally uti- mostly including Bacillariophyta (diatoms), Chloro-
lize amino acids as food. The biomass-based schemes phyta (green algae), Chrysophyta (golden algae), and
available for microbial production of chemicals can be Cyanophyta (blue–green algae). The fractionated com-
transformed into those based on proteins. However, the position of algae generally comprises 35–50% proteins
development of both fields is still in infancy. This review [15]. However, microalgae display high diversity in
is to provide an idea framework for future research terms of ecological, metabolic, chemical, and biologi-
efforts to this end. cal characteristics. Botryococcus braunii, for instance,
has hydrocarbons accounting for 75% of the total
Potential sources of protein waste weight. Diatom, Dunaliella salina, and Chlorella sp.
Protein source from crops are rich in lipid ranging from 30 to 75% [16]. Chloro-
The feedstock conventionally applied in biorefinery plat- phyta and Chlorophyceae contain 60% proteins which
forms mainly involves crops rich in sugars (e.g., sugar- can be finely processed into human nutrition source
beet and sugarcane), starch (e.g., cassava, maize, wheat, [17]. Chlorella strains cultivated in the photobioreac-
and sorghum), hemicellulose (e.g., switchgrass and cop- tors outdoors enable production of 32 tons protein per
pice trees), and oil (e.g., Jatropha seed, palm, rapeseed, year [18]. The composition of amino acids from micro-
soybean, and sunflower seed). Waste streams resulting algae is favorable for food, fishing, agriculture, and
from the production of vegetable oil and biodiesel with animal feed industry [17, 19]. As reported, the protein
oil crops comprise a very high protein content rang-
ing between 40 and 60% (w/w) of the mass fraction, and
their vital value is generally acknowledged as feedstuff. 60
44.6 46.7
Crude protein contents (%)

Nevertheless, Jatropha meal generated by the biodiesel 50

production has a potential application for the chemi- 35.9 35.9 37 37.8
cal production, because it is inedible without detoxifica- 40 35.6 37.5 36.3
tion. Recognized as the most abundant protein source, 29.4
30
distiller’s dried grains with solubles (DDGS) are the
nitrogen-rich residues derived from the alcoholic bever- 20 13.3
age fermentation with maize, wheat, and sorghum [11].
DDGS receive a substantial income from the animal feed 10
market, but have a low profit. The application of DDGS 0
for production of value-added chemicals seems to be an
S

S
ne GS

yb asse

yb cake

su eed e
ca lowe eal

ca ed m l
al l
s
ea

a
a
ve
ak
DG

su hum DG

las r me

incentive opportunity. Sugarcane vinasse in the bioetha-

rap n m

m
rca DD

ea
rap ed c
n
eD

rg t D

n
vi
ea

nol sector has a low content of proteins, and its economic

ea

e
av
ea
aiz

es

es

ss
wh

so

nf
m

so

value may be realized for an alternative application other

no
ga
so

than fertilizer [12]. As exploited for production of ligno-

Fig. 1  Crude protein content of biorefinery feedstock
cellulosic biofuel, maize stover, wheat straw, and sorghum
Li et al. Biotechnol Biofuels (2018) 11:256 Page 3 of 15

production of Chlorella reaches up to 400 g/g-biomass 34, 35], grass and leaves [25, 30, 31, 36, 37], seaweeds [30,
under the mixotrophic condition [20]. The protein con- 31, 38], and microalgae [30–32, 39, 40]. The treatment
tent of selected microalgae is summarized in Fig.  2, process generally consists of (1) cell disruption and frac-
including Chlamydomonas reinhardtii [21], C. pyrenoi- tioning, (2) protein recovery, and (3) protein hydrolysis.
dosa [22], Scenedesmus sp. NT1d, S. dimorphus NT8c, It has been reported to purify oil seed proteins with the
Tetrahedron caudatum NT5, Chlorella sp. NT8a, method of heat [41], urea [42], pH [43], and ethanol [44],
Graesiella emersonii NT1e [23], Nannochloropsis sp. and cereal proteins with heat [41], water soluble [45], salt
[24], C. vulgaris [20], Anabaena variabilis [25], Het- and ethanol [46]. Grass and leaf proteins are recovered
erochlorella luteoviridis, and D. tertiolecta [26]. C. vul- by heat [41] and acid/alkaline [38], seedweed proteins by
garis, D. bardawil, S. obliquus, and Spirulina platensis heat [41] and acid [43], and microalgal proteins by heat
have a well-balanced composition of amino acids [15]. [47] and acid/alkaline [38]. The purification efficiency
Microalgal proteins are expected to contribute around of implemented methods varies with distinct feedstock
30% of the animal feed market in the future [27]. (Fig.  3). Nevertheless, the alkali- or acid-based precipi-
The protein content of fungi and bacteria is within tation method is usually employed to separate proteins.
a range of 30–70% and 50–80%, respectively [28]. An effective method was reported to precipitate proteins
Microbes have been genetically manipulated for mass at pH 9 and 30 °C or pH 4.0–4.5 and 80–90 °C [48, 49].
production of amino acids and chemicals of specific One technology called ammonia fiber expansion (AFEX)
interest [29], which is unaffected by climate changes. illustrates efficient recovery of proteins from cellulosic
However, microbes contain a high level of nucleic acids biomass. The treatment process starts with the warm
and suffer a high risk of contamination with heavy met- ammonia solution for extraction of proteins. Proteins are
als and toxins [28]. Microbial proteins can provide an then obtained after drying [50]. Leaf protein processed
important and promising source if the contamination by AFEX provides a viable method of alternative choice
issue is well addressed. [51]. A convection method involves the use of a mechani-
cal mill to disrupt leaf cells, followed by removal of the
Protein recovery and hydrolysis juice with a screw press. The juice is subject to heat to
Recovery of protein waste from biomass feedstock is a withdraw the coagulated proteins which are isolated after
necessary step prior to isolation of amino acids. Figure 3 drying [52].
outlines the treatment schemes applied for various feed- There are many methods developed to separate pro-
stock including oil seeds [11, 30–33], cereals [11, 30, 31, teins from the waste stream in the biodiesel production
process. One approach employs a multistep extraction
method for recovery of Jatropha seed proteins [53]. Jat-
70 ropha seed kernels and husks are first processed by either
60
a milling machine or a screw press. The residue biomass
after the machine pressing is extracted with solvents, fol-
based on dry weight
Protein content (%)

50 lowed by subjecting the crude extracts to three stages of

40 cross-flow extraction. Proteins are finally isolated by pre-
cipitation at the acidic condition.
30 Disruption of cell walls presents to be the critical step
20 in recovery of proteins from microalgae. This task is
conventionally carried out by the chemical treatment
10
using acid–alkaline solution (e.g., 0.4 M HCl and 0.4 M
0 NaOH), or a two-phase system with polyethylene glycol
(PEG) and potassium carbonate [54]. However, physi-
5

1e
.

bilis
dtii

is

lecta
sa
d
8c

T8a

is
is sp
1

m NT

ovirid
vulga
noido

s NT

cal treatments usually favor the recovery of microal-

ii NT
T
inhar

varia
sp. N

sp. N

tertio
rops

gal proteins at a large scale. Many methods have been

datu

a lute
rphu

rson
as re
pyre

rella
aena
ochlo
mus

rella

developed, including high-pressure homogenization,

liella
n cau

eme
dimo

lorell
Chlo
mon
rella

liquid nitrogen grinding, ultrasonic crushing, osmotic

edes

Chlo

Anab

Duna
Nann
siella
edro
mus

cracking pulsed electric field, and microwave-assisted

Chlo
mydo

roch
Scen

extraction [55, 56]. Moreover, other cell compositions

Tetra
edes

Grae

Hete
Chla

associated with proteins are removed by precipitation

Scen

or fractionation to ensure a good quality of recovered

Fig. 2  Protein content of selected microalgae. The various levels of proteins [57]. Nevertheless, the application of microal-
proteins in selected microalgae are summarized gal proteins still remains to explore.
Li et al. Biotechnol Biofuels (2018) 11:256 Page 4 of 15

Fig. 3  Flow chart of protein extraction and amino-acid recovery from various biomass sources. The treatment processes applied for selected
feedstock are illustratively summarized. The numerical number in the parenthesis indicates the efficiency of the implemented methods

The hydrolysis of proteins is commonly carried out in Escherichia coli. The amination of glutamate medi-
with a prolonged treatment of acids or alkalis. How- ated by l-glutamine synthetase (GS) leads to glutamine.
ever, not all amino acids remain intact during the treat- Moreover, ammonia in low concentration is assimilated
ment process [58, 59]. A milder way lies in the use of to glutamate by the combined reaction of GS and gluta-
proteases. Alcalase is an alkaline protease and has been mate synthase (or glutamine: α-oxoglutarate aminotrans-
exploited for hydrolysis of proteins in poultry, shrimp ferase). Glutamate and glutamine serve as ammonia
waste, and wheat gluten [60–62]. In addition, proteins donor for reductive synthesis of more than 10 amino
extracted from wheat DDGS were treated with pro- acids. The catabolism of proteinogenic amino acids dif-
tease including Protex 14L, Protex 6L, and Protex 51P fers from their anabolic pathways due to the thermody-
[63]. The production of amino acids from sorghum was namic constraint and subtle molecular regulations. In
proven feasible by the amino-peptidase and the neutral general, the catabolic route for glucogenic amino acids
proteinase from Novozymes [64]. A patent discloses the leads to pyruvate and metabolite nodes in the TCA cycle
employment of peptidases to release free amino acids and for ketogenic amino acids ends with production of
from protein sources [65]. Lactic acid bacteria possess a acetyl-CoA and acetoacetyl-CoA (Fig.  4a). The catabo-
variety of peptidases [66], and have a potential applica- lism of amino acids varies in living cells. This review is
tion for production of chemicals (see “Discussion and mainly focused on the catabolic pathways in E. coli and
future perspectives”). Enzymatic reactions require a selected microbes.
controlled condition to optimally proceed, which may
be fulfilled by the pH-stat enzymatic hydrolysis [67]. Amino‑acid catabolism with single or two steps
In E. coli and Saccharomyces cerevisiae, glycine is oxi-
Catabolism of proteinogenic amino acids datively degraded to ­CO2, ammonia, and a methylene
The synthesis of 20 proteinogenic amino acids requires group by the glycine cleavage complex (namely, glycine
six precursor metabolites in the central metabolism, decarboxylase) [1, 68]. The methylene group enters
involving glycolysis, the tricarboxylic acid (TCA) cycle, one-carbon metabolism mediated by tetrahydrofolic
and the pentose phosphate (PP) pathway. Inorganic acid (THF). Consequently, the formation of methylene-
ammonia is assimilated by reductive amination of THF drives the serine synthesis by serine hydroxym-
α-oxoglutarate with l-glutamate dehydrogenase (GDH) ethyl transferase in E. coli. Alternatively, Clostridium
Li et al. Biotechnol Biofuels (2018) 11:256 Page 5 of 15

(See figure on next page.)

Fig. 4  Catabolism of proteinogenic amino acids. a Catabolic routes of amino acids linking to the central metabolism. b Involvement of H donors
and H acceptors in Stickland reactions. In the oxidation reaction, alanine, leucine, isoleucine, valine, phenylalanine, tryptophan, and histidine
function as H donors to produce acetate, 3-methylbutyrate, 2-methylbutyrate, 2-methylpropionate, phenylacetate, indolacetate, and glutamate,
respectively. In the reduction reaction, glycine, proline, phenylalanine, tryptophan, ornithine, and leucine function as H acceptors to produce
acetate, 5-aminovalerate, phenylpropionate, indolpropionate, 5-aminovalerate, and 4-methylvalerate, respectively

sticklandii employs a glycine degradation route consist- as the main pathway for degradation of glutamate to
ing of glycine decarboxylase and glycine reductase to α-ketoglutarate and ammonia [80].
produce acetate [69]. E. coli exhibits slow growth on glutamine. Glutamine is
Serine is deaminated to pyruvate by serine deaminase utilized by conversion of two glutamate based on gluta-
in E. coli [70] or serine dehydratase in S. cerevisiae [71]. mate synthase, followed by glutamate catabolism [81].
E. coli synthesizes two serine deaminases (encoded by Hydrolysis of proline to glutamate proceeds in two
sdaA and sdaB), but is unable to utilize serine as sole car- steps. E. coli synthesizes proline dehydrogenase (encoded
bon source. by putA) with a dual function which is responsible for
Cysteine is a sulfur-containing compound and this catabolic pathway [82].
degraded to pyruvate and ­H2S in E. coli. There involves E. coli has two aerobic degradation routes of thre-
cysteine desulfhydrase (encoded by cysK and cysM) onine [83]. The major route consists of threonine
in this catabolic reaction [72]. In addition, tryptopha- dehydrogenase (encoded by tdh or yiaY) and 2-amino-
nase (encoded by tnaA) displays a catalytic function of 3-ketobutyrate CoA ligase (encoded by kbl) and ena-
cysteine desulfhydrase and plays a key role in cysteine bles conversion of threonine to glycine and acetyl-CoA.
utilization. Its synthesis is induced in the presence of Another route comprises low-specificity l-threonine
tryptophan [73]. aldolase (encoded by ybjU) which generates glycine and
E. coli utilizes alanine in a unique way. l-Alanine is first acetaldehyde from threonine.
converted to d-alanine by alanine racemase (encoded
by dadX and alr). A subsequent reaction catalyzed by Amino‑acid catabolism with multiple steps
d-amino-acid dehydrogenase (encoded by dadA) of The catabolic pathway of histidine consists of four reac-
broad substrate specificity leads to deamination of d-ala- tions steps, which leads to the formation of glutamate
nine to pyruvate [74]. This catabolic pathway enables E. and formamide. This pathway exists in many microbes
coli to utilize both l-alanine and d-alanine. Alternatively, (such as B. subtilis) but not found in E. coli [84].
Bacillus subtilis obtains the energy for sporulation by Microbes utilize arginine through various pathways
conversion of l-alanine to pyruvate with alanine dehy- [85]. The catabolic reaction starts with arginine succinyl-
drogenase [75]. transferase or arginine decarboxylase in E. coli, arginine
The synthetic pathway of aspartate is reversible and oxidase in P. putida, arginine deiminase in lactic acid
mostly adopted for its utilization in living cells. By the bacteria, arginase in B. subtilis, and arginine:pyruvate
aspartate aminotransferase (encoded by aspC)-medi- transaminase in P. aeruginosa. The arginine succinyl-
ated transamination reaction, aspartate is converted transferase (AST) pathway is the major catabolic pathway
to oxaloacetate and α-ketoglutarate receives the amino of arginine in E. coli. The α-amino group of arginine is
group to give glutamate. Aspartate is also deaminated to first succinylated, followed by deamination of two amino
fumarate as catalyzed by aspartase in E. coli and lactic groups and transamination of the third amino group on
acid bacteria [76, 77]. the side chain. The succinyl group is detached from the
The degradation of asparagine is initiated by its conver- α-amino group of arginine through hydrolysis, finally
sion to aspartate as catalyzed by asparaginase. Further giving glutamate and succinate. The arginine deiminase
catabolism of aspartate proceeds with aspartase. E. coli (ADI) pathway found in C. sticklandii [69] and lactic acid
enables synthesis of two asparaginases encoded by ansA bacteria [66] produces intermediate metabolites includ-
and ansB [78]. ing citrulline, ornithine, and carbamoyl phosphate which
The utilization of glutamate is limited by its inefficient appear in the synthetic pathway of arginine. Carbamoyl
transport in E. coli. The AspC-catalyzed transamina- phosphate is further cleaved into ­CO2 and ammonia with
tion reaction mediates the conversion of glutamate to generation of ATP. This substrate-level phosphoryla-
α-ketoglutarate and aspartate. Aspartate is deaminated tion in arginine catabolism is coupled with the bacterial
to fumarate catalyzed by aspartase [79]. In S. cerevisiae, growth [66].
the NAD-dependent glutamate dehydrogenase serves Leucine, isoleucine, and valine are branch-chain amino
acids (BCAAs) which produce volatile compounds
Li et al. Biotechnol Biofuels (2018) 11:256 Page 6 of 15

a Alanine

Cysteine

Glycine Isoleucine

Serine Leucine

Threonine Threonine
Tyrosine
Pyruvate
Tryptophan Tryptophan
Leucine

Acetyl-CoA Acetoacetyl-CoA Lysine

Phenylalanine

Citrate Tryptophan
Asparagine Arginine
Oxaloacetic acid cis-Acotinate
Aspartate
Histidine

Isocitrate
Proline

Malate Glutamine

α-ketoglutarate Glutamate

Phenylalanine
Fumarate
Tyrosine Succinyl-CoA Isoleucine

Succinate Methionine

Theronine

Valine
b

H-donor Product
Alanine CO2 + NH3 Acetate
Leucine 3-Methylbutyrate
Isolucine 2-Methylbutyrate
Valine 2-Methylpropionate
Phenylalanine Phenylacetate
Tryptophan Indolacetate
Histidine Glutamate
H
Acetate Glycine
5-Aminovalerate Proline
Phenylpropionate Phenylalanine
Indolpropionate Trytophane
5-Aminovalerate Ornithine
4-Methylvalerate NH3 Leucine

Product H-acceptor
Li et al. Biotechnol Biofuels (2018) 11:256 Page 7 of 15

involving acids, aldehydes, alcohols, and esters. Their Bio‑based production of fuels and chemicals
degradation starts with the transamination reaction from amino acids
using α-oxoglutarate as the amino acceptor to pro- Biotransformation approach
duce α-oxoisocaproate, α-oxomethylvalerate, and Protein hydrolysates contain a mixture of 20 amino
α-oxoisovalerate, respectively. Aminotransferases such acids. Isolation of amino acids is required for perform-
as AraT (EC 2.6.1.1) and BcaT (EC 2.6.1.42) in lactic ing biotransformation. Many methods for separation of a
acid bacteria and BAT1 in S. cerevisiae are responsible single amino acid from a mixture have been developed,
for this transamination reaction [86, 87]. The degrada- and each has its own advantage and disadvantage. By the
tion pathways for conversion of these α-oxoacids to alde- electrodialysis method, amino acids are separated into
hydes, carboxylic acids, and hydroxyacids are common the acidic, basic, and neutral groups [94]. The potential
in most microbes. Alternatively, the common path- problem is the interaction of some amino acids with the
way for conversion of leucine, isoleucine, and valine to ion-exchange membrane. A recent study has reported
3-methylcrotonyl-CoA, (E)-2-methylcrotonoyl-CoA, and the use of ethanol to fractionally precipitate amino acids
methylacrylyl-CoA proceeds with BCAAs aminotrans- [57]. Groups of amino acids are separated from a mix-
ferase, branched-chain α-ketoacid dehydrogenase, and ture, which needs extra work for the complete separation
2-methylacyl-CoA dehydrogenase, respectively. The of amino acids. The implementation of chromatography
subsequent cleavage of these acyl-CoA derivatives goes appears useful for isolation of individual amino acids
through distinct routes, consequently leading to propio- [95]. However, these mentioned methods are generally
nyl-CoA, acetyl-CoA, or acetoacetate. impractical due to a high cost associated with the scale-
Tryptophan can be degraded in various ways. E. coli up operation and waste management.
synthesizes tryptophanase which catalyzes the conver- A variety of amino group-containing compounds are
sion of tryptophan to indole, pyruvate, and ammonia idea candidates for production from amino acids with a
[88]. The catabolic pathway involving homogentisate as simple reaction scheme. Learning from the amino-acid
an intermediate is employed by animals, fungi, and some catabolism of microbes, the deamination, decarboxyla-
bacteria for tyrosine degradation. Further hydrolysis of tion, and hydrolysis reactions provides a basis for the pro-
homogentisate produces fumarate and acetoacetate. The duction scheme (Table 1). A good example illustrates the
degradation of phenylalanine can proceed with the same arginase-catalyzed conversion of arginine to ornithine
pathway after its hydroxylation to tyrosine by phenylala- [96]. The starting material for Nylon-4,6 can be obtained
nine hydroxylase [89]. by further decarboxylation of ornithine to 1,4-diaminob-
The degradation pathway of lysine diversifies. P. putida utane using ornithine decarboxylase [97]. This two-step
catabolizes lysine to glutarate via the δ-aminovalerate reaction is limited by ornithine decarboxylase [total turn-
pathway [90]. Glutarate is activated to glutaryl-CoA over number (TTN) of ~ 105] due to its lower operational
which is cleaved to C ­ O2 and acetyl-CoA in several steps stability than arginase (TTN of ~ 109). The phenylalanine
[91]. ammonia lyase (PAL)-mediated reaction produces cin-
Note that S. cerevisiae has evolved a generalized path- namic acid from phenylalanine [98]. The importance of
way for utilization of amino acids discovered by Ehrlich cinnamic acid is acknowledged as the precursor for the
[92]. The Ehrlich pathway starts with the transamination synthesis of styrene (> 1.7 × 107  tons/year). PAL prevails
reaction by conversion amino acids to their respective in yeast and is subject to oxidation. The reaction scheme
α-keto acids. Subsequent decarboxylation of α-keto acids is usually conducted with suspended whole cells under
produces aldehydes, known as fusel aldehydes. Fusel the anaerobic and static condition. A similar idea can also
aldehydes are finally reduced to fusel alcohols or oxi- be applied for production of β-alanine by decarboxyla-
dized to fusel acids, which serve as flavor compounds or tion of aspartate with aspartate α-decarboxylase [99].
precursors of flavor compounds. The Stickland reaction β-Alanine has a potential application for the synthesis of
prevails in clostridial species that ferment the amino- acrylonitrile and acrylamide (> 0.5 × 106  tons/year). This
acid mixture [93]. In this reaction, some amino acids enzymatic reaction generates C ­ O2 which causes a pH
preferably function as H donors, while others H accep- shift and the malfunction of the fixed-bed reactor. The
tors (Fig. 4b). It is carried out by coupling the oxidation oxidation and decarboxylation of lysine lead to 5-ami-
reaction of one amino acid with the reduction reaction novaleric acid and 5-diaminopentane, respectively. The
of another. For instance, the oxidation of glycine pair- former reaction proceeds with lysine oxidase, while the
ing with the reduction of glycine produces acetate and latter with lysine decarboxylase [100–102]. The potential
ammonia. This reaction is featured with conservation of application of 5-aminovaleric acid and 5-diaminopen-
ATP via the substrate-level phosphorylation. tane is their use for production of nylon and polyamide.
However, the enzymatic reaction is less efficient and
Li et al. Biotechnol Biofuels (2018) 11:256 Page 8 of 15

Table 1  Summary of selected biotransformation of amino acids into chemicals

Amino acid Product Enzymatic reaction Enzyme

Arginine Ornithine Arginase

Aspartic acid β-Alanine Aspartate α-decarboxylase

Glutamic acid α-Ketoglutaric acid Glutamate deaminase

Glutamic acid N-Methyl- and Glutamate decarboxylase and

N-vinyl-pyrrolidone NADH oxidase

Lysine 5-Aminovaleric acid Lysine oxidase

5-Diaminopentane Lysine decarboxylase

Phenylalanine Cinnamic acid Phenylalanine ammonia lyase

Alcohol and alanine Primary amines Alcohol dehydrogenase (ADH-

hT), ω-transaminase (ωTA)
and l-alanine dehydrogenase
(AlaDH)

l-Amino acids Enantio-compounds l-Amino acid oxidases (l-AAD),

isocaproate reductase (Hic)
and formate dehydrogenase
(FDH)

conducted for several days to achieve a conversion yield gives a production rate of 34.3 g/L/h. The deamination of
of 95%. Glutamic acid is a non-essential amino acid and glutamate by glutamate deaminase gives α-ketoglutaric
the most abundant amino acid found in most of feedstock acid [104, 105]. Interestingly, α-ketoglutaric acid can be
proteins. Through the decarboxylation reaction, gluta- polymerized into poly(triol α-ketoglutarate), a biode-
mate is converted to γ-aminobutyric acid by glutamate gradable material. However, this enzyme displays insta-
decarboxylase (GAD) [103]. The synthetic routes start- bility to lose 75% of the activity after the four times reuse
ing from γ-aminobutyric acid to N-methylpyrrolidone of the immobilized cells.
(NMP) and N-vinylpyrrolidone (NVP) are considered Primary amines are important for the synthesis of
environmentally favorable. NMP and NVP are useful azo dyes, antioxidants, or rubber products. They can be
intermediates for the synthesis of many bulk chemicals. obtained from primary alcohols using one-pot enzymes
The GAD-mediated reaction is relatively efficient with a including thermostable alcohol dehydrogenase (ADH-
complete conversion of glutamic acid within 3  h, which hT), ω-transaminase (ωTA), and l-alanine dehydrogenase
Li et al. Biotechnol Biofuels (2018) 11:256 Page 9 of 15

(AlaDH) [106]. ADH-hT participates in the oxidation ilvE, avtA, and sdaB. The first cycle operates using
reaction, while ωTA catalyzes the amination reaction. LeuDH to deaminate isoleucine and leucine to 2-keto
The oxidation and amination reactions are continued to methylvalerate and 2-keto isocaproate, respectively. The
occur by cycling l-alanine/pyruvate and N ­ AD+/NADH second cycle driven by AvtA converts valine to 2-keto
with the aid of AlaDH. The conversion reaction can be isovalerate, and pyruvate accepts the amino acceptor of
driven to completion (up to 99%) using ammonia as an valine. Through the IlvE-catalyzed transamination reac-
amino-donor in the presence of 1,2-dimethoxyethane tion, these three α-keto acids receive the amino group
at room temperature. An alternative approach to obtain from glutamate to form isoleucine, leucine, and valine,
amine has been illustrated with galactose oxidase from respectively. The third cycle proceeds by SdaB-mediated
Fusarium sp., and a ω-TA from B. megaterium, P. putida, deamination of serine to pyruvate. Re-synthesis of ser-
Paracoccus denitrificans, and Vibrio fluvialis [107]. Like ine occurs by gluconeogenic conversion of pyruvate to
AlaDH, either formate dehydrogenase or glucose dehy- 3-phosphoglycerate which serves as the precursor for
drogenase was employed to complete the l-alanine/ serine synthesis via the native synthetic pathway consist-
pyruvate and ­NAD+/NADH cycle [108]. The continued ing of serA, serB, and serC. These combined strategies
operation of these reactions is driven by regeneration of finally resulted in the production of biofuels (isobutanol,
­NAD+ which is added as a cofactor. 2-methyl-1-butanol, and 3-methyl-1-butanol) account-
l- or d-Hydroxy acids mostly used for enantio-drugs ing for 56% the theoretical yield. Another merit of this
can be synthesized by a designed cascade reaction, approach is manifested by removal of the ammonia from
namely one-pot simultaneous multi-enzyme system amino acids, and the recycled nitrogen may provide the
[109]. The (R)- and (S)-enantiomers of amino acids are need of fertilizer for crops. This reduces the dependence
converted to enantiopure form (> 99% ee) by l-amino- of chemical fertilizer stemming from the environmen-
acid oxidases (l-AAD) [110]. A subsequent reaction pro- tally unfavorable Haber–Bosch process, which eventu-
ceeds as an asymmetric reduction which consists of the ally ameliorates the climate change [114]. Recently, the
coupled reaction of isocaproate reductase (Hic) and for- same idea has been applied for production of biofuels
mate dehydrogenase (FDH) [111]. The implementation of from DDGS by a bacterial consortium which consists of
l- or d-Hic enables selective production of the hydroxy two strains designed for selective utilization of carbohy-
acid enantiomers. The regeneration of NADH cofactor is drates and amino acids, respectively [115]. The strategy
carried out by FDH, which is widely applied in industry. by deamination of amino acids has also been exploited
for the production of ammonia [116].
Pathway engineering approach Although feasible, the amino acid-based produc-
Naturally occurring microbes exhibit a different degree tion scheme of biofuels by genetically modified E. coli
of amino-acid utilization as the carbon and nitrogen is afflicted with the need for protein hydrolysate. The
source. Nevertheless, the production of chemicals by implementation of a native protease-secreting microbe
microbes using protein waste can be implemented with- such as B. subtilis seems to provide a solution to this issue
out the need for isolation of amino acids. This idea has by integration of protein hydrolysis and amino-acid fer-
been well illustrated by pathway engineering of E. coli to mentation in one step. The strategy exploited for pathway
rewire and optimize its amino-acid catabolism [112]. E. engineering of B. subtilis was carried out in several steps
coli was first evolved to utilize 13 individual amino acids [117]. In essence, three transamination–deamination
after several rounds of chemical mutagenesis. To produce pathways for cycling BCAAs were established by recruit-
isobutanol, the evolved strain was endowed with the syn- ment of T. intermedium leuDH. Two cycle pathways gen-
thetic pathway. The microbial consumption of proteins erally resemble those in E. coli. One involves YbgE (like
may be restricted upon induction of quorum sensing in E. coli IlvE) and LeuDH to cycle isoleucine, leucine, and
cells grown on the protein-rich medium [113]. The regu- valine. Valine is cycled in another route by coupling YbgE
latory circuit of amino-acid catabolism was then disabled with AlaT (like E. coli AvtA). The third cycle proceeds
by deletion of the quorum-sensing genes luxS or lsrA, with deamination of glutamate by endogenous RocG and
consequently increasing the isobutanol production. Fur- re-synthesis of glutamate by YbgE-mediated transami-
thermore, a driving force to drain more amino acids was nation. Moreover, the regulatory circuit involved in the
created by blockage of the nitrogen assimilation pathways related pathways of BCAAs was decoupled by removal
involving GDH and GS (Fig. 5). This approach led to the of the global transcriptional regulator CodY. Inactivation
accumulation of glutamate and BCAAs. Three transami- of BkdB to nullify the degradation pathway of BCAAs led
nation–deamination cycles were generated for utilization to accumulation of 2-keto acid pools. Finally, the engi-
of BCAAs by overexpression of leuDH from Thermoac- neered B. subtilis enabled production of biofuels with
tinomyces intermedium and endogenous genes involving 18.9% of the theoretical yield after recruitment of the
Li et al. Biotechnol Biofuels (2018) 11:256 Page 10 of 15

Asn Arg Pro

Cys Ala Thr

NH3 NH3
NH3
NH3 NH3
Pyr Asp Glu Glu
Pyr OAA
SdaB SerB
Pyr Ser 2-KG
SerA SerC
Pyr 3-PG
2-KG
AlsS Glu Glu
Acetolactate
IlvCD 2-KG Glu Glu
LeuABCD Pyr
KIV/KIC/KMV NH3 NH3
GDH
KivD
Glu Glu
Aldehyde Pyr Ala
YqhD NH3 GS 2-KG
AvtA
IlvE
iBOH/2MB/3MB KMV/KIC
Gln
KIV
Val LeuDH
NH3 IlvE Ile/Leu NH3

2-KG Glu
cell membrane

Fig. 5  Rewiring of metabolic pathways in E. coli for conversion amino acids to biofuels and ammonia. The rational design of catabolic pathways for
amino acids leads to the accumulation of intracellular metabolites involving pyruvate, α-oxoglutarate, glutamate, and ammonia. The deleted genes
are marked with “X”. OAA oxaloacetate, Pyr pyruvate, 2-KG α-oxoglutarate, KIV 2-ketoisovalerate, KIC 2-ketoisocaproate, KMV 3-ketomethylvalerate,
iBOH isobutanol, 2-MB 3-methyl-1-butanol, 3MB 3-methyl-1-butanol

Ehrlich pathway comprising Lactococcus lactis kivD and microbial production of C4 and C5 fusel alcohols from
E. coli yqhD. DDGS [115]. An E. coli strain which metabolizes glucose
and xylose was equipped with the synthetic pathway of
Discussion and future perspectives fusel alcohols by overexpressing Als, IlvCD, KivD, and
The world is now entering a new era of the bio-based YqhD (refer to Fig. 5). In addition to the synthetic path-
economy that is marked with sustainability and ecologi- way of fusel alcohols, the catabolic pathways of amino
cal soundness. To support the growth of this economy acids were rewired in another E. coli strain for high utili-
system, the biorefinery platforms need to be continu- zation of amino acids according to the reported approach
ously advanced. The historical production of bio-based [112]. Sugars and amino acids are released in DDGS
fuels and products has been mainly carried out with by pretreatment with dilute sulfuric acid and Pronase.
sugar feedstock. However, the cost of bio-based products Consequently, co-culturing of the two strains on DDGS
generally exceeds that of petrochemical counterparts. hydrolysate enables production of 10.3 g/L fusel alcohols.
A considerable volume of proteins will be generated in The production of putrescine by recombinant C. glutami-
the waste streams associated with the sugar-based pro- cum presents another example. By expressing ornithine
duction process. Therefore, the economical viability of decarboxylase, this strain was grown on glucose, while
the biorefinery platform can be realized by incorpora- supplemented with l-arginine as a precursor [118]. An
tion of the protein-based production scheme into the earlier study proposed a process to fractionate sugarcane
existing production scheme based on sugars. This sce- leaves and tops for simultaneous production of electric-
nario is well exemplified by a recent study reporting the ity, single cell proteins, and leaf proteins [119]. It would
Li et al. Biotechnol Biofuels (2018) 11:256 Page 11 of 15

be appealing to integrate this process with a production modified for utilization of surplus or non-essential amino
scheme of chemicals based on leaf proteins. Neverthe- acids. Recovery of the produced amino acid is made pos-
less, the development of the protein-based biorefinery is sible with crystallization [124]. In addition, the stability
still immature and fallen far behind the biomass-based of enzymes appears to be another challenge that limits
platform. the development of biotransformation. The method using
The biotransformation method provides a simple way directed evolution or/and immobilization would be use-
for production of bulk chemicals from amino acids. ful to address this issue [125, 126].
However, isolation of a single amino acid from protein Naturally existing microbes are evolved to utilize part
hydrolysates is required and remains technically difficult. of amino acids and produce fermentation products such
One potential solution is provided by the microbial pro- as hydrogen and n-butanoate/acetate [127, 128]. As
duction of cyanophycin [120, 121]. Cyanophycin consists described in “Catabolism of proteinogenic amino acids”
of a poly-l-aspartic acid backbone with l-arginine side section, it seems possible to engineer a microbe for uti-
chain. The employment of recombinant strains grown lization of all amino acids by integration of the catabolic
on sugars enables mass production of insoluble cyano- pathways from a variety of strains. One of various cata-
phycin by direct utilization of aspartic acid and arginine bolic pathways for a specific amino acid can be chosen
present in the medium. Either aspartic acid or arginine for design according to the engineering purpose. The
recovered from the hydrolysis treatment of isolated catabolic routes of amino acids interconnect the cen-
cyanophycin is readily applicable for the biotransforma- tral metabolism at distinct nodes (Fig. 4a). To direct the
tion production. Apparently, this approach provides a carbon flux to the desired pathway of a specific product,
useful route for selective separation of aspartic acid and the task necessitates a rational design of pathways and is
arginine from others in protein waste. The industrial pro- highly delicate and complicated to fulfill. A consolidated
duction of l-glutamic acid by microbial fermentation has platform for production of chemical is appealing with-
been practiced over 50  years ago since its birth [122]. out the need of processing proteins into amino acids. An
Until now, almost all proteinogenic amino acids can be illustrative example employs Lactococcus lactis (naturally
produced with rationally designed microbes on indus- secreting proteases) for nisin production based on defat-
trial scale [123]. Based on this well-established fermen- ted soybean meal [129]. Amino acids from intracellular
tation scheme, the amino-acid producer strain may be hydrolysis of small peptides were utilized to synthesize

O O OH OH
OH HO
HO OH
OH
HO
O O
OH OH

Adipic acid Muconic acid Glucose Sucrose Sorbitol

O OH OH

HO
C
C
OH
D-Glucaric acid Glucose-6-phosphate
OH OH O

Fructose-6-phosphate

Fructose-1,6-bisphosphate
HO OH

Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate 1,3-Propanediol

CH3
H2C
CH3 CH3 CH3 OH
H2
C
Farnesene H3C OH O
CH3

CH2 PEP Ethanol O Acrylic acid

H2C
Isoprene
OH

CH3 O OH Isobutanol Pyruvate Lactate OH

Acryloyl-CoA n-Propanol
OH
H
O OH
n
Polyhydroxyalkanoates O

Acetoacetyl-CoA Acetyl-CoA 3-Hydroxypropionic acid 1,2-Propanediol

HO OH
OH n-Butanol n-Butyraldehyde H3C OH
HO
O Acetate
O
O OH
O OH
O
H Citrate HO OH
Isopropanol H O
OH
OH Oxaloacetic acid cis-Acotinate Itaconic acid HO

Glyoxylate Isocitrate ethylene

H2C CH2
Acetyl-CoA
Malate
Glycolic acid α-ketoglutarate O

O
Caprolactam NH

OH
Fumarate HO
Succinyl-CoA 1, 4-Butanediol
OH
Succinate HO

O 5-Aminolevulinic Acid
HO
OH O

O
H2N OH
O

Fig. 6  Biomass-derived building blocks of industrial interest. The schematic drawing illustrates the synthetic pathways leading to building blocks of
industrial interest
Li et al. Biotechnol Biofuels (2018) 11:256 Page 12 of 15

nisin. A synthetic consortium may be designed by co-cul- 4

 Department of Medical Laboratory Science and Biotechnology, China Medi‑
cal University, No. 91, Hsueh‑Shih Road, Taichung 40402, Taiwan. 5 Depart‑
turing of one protease-secreting strain and another strain ment of Chemical Engineering, Feng Chia University, 100 Wenhwa Road,
specialized in utilization of amino acids. This approach Taichung 40724, Taiwan. 6 Department of Health and Nutrition Biotechnology,
will simplify the genetic manipulation and ameliorate Asia University, Taichung 41354, Taiwan. 7 Department of Medical Research,
China Medical University Hospital, Taichung 40447, Taiwan.
the metabolic stress incurred by overexpression of many
enzymes. Competing interests
Figure 6 shows the biomass-derived building blocks of The authors declare that they have no competing interests.
chemicals that receive industrial interest. All of them are Availability of data and materials
either in development or in pipeline. The list includes C-2 Not applicable.
products (e.g., ethanol [130], ethylene [131], and glycolic
Ethics approval and consent to participate
acid [132]), C-3 products (e.g., lactate [133], 1,3-pro- Not applicable.
panediol [134], isopropanol [135], 3-hydroxy propanoic
acid [136], 1,2 propanediol [137], acrylic acid [138], and Funding
This work is supported by Ministry of Science and Technology (MOST
n-propanol [139]), C-4 products (e.g., n-butanol [140], 105-2221-E-035-085-MY3 and 105-2221-E-039-010-MY3), Taiwan.
isobutanol [141], n-butyraldehyde [142], 1,4-butanediol
[143], and succinate [144]), and products with a high Publisher’s Note
carbon number (n > 6) (e.g., muconic acid [145], itaconic Springer Nature remains neutral with regard to jurisdictional claims in pub‑
acid [146], isoprene [147], 5-aminolevulinic acid [148], lished maps and institutional affiliations.
adipic acid [149], glucaric acid [150], caprolactam [151], Received: 10 May 2018 Accepted: 22 August 2018
α-farnesene [152], and polyhydroxyalkanoates [153]). In
general, the synthetic routes of these terminal products
starting with sugars can be transformed into those based
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