Available online at www.sciencedirect.

com

ScienceDirect

Biobased organic acids production by metabolically

engineered microorganisms
Yun Chen1,2 and Jens Nielsen1,2,3

Bio-based production of organic acids via microbial chemicals through metabolic engineering [1,2]. Further
fermentation has been traditionally used in food industry. With developments in the field of systems and synthetic biol-
the recent desire to develop more sustainable bioprocesses for ogy, along with evolution and selection has allowed great
production of fuels, chemicals and materials, the market for advances in our engineering capability to develop highly
microbial production of organic acids has been further efficient microorganisms that can produce chemicals of
expanded as organic acids constitute a key group among top interest [3,4].
building block chemicals that can be produced from renewable
resources. Here we review the current status for production of With the desire to develop more sustainable processes for
citric acid and lactic acid, and we highlight the use of modern production of fuels, chemicals and materials, the chemical
metabolic engineering technologies to develop high industry has started to exploit these technologies to
performance microbes for production of succinic acid and 3- develop novel bioprocesses for production of both fine
hydroxypropionic acid. Also, the key limitations and challenges and commodity chemicals. Organic acids constitute a key
in microbial organic acids production are discussed. group among top platform chemicals that can be pro-
Addresses
duced by microbial fermentation. The current market for
1
Department of Biology & Biological Engineering, Chalmers University of microbial organic acid production is still moderate and
Technology, SE412 96 Gothenburg, Sweden often associated with food applications. However, one can
2
Novo Nordisk Foundation Center for Biosustainability, Chalmers envision its future market potential with this transition
University of Technology, SE412 96 Gothenburg, Sweden
3 towards their use as building blocks for synthesis of a wide
Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, DK2970 Hørsholm, Denmark range of chemicals. Here, we review recent advances in
metabolic engineering to develop microbial cell factories
Corresponding author: Nielsen, Jens ([email protected]) for the production of industrially important organic acids,
including citric acid and lactic acid as examples of indus-
Current Opinion in Biotechnology 2016, 37:165–172
trially large-scale implemented production of organic
acids, and succinic acid and 3-hydroxypropionic acid as
This review comes from a themed issue on Food biotechnology
representations at the stage of commercialization, with a
Edited by Yong-Su Jin and Jin-Ho Seo particular emphasis on the latest concerning product
For a complete overview see the Issue and the Editorial yield, productivity and achieved product concentrations.
Available online 31st December 2015 Also, we discuss the key limitations and challenges in the
http://dx.doi.org/10.1016/j.copbio.2015.11.004
field of microbial organic acid production.
0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Citric acid
Citric acid is a successful example of a commercially bio-
produced organic acid with annual production estimated
at 1.75 million tons in 2011 and having a continuous
increasing demand/consumption [5]. Besides its predom-
Introduction inant use in the food and beverage industry, citric acid has
Since ancient times microbial fermentations have been been also used in pharmaceuticals and a range of technical
traditionally used in food and feed applications, such as and industrial segments as well. The current industrial
for production of organic acids, alcohols, amino acids and production relies on Aspergillus niger through submerged
vitamins. The introduction of citric acid production via fermentation of starch-based or sucrose-based media [6].
microbial fermentation around 1920 marks the foundation Industry is seeking newer cheap and economic process
of industrial-scale microbial fermentation for chemicals technologies to enhance the competitiveness to cope with
production. This was further cemented with the intro- the current adverse market conditions. A cost reduction,
duction of first penicillin production and later production in this light, can be achieved by shifting towards using less
of many other antibiotics, and with the introduction of expensive substrates such as agro-industrial wastes and
industrial fermentations for the production of enzymes in their by-products [7]. However, agro-industrial wastes are
the 1970s. With the emergence of genetic engineering it often complex, which may require pretreatment to ensure
became possible to produce compounds that are not proper nutrient availability and rheological properties,
native to microbes and enabled construction of microbial and this may further complicate the downstream proces-
cell factories that can be used for the production of sing. Furthermore, for production of chemicals to be used

www.sciencedirect.com Current Opinion in Biotechnology 2016, 37:165–172

166 Food biotechnology

for polymer production, where a high purity of the chem- Lactic acid
ical is required, the use of complex substrates may in- Lactic acid has traditionally been produced through fer-
crease the costs of purification. Alternatively, the use of mentation and used widely in food, cosmetic, pharma-
yeast for instance Yarrowia lipolytica has also been devel- ceutical and leather industries. The main current
oped for the production of citric acid from various carbon application of lactic acid is however as a chemical inter-
sources, for example, inulin through surface displaying an mediate for the production of polylactic acid, which is one
exo-inulinase [8]. Further engineering the strain by of the most promising biodegradable polymers. The
down-regulation of the ATP-citrate lyase (ACL, global annual production of lactic acid has been estimated
Figure 1) and enhancing the expression of isocitrate lyase at 370,000 tons in 2011, ranking among the high-volume
(ICL, Figure 1) has achieved citric acid production at a products being biologically produced [11]. Biological
titer of 84 g/L from 10% inulin [9]. Despite this impres- production of lactic acid was traditionally carried out
sive titer, it is still significantly lower than what has been using lactic acid bacteria, but this process is not economi-
achieved using A. niger grown on glucose, that is, a yield of cal viable for large industrial-scale fermentations owing to
0.88 g/g glucose and a titer of 240 g/L [10], but it demon- the requirements of nutritionally rich media and moder-
strated that metabolic engineering offers a potentially ate pH conditions [12]. Other natural producers such as
useful approach for improving yields and fermentation Rhizopus oryzae have been exploited for lactic acid pro-
rates in Y. lipolytica. duction and a high titer of 231 g/L of lactic acid with yield

Figure 1

Sugars

Glycerol

O
LDH O PDC1
OH OH

OH O
Ethanol
Lactic acid Pyruvic acid

Acetyl-CoA

Oxaloacetate
O OH Acetyl-CoA
O O
ACL
HO OH
OH
Reductive pathway

Malate Citric acid Oxaloacetate

Glyoxylate
ICL Isocitrate

Fumarate

O
HO
OH
O
Succinic acid
Current Opinion in Biotechnology

Overview of the metabolic pathways for production of lactic acid, citric acid and succinic acid from sugars. ACL, ATP-citrate lyase; ICL, isocitrate
lyase; LDH, lactate dehydrogenase; PDC1, pyruvate decarboxylase 1.

Current Opinion in Biotechnology 2016, 37:165–172 www.sciencedirect.com

 Bio-production of organic acids Chen and Nielsen 167

and productivity of 0.92 g/g glucose and 1.83 g/L/h has have contributed to the application of metabolic engineer-
been reported using immobilized R. oryzae in a fed-batch ing to improve the rumen bacteria performance. The use of
13
culture [13]. However, the use of R. oryzae has not been C metabolic flux analysis was employed to identify
proven to be commercially relevant as it requires aeration, undesired fluxes in Basfia succiniciproducens, and subse-
which increases costs of operation, and maintenance of quently engineering of the strain by deletion of pflD and
pH above 4.5 for efficient production. ldhA resulted in a succinic acid yield of 0.71 g/g glucose
[16]. An engineered Mannheimia succiniciproducens has also
Owing to its robustness such as tolerance to low pH and been reported to produce homo-succinic acid of 90–100 g/
less stringent nutritional requirements, Saccharomyces cer- L with yield and productivity of 0.9–1.0 g/g glucose or
evisiae has been explored for lactic acid production. Intro- sucrose, 5–30 g/L/h, respectively [17]. Currently, BASF/
duction of L-lactate dehydrogenase gene from Pelodiscus Corbion-Purac (joint venture Succinity) is in the process of
sinensis (LDH, Figure 1) and fine tuning its expression commercializing biobased production of succinic acid
enabled S. cerevisiae to accumulate 27.6 g/L of L-lactic using B. succiniciproducens. A drawback of the use of rumen
acid [14]. Further rewiring cellular metabolic fluxes to the bacteria is that a complex medium is required as these
production of lactic acid by reducing competing pathways organisms are vitamins and amino acids auxotrophs.
that lead to ethanol and glycerol formation (Figure 1)
increased lactic acid production up to 35 g/L. Moreover, The commonly used bacterium Escherichia coli has been
manipulating the availability of intracellular redox by metabolically engineered for succinic acid production. A
deleting the external NADH dehydrogenase genes has relatively high succinic acid yield of 1.0 g/g glucose with
proven to be important to achieve high L-lactic acid titer and productivity of 71.6 g/L and 0.75 g/L/h, was
production, resulting in a strain capable of producing achieved by elimination of the side-product formation
117 g/L of L-lactic acid in a fed-batch mode with pH and reinforcement of the reductive pathway of the TCA
controlled at 3.5 [14]. Cargill has developed a yeast-based cycle (Figure 1) [18]. Myriant is working on the commer-
process (undisclosed yeast species) for lactic acid produc- cialization of succinic acid production using E.
tion that was commercially implemented in 2008 [11]. coli. Another commonly applied bacterium Corynebacteri-
Their yeast was engineered by deletion of pyruvate um glutamicum has also been studied for biobased produc-
decarboxylase 1 gene and with integration of the L-lactate tion of succinic acid. The highest final succinic acid
dehydrogenase gene from Lactobacillus (LDH, Figure 1). concentration published is 146 g/L with yield and pro-
These engineering steps, in combination with traditional ductivity of 0.92 g/g glucose and 3.2 g/L/h, achieved in a
mutagenesis and selection, including evolution, were fed-batch process by using this organism [19]. The chal-
employed to develop improved strains that could produce lenge of using this organism is that a two stage cultivation
up to 135 g/L of lactic acid at pH 3. Under this condition process is required for shifting aerobic growth to anaero-
about 90% of the lactic acid produced were in free acid bic production phase, and it would be difficult to imple-
form, thereby significantly reducing the use of calcium ment this system, such as washing and resuspension of
hydroxide and sulfuric acid, and in turn contributing cells [19,20], in a large scale industrial fermentation.
significantly to cost reduction in the downstream process
compared with bacterial production systems. S. cerevisiae has also been developed to produce succinic
acid and it has the advantage that it allows low pH
Succinic acid fermentation. DSM/Roquette (joint Venture Reverdia)
Succinic acid is predominantly produced from petro- constructed an efficient S. cerevisiae strain able to produce
chemicals at a scale of 30,000–50,000 tons per year [15] 100 g/L of succinic acid at pH 3 [17]. The advantage of
and mainly used as a chemical intermediate for the low pH fermentation is the cost reduction in the pH
production of fine chemicals, such as perfume esters or titrant and downstream processing. Moreover, low pH
as a neutralizing agent used in the food industry. Cur- fermentation to free succinic acid with direct crystalliza-
rently, there is a transition towards a much larger scale tion has been shown to be the most attractive from an
using microbial fermentation, and several companies or environmental perspective, compared with other fermen-
consortia have started or are about to start industrial tation routes and petrochemical processes [21]. In this
production of succinic acid. It is hereby expected that light, recently isolated yeast Issatchenkia orientalis SD108,
succinic acid will become cheaper and could serve as a that is tolerant to low pH and high concentrations of
building block for many bulk chemicals, such as 1,4- organic acids, has been engineered through enhancing the
butanediol, gamma-butyrolactone, tetrahydrofuran, n- reductive TCA cycle to produce succinic acid (Figure 1)
methylpyrrolidone or the biodegradable polymers poly- [22]. Bioamber/Mitsui have switched from their engi-
butylene succinate and polyester polyols. neered E. coli system to low pH yeast technology using
the yeast Candida krusei, which they licensed from Cargill
Several rumen bacteria have received considerable interest [15]. Although low pH fermentation is advantageous for
as they naturally secrete substantial amounts of succinate. downstream processing, a big challenge is the effect of
Genome sequencing and systems-wide characterization low pH on the overall fermentation performance that

www.sciencedirect.com Current Opinion in Biotechnology 2016, 37:165–172

168 Food biotechnology

requires dedicated strain and fermentation process devel- as diapers and paints more sustainable [23]. Because of
opment. the high industrial relevance, various industrial parties,
such as OPX Biotechnologies-Dow Chemical, Cargill-
3-Hydroxypropionic acid Novozymes-BASF, have been developing biobased pro-
3-Hydroxypropionic acid (3-HP) can serve as a platform for cesses for the production of 3-HP with the objective to
the production of 3-carbon intermediates, which are today derived acrylic acid from different renewable feed-stocks
produced in the petrochemical industry, and in particular such as glycerol and sugars.
because it can easily be converted to acrylic acid upon
dehydration. A variety of bulk chemicals include acrylam- Production of 3-HP from glycerol
ide, acrylonitrile, ethyl 3-HP, 1,3-propanediol, and malonic Microbial conversion of glycerol into 3-HP has been
acid can also be easily produced from 3-HP. The demand reported most extensively in bacterial systems, such as
for acrylic acid is increasing every year due to its various E. coli and Klebsiella pneumonia [24–26]. Both CoA-de-
applications in superabsorbent, adhesive, surface coating, pendent and CoA-independent pathways have been de-
and paint. With a worldwide production of more than scribed for the production of 3-HP with glycerol as carbon
5 million tons, the current market for acrylic acid is esti- substrate, nevertheless, the CoA-dependent pathway has
mated to be more than USD 11 billion. Its annual world been less explored as it is more complicated compared
demand is anticipated to reach 8 million tons and the with the CoA-independent pathway. The CoA-indepen-
market size is expected to reach USD 18 billion in 2020 dent pathway needs two enzymatic steps: the conversion
(https://www.alliedmarketresearch.com/acrylic-acid- of glycerol into 3-hydroxypropionaldehyde (3-HPA) by
market). glycerol dehydratase and the oxidation of 3-HPA to yield
3-HP by aldehyde dehydrogenase (Figure 2).
Currently, acrylic acid is produced through oxidation of
petroleum-based propylene in a two-step process via To develop a recombinant E. coli strain for high-level
acrolein, achieving about 90% yield overall. Unfortunate- production of 3HP from glycerol, previous efforts have
ly, petrochemical carbon sources are not renewable and been focused on stabilizing glycerol dehydratase and
the cost will rise when it comes to the short supply of balancing the enzyme activity between dehydratase
petroleum. With a biobased process for the production of and dehydrogenase. A recent study has demonstrated
3-HP it will be possible to make consumer products such that the second enzyme is crucial for 3-HP production

Figure 2

OH
Sugars Propylene HO OH

NAD+
O Glycerol
+
NADH NAD
NADH ATP OH O B12
Pyruvate OH
Lactic acid Acrolein
ATP

O OH
Oxaloacetate Acetyl-CoA O
ATP 3-Hydroxypropionaldehyde
OH (3-HPA)
Acrylic acid NAD+
Aspartate Malonyl-CoA
NADPH NADH

NADP+ O O
O
O OH HO OH
H 2N OH
Malonic 3-Hydroxypropionic acid
beta-Alanine semialdehyde NADPH +
NADP (3-HP)
Current Opinion in Biotechnology

Schematic representation of various biological routes for production of 3-hydroxypropionic acid and chemical catalysis routes for acrylic acid.
Reactions encoded by genes obtained from heterologous organisms are indicated by blue (glycerol pathway), red (b-alanine pathway) or green
(malonyl-CoA pathway). Reactions through chemical catalysis are donated by orange color arrows.

Current Opinion in Biotechnology 2016, 37:165–172 www.sciencedirect.com

 Bio-production of organic acids Chen and Nielsen 169

as the accumulation of the intermediate product 3-HPA hold as they conclude that the glycerol oversupply turned
is toxic to the host cells [27]. By selecting the most out to be only temporary [33].
efficient aldehyde dehydrogenase, GabD4 derived from
Cupriavidus necator, combined with directed mutagene- Production of 3-HP from sugar
sis this enzyme activity was further increased and the A number of potential pathways have been described for
highest 3-HP titer and productivity was achieved, up to 3-HP production from glucose, mainly through malonyl-
71.9 g/L of 3-HP with a productivity of 1.8 g/L/h using CoA, b-alanine or lactate [26], and examined mainly via
glucose and glycerol as co-carbon sources. One draw- employing S. cerevisiae.
back with this system is the requirement of adding the
enzyme cofactor vitamin B12 to the culture medium, Malonyl-CoA pathway
which is not feasible at an industrial scale. Although Heterologous expression of malonyl-CoA reductase de-
E. coli has been reported to produce vitamin B12 by rived from Chloroflexus aurantiacus in E. coli and S. cere-
heterologous expressing the biosynthetic pathway from visiae have been described for the production of 3-HP. A
Pseudomounas denitrificans [28], involving more than recent patent by OPX Biotechnologies showed that one
25 genes represents a big challenge for incorporating extensively engineered E. coli strain was able to produce
and balancing this pathway with 3-HP production will 48.4 g/L of 3-HP with a final yield of 0.53 g/g glucose in
require extensive engineering. aerated fed-batch fermentation [34]. This was achieved
by enhancing the availability of malonyl-CoA through
K. pneumonia has been suggested as an interesting alter- attenuation of fatty acid biosynthesis combined with
native host because it can synthesize vitamin B12 natu- overexpression of the native acetyl-CoA carboxylase gene
rally. However, the use of K. pneumonia as a host strain for and improving the carbon flux towards acetyl-CoA
3-HP production can be challenging, as on one hand, through elimination of byproducts such as lactate, etha-
efficient regeneration of NAD+ from NADH via the nol, acetate and methylglyoxal. As this bi-functional
electron transport chain needs aerobic conditions; on malonyl-CoA reductase requires NADPH, improving
the other hand, the production of vitamin B12 significantly precursor supply coupled with enhanced redox co-factor
decreases with increasing aeration and the glycerol dehy- has been shown in S. cerevisiae to improve 3-HP produc-
dratase DhaB is also inactivated by oxygen. The crucial tion by fivefold, up to 0.46 g/L [35]. Furthermore, in-
role of aeration conditions was demonstrated in one study, creasing NADPH supply by replacement of native NAD
where optimization of the aeration rate was able to dependent glyceraldehyde-3-phosphate dehydrogenase
improve the production of 3-HP up to 48.9 g/L, with a (GAPDH) with exogenous NADP dependent GAPDH,
yield and productivity of 0.40 g/g glycerol and 1.75 g/L/h, combined with improving metabolic flux towards 3-HP
respectively [29]. But 25.3 g/L 1,3-propanediol was accu- via overexpression of Pdc, Ald, Acs, Acc genes in S.
mulated at the same time, owing to the requirements for cerevisiae resulted in a 3-HP titer of 9.5 g/L with a yield
NAD+ regeneration. To address this problem, nitrate, of 0.09 g/g glucose [36]. Although the titer and yield still
which can serve as an electron acceptor, was used as a lag behind that achieved with E. coli, this is promising as it
nitrogen source and this prevented byproducts accumu- allows for the use of low pH fermentation.
lation during anaerobic cultivation of K. pneumonia for 3-
HP production [30]. The reduced carbon flux towards 3- Studies on both host systems have highlighted that en-
HP production was also observed and hence additional richment of the intracellular malonyl-CoA pool is crucial
studies are required to optimize this system. for efficient 3-HP production when employing the mal-
onyl-CoA pathway. For this purpose, one interesting
Glycerol is produced abundantly today as a by-product study showed that in S. cerevisiae acetyl-CoA carboxylase,
from processing triglycerides from fats and plant oils into the key enzyme for malonyl-CoA synthesis can be upre-
biodiesel. A surplus and dramatic decrease in crude gulated by abolishing phosphorylation inhibition and
glycerol prices have stimulated many researches to eval- improved 3-HP production by threefold with this strategy
uate microbial conversion of glycerol into valuable che- alone [37]. Other strategies for engineering malonyl-CoA
micals. But the use of plant and cooking oils for biodiesel supply, such as the use of synthetic antisense RNAs [38]
production has been criticized due to the high costs of the or malonyl-CoA-responsive biosensors [39] have been
vegetable biodiesel process as well as the limited reduc- developed in E. coli, and this could be further exploited
tion in greenhouse gas emission by this process compared for 3-HP improving production in both cell factories.
with petroleum [31,32]. Therefore, the current biodiesel
production is not probably to be part of the future biofuel b-Alanine pathway
industry and hence the supply and cost of glycerol will be The use of either b-alanine/pyruvate aminotransferase
an issue. This problem has been addressed by Arkema, a (BAPAT) or b-alanine/a-ketoglutarate aminotransfer-
specialty chemical company that had developed a process ase (4,4-aminobutyrate transaminase) has been de-
ready for full-scale manufacturing acrylic acid from bio- scribed for 3-HP production (Figure 2), however,
based glycerol via acrolein (Figure 2), but then put it on only proof of concept production was shown. Recently

www.sciencedirect.com Current Opinion in Biotechnology 2016, 37:165–172

170 Food biotechnology

the identification of a novel b-alanine/pyruvate amino- [48,49], and (3) simultaneous co-utilization of glucose
transferase, BcBAPAT derived from Bacillus cereus, was and xylose that are two abundant sugars in lignocellulosic
disclosed and when expressed in a S. cerevisiae this cell hydrolysates [50]. Furthermore, using a cheaper feed-
factory could produce 13.7 g/L 3-HP with a yield and stock such a lignocellulosic hydrolysates may be offset by
productivity of 0.07 g/g glucose and 0.17 g/L/h, respec- the requirement for additional downstream processing
tively [40]. This was obtained through overexpression costs, in particular if the chemical is to be used for
of yeast native PYC and AAT, and introduction of panD production of polymers where it is important to have a
from Tribolium castaneum, bapat from B. cereus, ydfG from pure product. Despite progress in recent years, applica-
E. coli to channel the carbon flux towards 3-HP. Except tion of non-food biomass as feedstock has to advance
for recycling of L-alanine discussed in the study, one further before it is economically competitive with the use
potential drawback is redox co-factor imbalance in this of glucose or sucrose.
pathway starting from glucose as the ydfG used encodes
a NADP dependent 3-hydroxypropanoate dehydroge- To facilitate downstream processing the final product
nase. This would imply a room for additional improve- concentrations need to be high, particularly for commod-
ments, as implemented in other pathways [35,36]. ity chemicals, the minimum for commercialization is
probably to be above 50 g/L [51]. This concentration is
Lactate pathway well above the toxic level of the organic acid for most
Cargill has been developing biobased production of 3-HP microorganisms. It is often difficult to rationally engineer
through the lactate intermediate pathway [41,42]. Re- improved tolerance into industrial model organisms due
combinant yeast and E. coli strains were reported to our poor understanding of the underlying molecular
produce 3-HP via the lactate pathway, however, no mechanisms of toxicity. One strategy to circumvent this
details on the yields and titers were given. The most issue is the use of adaptive laboratory evolution [52–54].
significant problem for this pathway is that it is thermo- With rapid developments in DNA sequencing technolo-
dynamically unfavorable under fermentation conditions, gies that enable the cost-effective genome re-sequencing,
as the dehydration of lactoyl-CoA to acryloyl-CoA will not it is now possible to investigate the links between geno-
be activated by an electron-withdrawing group and thus type and phenotype, and hereby the causal mutations in
necessitates an oxygen-sensitive radical mechanism [43]. the evolved tolerant phenotypes can be identified and
Moreover, it is probably to result in a mixture of lactate reverse engineered in the host. By using this strategy a
and 3-HP from which it will be difficult to purify 3-HP glutathione-dependent mechanism was identified to
selectively. Besides Cargill’s process for microbial con- improve 3-HP tolerance in S. cerevisiae [55]. Appling this
version of lactate to 3-HP followed by chemical catalysts approach to an E. coli MG 1655 derivate has yielded one
to acrylic acid, Massachusetts-based Myriant is develop- strain with not only improved tolerance to exogenous
ing a chemical route to acrylic acid that runs through octanoic acid, but also enhanced production of carboxylic
sugar-derived lactic acid (Figure 2), which is thought to be acids [56].
cheaper and enables making very high yields and con-
centrations [33]. However, routes for dehydration of Another effect of organic acids production is acidification
lactate by chemical catalysts also suffer from a low equi- that often requires large amounts of base to maintain the
librium ratio of acrylate/lactate under ambient conditions appropriate pH demand. Given that the pKa values for
[44]. most organic acids are typically 3–5, the pH of a fermen-
tation culture is expected to decrease to around 2.0 for
Challenges and perspective acid titers of 50 g/L. Choosing acid-tolerant strain as the
For large-scale organic acid production by microbial fer- host for development efforts is an alternative option to pH
mentation the costs of feedstock and downstream proces- titration, as it minimizes the consumption of bases for pH
sing are crucial constraints to make a biobased process control and thus reduces the cost for downstream proces-
economically viable [45]. Therefore, it is important to sing. However, direct production of organic acids at low
develop high performance strains in terms of titer, yield pH would need additional expense for maintenance
and productivity. Furthermore, in order to maintain a requirements for the cells, which might have an impact
sufficient margin between cost of feedstock and revenues on the overall fermentation performance [57,58]. Alter-
from product it may be necessary in the future to apply native technologies such as in situ removal of acid by
non-edible plant biomass, such as waste stream from extraction could be used with the integration of produc-
agriculture, forestry and paper milling as feedstock, since tion process [59].
they have the potential to be generally cheaper. However,
to efficiently utilize the sugars present in such plant Even though several hurdles still remain to accelerate
materials several challenges remain that are all being commercialization of microbial organic acid production,
addressed by different studies: (1) increasing the toler- with several important organic acids already being com-
ance to inhibitors that are derived from chemical hydro- mercialized or in advanced stages of industrial develop-
lysis of lignocellulose [46,47], (2) pentose utilization ment, it is expected that microbial production of organic

Current Opinion in Biotechnology 2016, 37:165–172 www.sciencedirect.com

 Bio-production of organic acids Chen and Nielsen 171

acid from biomass will become more important in the 10. Soccol CR, Vandenberghe LPS, Rodrigues C, Pandey A: New
perspectives for citric acid production and application. Food
future. Competition is currently on the production costs, Technol Biotechnol 2006, 44:141-149.
but a desire to establish a more sustainable society will 11. Miller C, Fosmer A, Rush B, McMullin T, Beacom D, Suominen P:
generally favor biobased organic acid production com- 3.17 — industrial production of lactic acid. In Comprehensive
pared with petrochemical. Whether exploiting natural Biotechnology, edn 2. Edited by Moo-Young M. Academic Press;
2011:179-188.
producers or choosing commonly applied industrial plat-
form strains as cell factories for organic acid production, it 12. Sauer M, Porro D, Mattanovich D, Branduardi P: Microbial
production of organic acids: expanding the markets. Trends
will require significant engineering efforts to enhance Biotechnol 2008, 26:100-108.
strain performance and thus achieve the production 13. Yamane T, Tanaka R: Highly accumulative production of L(+)-
metrics needed for commercialization. Fortunately, ad- lactate from glucose by crystallization fermentation with
vanced tools that have been continuously developed in immobilized Rhizopus oryzae. J Biosci Bioeng 2013, 115:90-95.
synthetic biology, such as tools for genome editing, ex- 14. Lee JY, Kang CD, Lee SH, Park YK, Cho KM: Engineering
pression controlling and high-throughput screening [60– cellular redox balance in Saccharomyces cerevisiae for
improved production of L-lactic acid. Biotechnol Bioeng 2015,
62] expand our engineering capability to speed up cell 112:751-758.
factory development processes. With aids of these ad- 15. Jansen ML, van Gulik WM: Towards large scale fermentative
vanced technologies metabolic engineering will continu- production of succinic acid. Curr Opin Biotechnol 2014,
ously play an important role in accelerating strain 30:190-197.
development for biobased organic acid production, and 16. Becker J, Reinefeld J, Stellmacher R, Schafer R, Lange A,
contribute to establish a more sustainable society.  Meyer H, Lalk M, Zelder O, von Abendroth G, Schroder H et al.:
Systems-wide analysis and engineering of metabolic pathway
fluxes in bio-succinate producing Basfia succiniciproducens.
Acknowledgments Biotechnol Bioeng 2013, 110:3013-3023.
A great example of the use of systems biology to identify the metabolic
Work in our group is supported by the Knut and Alice Wallenberg engineering targets.
Foundation, the Novo Nordisk Foundation, Vetenskapsrådet, FORMAS 17. Choi S, Song CW, Shin JH, Lee SY: Biorefineries for the
and Ångpanneföreningens Forskningsstiftelse. We would like to thank Dr. production of top building block chemicals and their
Boyang Ji, Yongjin Zhou, Mingtao Huang, Zongjie Dai and Jiufu Qin for derivatives. Metab Eng 2015, 28:223-239.
crucial reading of the manuscript.
18. Jantama K, Zhang X, Moore JC, Shanmugam KT, Svoronos SA,
Ingram LO: Eliminating side products and increasing succinate
yields in engineered strains of Escherichia coli C. Biotechnol
References and recommended reading Bioeng 2008, 101:881-893.
Papers of particular interest, published within the period of review,
have been highlighted as: 19. Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H: An
efficient succinic acid production process in a metabolically
 of special interest engineered Corynebacterium glutamicum strain. Appl
 of outstanding interest Microbiol Biotechnol 2008, 81:459-464.
20. Litsanov B, Brocker M, Bott M: Toward homosuccinate
1. Keasling JD: Manufacturing molecules through metabolic fermentation: metabolic engineering of Corynebacterium
engineering. Science 2010, 330:1355-1358. glutamicum for anaerobic production of succinate from
glucose and formate. Appl Environ Microbiol 2012, 78:
2. Bailey JE: Toward a science of metabolic engineering. Science 3325-3337.
1991, 252:1668-1675.
21. Cok B, Tsiropoulos I, Roes AL, Patel MK: Succinic acid
3. Li M, Borodina I: Application of synthetic biology for production production derived from carbohydrates: an energy and
of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast greenhouse gas assessment of a platform chemical toward a
Res 2014. bio-based economy. Biofuel Bioprod Bior 2014, 8:16-29.
4. Cho C, Choi SY, Luo ZW, Lee SY: Recent advances in microbial 22. Xiao H, Shao Z, Jiang Y, Dole S, Zhao H: Exploiting Issatchenkia
production of fuels and chemicals using tools and strategies orientalis SD108 for succinic acid production. Microb Cell Fact
of systems metabolic engineering. Biotechnol Adv 2014. 2014, 13:121.
5. Kobayashi K, Hattori T, Hayashi R, Kirimura K: Overexpression of
the NADP+-specific isocitrate dehydrogenase gene (icdA) in 23. Adom F, Dunn JB, Han J, Sather N: Life-cycle fossil energy
citric acid-producing Aspergillus niger WU-2223L. Biosci consumption and greenhouse gas emissions of bioderived
Biotechnol Biochem 2014, 78:1246-1253. chemicals and their conventional counterparts. Environ Sci
Technol 2014, 48:14624-14631.
6. Angumeenal AR, Venkappayya D: An overview of citric acid
production. LWT – Food Sci Technol 2013, 50:367-370. 24. Kumar V, Ashok S, Park S: Recent advances in biological
production of 3-hydroxypropionic acid. Biotechnol Adv 2013,
7. Dhillon GS, Brar SK, Kaur S, Verma M: Screening of agro- 31:945-961.
industrial wastes for citric acid bioproduction by Aspergillus
niger NRRL 2001 through solid state fermentation. J Sci Food 25. Valdehuesa KN, Liu H, Nisola GM, Chung WJ, Lee SH, Park SJ:
Agric 2013, 93:1560-1567. Recent advances in the metabolic engineering of
microorganisms for the production of 3-hydroxypropionic
8. Liu XY, Chi Z, Liu GL, Wang F, Madzak C, Chi ZM: Inulin acid as C3 platform chemical. Appl Microbiol Biotechnol 2013,
hydrolysis and citric acid production from inulin using the 97:3309-3321.
surface-engineered Yarrowia lipolytica displaying inulinase.
Metab Eng 2010, 12:469-476. 26. Chen Y, Nielsen J: Advances in metabolic pathway and strain
engineering paving the way for sustainable production of
9. Liu XY, Chi Z, Liu GL, Madzak C, Chi ZM: Both decrease in ACL1 chemical building blocks. Curr Opin Biotechnol 2013, 24:
gene expression and increase in ICL1 gene expression in 965-972.
marine-derived yeast Yarrowia lipolytica expressing INU1
gene enhance citric acid production from inulin. Mar Biotechnol 27. Chu HS, Kim YS, Lee CM, Lee JH, Jung WS, Ahn JH, Song SH,
(NY) 2013, 15:26-36.  Choi IS, Cho KM: Metabolic engineering of 3-hydroxypropionic

www.sciencedirect.com Current Opinion in Biotechnology 2016, 37:165–172

172 Food biotechnology

acid biosynthesis in Escherichia coli. Biotechnol Bioeng 2015, 46. Wang X, Yomano LP, Lee JY, York SW, Zheng H, Mullinnix MT,
112:356-364. Shanmugam KT, Ingram LO: Engineering furfural tolerance in
This study demonstrates the importance of aldehyde dehydrogenase for Escherichia coli improves the fermentation of lignocellulosic
3-HP production via glycerol pathway, resulting the highest titer and sugars into renewable chemicals. Proc Natl Acad Sci U S A
productivity for microbial 3-HP production reported thus far. 2013, 110:4021-4026.
28. Ko Y, Ashok S, Ainala SK, Sankaranarayanan M, Chun AY, 47. Kim SK, Jin YS, Choi IG, Park YC, Seo JH: Enhanced tolerance of
Jung GY, Park S: Coenzyme B12 can be produced by Saccharomyces cerevisiae to multiple lignocellulose-derived
engineered Escherichia coli under both anaerobic and aerobic inhibitors through modulation of spermidine contents. Metab
conditions. Biotechnol J 2014, 9:1526-1535. Eng 2015, 29:46-55.
29. Huang Y, Li Z, Shimizu K, Ye Q: Co-production of 3- 48. Young EM, Tong A, Bui H, Spofford C, Alper HS: Rewiring yeast
hydroxypropionic acid and 1,3-propanediol by Klebseilla  sugar transporter preference through modifying a conserved
pneumoniae expressing aldH under microaerobic conditions. protein motif. Proc Natl Acad Sci U S A 2014, 111:131-136.
Bioresour Technol 2013, 128:505-512.
49. Demeke MM, Dietz H, Li Y, Foulquie-Moreno MR, Mutturi S,
30. Ashok S, Mohan Raj S, Ko Y, Sankaranarayanan M, Zhou S, Deprez S, Den Abt T, Bonini BM, Liden G, Dumortier F et al.:
Kumar V, Park S: Effect of puuC overexpression and nitrate Development of a D-xylose fermenting and inhibitor tolerant
addition on glycerol metabolism and anaerobic 3- industrial Saccharomyces cerevisiae strain with high
hydroxypropionic acid production in recombinant Klebsiella performance in lignocellulose hydrolysates using metabolic
pneumoniae DglpKDdhaT. Metab Eng 2013, 15:10-24. and evolutionary engineering. Biotechnol Biofuels 2013, 6:
89.
31. Caspeta L, Buijs NAA, Nielsen J: The role of biofuels in the future
energy supply. Energy Environ Sci 2013, 6:1077-1082. 50. Farwick A, Bruder S, Schadeweg V, Oreb M, Boles E: Engineering
32. Caspeta L, Nielsen J: Economic and environmental impacts of  of yeast hexose transporters to transport D-xylose without
microbial biodiesel. Nat Biotechnol 2013, 31:789-793. inhibition by D-glucose. Proc Natl Acad Sci U S A 2014,
111:5159-5164.
33. Tullo AH: Hunting for biobased acrylic acid: companies vie for a [48,50] great examples that illustrate reprogramming transporter proper-
nonpetrochemical route into this lucrative industry. Chem Eng ties via rationally altering a specific sequence motif or mutagenesis
News 2013, 91:18-19. combined with screening.
34. Lynch MD, Gill RT, Lipscomb TEW: Methods for producing 3- 51. Van Dien S: From the first drop to the first truckload:
hydroxypropionic acid and other products. 2014. commercialization of microbial processes for renewable
chemicals. Curr Opin Biotechnol 2013, 24:1061-1068.
35. Chen Y, Bao J, Kim IK, Siewers V, Nielsen J: Coupled incremental
precursor and co-factor supply improves 3-hydroxypropionic 52. Portnoy VA, Bezdan D, Zengler K: Adaptive laboratory
acid production in Saccharomyces cerevisiae. Metab Eng 2014, evolution — harnessing the power of biology for metabolic
22:104-109. engineering. Curr Opin Biotechnol 2011, 22:590-594.
36. Jensen NB, Borodina I, Chen Y, Maury J, Kildegaard KR, Förster J, 53. Ling H, Teo W, Chen B, Leong SS, Chang MW: Microbial
Nielsen J: Microbial production of 3-hydroxypropionic acid. 2014, tolerance engineering toward biochemical production: from
WO2014198831 A1. lignocellulose to products. Curr Opin Biotechnol 2014, 29:
99-106.
37. Shi S, Chen Y, Siewers V, Nielsen J: Improving production of
malonyl coenzyme A-derived metabolites by abolishing Snf1- 54. Caspeta L, Chen Y, Ghiaci P, Feizi A, Buskov S, Hallstrom BM,
dependent regulation of Acc1. mBio 2014, 5 e01130-14. Petranovic D, Nielsen J: Altered sterol composition renders
yeast thermotolerant. Science 2014, 346:75-78.
38. Yang Y, Lin Y, Li L, Linhardt RJ, Yan Y: Regulating malonyl-CoA
metabolism via synthetic antisense RNAs for enhanced 55. Kildegaard KR, Hallstrom BM, Blicher TH, Sonnenschein N,
biosynthesis of natural products. Metab Eng 2015, 29:217-226. Jensen NB, Sherstyk S, Harrison SJ, Maury J, Herrgard MJ,
39. Xu P, Li L, Zhang F, Stephanopoulos G, Koffas M: Improving fatty Juncker AS et al.: Evolution reveals a glutathione-dependent
 acids production by engineering dynamic pathway regulation mechanism of 3-hydroxypropionic acid tolerance. Metab Eng
and metabolic control. Proc Natl Acad Sci U S A 2014, 2014, 26C:57-66.
111:11299-11304. 56. Royce LA, Yoon JM, Chen Y, Rickenbach E, Shanks JV,
Description of a method using transcriptional regulator FapR as a bio- Jarboe LR: Evolution for exogenous octanoic acid tolerance
sensor to dynamically control gene expressions involved in the supply improves carboxylic acid production and membrane integrity.
and consumption of malonyl-CoA. Metab Eng 2015, 29:180-188.
40. Borodina I, Kildegaard KR, Jensen NB, Blicher TH, Maury J,
57. van Maris AJ, Konings WN, van Dijken JP, Pronk JT: Microbial
Sherstyk S, Schneider K, Lamosa P, Herrgard MJ, Rosenstand I
export of lactic and 3-hydroxypropanoic acid: implications
et al.: Establishing a synthetic pathway for high-level
for industrial fermentation processes. Metab Eng 2004, 6:
production of 3-hydroxypropionic acid in Saccharomyces
245-255.
cerevisiae via beta-alanine. Metab Eng 2015, 27:57-64.
41. Gokarn R, Selifonova OV, Gort S, Selmer T, Buckel W: 3- 58. Abbott DA, Zelle RM, Pronk JT, van Maris AJ: Metabolic
Hydroxypropionic acid and other organic compounds. US engineering of Saccharomyces cerevisiae for production of
patent 2011, US 8,076,120B2. carboxylic acids: current status and challenges. FEMS Yeast
Res 2009, 9:1123-1136.
42. Gokarn RR, Selifonova, OV, Jessen HJ, Gort SJ, Selmer T, Buckel
W: 3-hydroxypropionic acid and other organic compounds. 59. Lopez-Garzon CS, Straathof AJ: Recovery of carboxylic acids
2015, US 8,999,700B2.  produced by fermentation. Biotechnol Adv 2014, 32:873-904.
Gives a good overview of the options for recovering fermentative car-
43. Bar-Even A, Flamholz A, Noor E, Milo R: Rethinking glycolysis: boxylic acids.
on the biochemical logic of metabolic pathways. Nat Chem Biol
2012, 8:509-517. 60. Zeitoun RI, Garst AD, Degen GD, Pines G, Mansell TJ, Glebes TY,
Boyle NR, Gill RT: Multiplexed tracking of combinatorial
44. Straathof AJ, Sie S, Franco TT, van der Wielen LA: Feasibility of genomic mutations in engineered cell populations. Nat
acrylic acid production by fermentation. Appl Microbiol Biotechnol 2015, 33:631-637.
Biotechnol 2005, 67:727-734.
61. Redden H, Morse N, Alper HS: The synthetic biology toolbox for
45. Straathof AJJ: 2.57 — the proportion of downstream costs in tuning gene expression in yeast. FEMS Yeast Res 2014.
fermentative production processes. In Comprehensive
Biotechnology, edn 2. Edited by Moo-Young M. Academic Press; 62. David F, Siewers V: Advances in yeast genome engineering.
2011:811-814. FEMS Yeast Res 2014.

Current Opinion in Biotechnology 2016, 37:165–172 www.sciencedirect.com