Accepted Manuscript

Title: The flexible feedstock concept in Industrial

Biotechnology: metabolic engineering of Escherichia coli,
Corynebacterium glutamicum, Pseudomonas, Bacillus and
yeast strains for access to alternative carbon sources

Author: Volker F. Wendisch Luciana Fernandes Brito Marina

Gil Lopez Guido Hennig Johannes Pfeifenschneider Elvira
Sgobba Kareen H. Veldmann

PII: S0168-1656(16)31432-8
DOI: http://dx.doi.org/doi:10.1016/j.jbiotec.2016.07.022
Reference: BIOTEC 7634

To appear in: Journal of Biotechnology

Received date: 25-5-2016

Revised date: 25-7-2016
Accepted date: 28-7-2016

Please cite this article as: Wendisch, Volker F., Brito, Luciana Fernandes, Gil
Lopez, Marina, Hennig, Guido, Pfeifenschneider, Johannes, Sgobba, Elvira, Veldmann,
Kareen H., The flexible feedstock concept in Industrial Biotechnology: metabolic
engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus
and yeast strains for access to alternative carbon sources.Journal of Biotechnology
http://dx.doi.org/10.1016/j.jbiotec.2016.07.022

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The flexible feedstock concept in Industrial Biotechnology: metabolic engineering of Escherichia coli,
Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources

Volker F. WENDISCH* volker.wendisch@uni-bielefeld.de, Luciana Fernandes BRITO, Marina GIL LOPEZ, Guido
HENNIG, Johannes PFEIFENSCHNEIDER, Elvira SGOBBA, Kareen H. VELDMANN

Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany

*
Corresponding author at: Genetics of Prokaryotes, Faculty of Biology & Center for Biotechnology, Bielefeld
University, Universitätsstr. 25, 33615 Bielefeld, Germany
Highlights

 Flexible feedstock concept implemented for industrial biotechnology

 Biotechnological production hosts engineered for utilization of alternative carbon sources
 Second generation feedstocks made accessible for chemicals and biofuels production
 Strain development for utilization of lignocellulosics and other complex polymers
 Metabolic engineering for value-added compounds from agro-waste
ABSTRACT

Most biotechnological processes are based on glucose that is either present in molasses or generated

from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance

for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and

amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into

account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and

Baker’s yeast used in these large scale processes have been engineered for efficient utilization of

alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second

and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to

broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include

non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures

of native and non-native carbon sources present in biomass hydrolysates will be summarized and a

perspective on how to further increase feedstock flexibility for the realization of biorefinery processes

will be given.

Keywords: flexible feedstock concept; metabolic engineering; strain development; lignocellulosics; biofuels; bio-

based value-added chemicals; second generation feedstocks; E. coli; C. glutamicum; Bacillus; Pseudomonas; yeast;

biorefinery; hydrolysates; methanol; CO2

 1. Introduction

Biotechnological production of value-added compounds ranging from pharma proteins to bulk chemicals

mostly relies on glucose as carbon source in production media. At large production scales, price,

availability and competing uses of carbon sources are key factors for economic success of

biotechnological processes. Glucose is a component of molasses and it can be obtained from starch by

enzymatic hydrolysis. In 2014, in Brazil alone about 632,127,000 tons of sugar cane were harvested and

bio-ethanol production amounted to 28,394,000 m3 (http://www.unicadata.com.br; last accessed 23

May 2016). For the annual fermentative production of about 5 million tons of amino acids about twice

as much sugar is consumed (Wendisch, Jorge, Pérez-García, & Sgobba, 2016). At these scales, competing

uses of sugars in human and animal nutrition are an important issue and, thus, alternative carbon

sources for biotechnological processes are sought after.

The microbial production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker’s yeast differ

with respect to their natural ability to consume industrially relevant carbon sources for growth

(Blombach & Seibold, 2010; Kircher, 2015; Kondo, Ishii, Hara, Hasunuma, & Matsuda, 2013; Lan & Liao,

2013; van Maris et al., 2006). The carbon sources considered in this review are polymers such as

cellulose, starch, chitin or algal polysaccharides, monomeric components of lignocellulosic hydrolysates

such pentose sugars or hexouronic acids, glycerol from the Biodiesel process, amino sugars, organic

acids and C1 compounds. Utilization of biomass by strains able to grow with monomeric, but not

polymeric substances requires prior hydrolysis or pretreatment of the biomass, a process that involves

chemical and/or enzymatic treatment and that is prone to catalyst deactivation (Lange, 2015).

Development of strains able to directly utilize polymeric substances from biomass may include secretion

and/or cell-surface display of depolymerizing enzymes (Figure 1) (Kondo et al., 2013; Tanaka & Kondo,

2015). Alternatively, polymers may be imported into the cell prior to intracellular hydrolysis (Figure 1).

The ability of co-utilization of carbon sources is highly relevant for nearly all hydrolysates since they are

composed of a blend of carbon substrates, but this is rarely found in the microbial production hosts
(Blombach & Seibold, 2010; Xia, Eiteman, & Altman, 2012). Metabolic engineering efforts to broaden

the substrate scope of these organisms with respect to access to non-native carbon sources and to allow

simultaneous consumption of several carbon sources present in blends will be reviewed. The metabolic

engineering strategies for access to carbon sources are discussed per carbon source in the text and are

summarized per microorganism in Table 1 (E. coli), Table 2 (C. glutamicum), Table 3 (pseudomonads),

Table 4 (B. subtilis), and Table 5 (yeasts).

2. Utilization of alternative carbon sources derived from complex polymers

A wide variety of biomass resources are available for conversion into bioproducts (Smith, Anderson,

Senior, & Aidoo, 1987). These may include whole plants, plant parts (e.g. seeds, stalks), plant

constituents (e.g. starch, lipids, protein and fibre), processing by-products (distiller’s grains, corn

solubles), materials of marine origin, municipal and industrial wastes (Smith et al., 1987). This chapter

will investigate how to access recalcitrant polymers for utilization of their monomers.

2.1. Starch

Starch is a carbohydrate extracted from diverse agricultural raw materials. Starch is present in significant

amounts in food and non-food materials. It is also an abundant and inexpensive substrate as an

alternative to glucose in large-scale fermentation of microorganisms. Often the addition of amylolytic

enzymes to the culture broth is required for access of microorganisms to starch. Direct access to starchy

materials as carbon sources for cultivation typically is not found for industrially relevant

microorganisms. Thus, the introduction of heterologous genes encoding amylases and other starch

hydrolyzing enzymes into industrially relevant microorganisms has been studied intensively.

For the utilization of starch as a sole carbon source by alcohol producing yeasts, the heterologous

expression of amylase genes driven by inducible promoters has been employed since decades (Innis et
al., 1985). A cell surface-engineered Saccharomyces cerevisiae displaying the heterologous amylolytic

enzyme on its cell wall leading to growth with starch as sole carbon source has been described (Table 5;

Murai et al., 1997). Recently, alternative platforms for recombinant amylase production by yeasts were

developed. When the Aspergillus tubingensis amyA and glaA genes were co-expressed in S. cerevisiae

(Table 5), the recombinant yeast could use raw corn starch as sole carbon source and convert it to

ethanol (Viktor, Rose, van Zyl, & Viljoen-Bloom, 2013). The gene encoding the thermostable α-amylase

in Geobacillus stearothermophilus was cloned into the methylotrophic Pichia pastoris, which is widely

used for the production of industrially important proteins. Since the natural thermophilic G.

stearothermophilus is not considered suitable because of low yield and high production costs, the P.

pastoris methanol inducible expression system is promising for industrial applications, especially in

liquefying saccharification due to the high levels of extracellular thermostable amylase (Gandhi, Salleh,

Rahman, Chor Leow, & Oslan, 2015). Furthermore, P. pastoris has been engineered to express α-

amylase gene amyE from B. subtilis constitutively (Table 5), thus, without the need of addition of

methanol as inducer to the production medium (Arruda et al., 2016).

When bacterial cells are used in biotechnological industry, the glucose derived from starch hydrolysates

can be used in the production of added-value products and the introduction of starch utilizing organisms

in this process avoids the cost-intensive enzymatic hydrolysis of the starch. In first engineering

approaches towards direct utilization of starch in C. glutamicum, a L-lysine producing strain expressing

α-amylase gene from Streptomyces griseus was constructed to synthetize and secrete α-amylase into

the medium (Seibold, Auchter, Berens, Kalinowski, & Eikmanns, 2006). The recombinant strain produced

L-lysine from starch as fast as from glucose, however, growth was only achieved when small amounts of

glucose were present and dextrins remained in the medium (Table 2; Seibold et al., 2006). Moreover, C.

glutamicum engineered for the utilization of starch by a surface displayed fusion protein containing the

Streptococcus bovis α-amylase and PgsA from B. subtilis was able to produce L-lysine directly from starch

(Tateno, Fukuda, & Kondo, 2007). Starch-based production of L-glutamate (Yao et al., 2009), cadaverine
(Tateno et al., 2009) and organic acids such as lactate and succinate (Tsuge et al., 2013) by engineered C.

glutamicum has been reported as well (Table 2). Recently, the C. glutamicum amylomaltase CgAM

(Srisimarat, Murakami, Pongsawasdi, & Krusong, 2013) was used in E. coli and shown to support growth

with different sources of starch (Table 1; Vongpichayapaiboon et al., 2016). Starch-based production of

polyhydroxybutyrate by E. coli became possible upon expression of the amylase gene from Paenibacillus

sp. and the polyhydroxybutyrate synthesis genes from Ralstonia eutropha (Bhatia et al., 2015). Direct

production of itaconic acid was achieved in E. coli using soluble starch as single substrate and α-

amylases from B. amyloliquefaciens and S. bovis (Okamoto et al., 2015). Moreover, E. coli is routinely

used for the heterologous expression of amylases (and other enzymes) to facilitate their biochemical

characterization, e.g. the extracellular, extremely thermostable α-amylase from the hyperthermophilic

archaeon Pyrococcus furiosus (Peng et al., 2016) and the maltohexaose-forming exoamylase AmyM from

Corallococcus sp. (Li et al., 2015).

2.2. Cellulose and other glucans

Cellulose is the major polymeric component of plant matter and is the most abundant polysaccharide on

Earth. Cellulosic biomass is one of the most abundant waste materials, however, its utilization is difficult

due to its complex structure. The complete and efficient degradation of cellulose requires the synergistic

action of the cellulolytic enzymes endoglucanases, cellobiohydrolases, and β-glucosidases. Cellulose is

degraded by endoglucanases and cellobiohydrolases, resulting in cellobiose and cellooligosaccharides,

which can be converted to glucose by β-glucosidases (Bayer, Chanzy, Lamed, & Shoham, 1998). The

recently discovered cooper-containing lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative

cleavage of polysaccharide chains facilitating break-down of recalcitrant polysaccharides such as

cellulose (Frandsen et al., 2016).

B. subtilis can grow in cellulosic material, but not efficiently. B. subtilis utilized cellulose when its

secretory glycoside hydrolase family 5 endoglucanase (Bscel5) was overproduced (Table 4). This strain
produced lactate from cellulose since the α-acetolactate synthase gene in the 2,3-butanediol pathway

was inactivated (Zhang, Sathitsuksanoh, Zhu, & Percival Zhang, 2011). Productivity was improved in B.

subtilis cells expressing recombinant β-glucosidase from Thermobifida fusca or a fusion of β-glucosidase

from T. fusca and cellulase from Bacillus sp. (Table 4; Tanimura et al., 2016). Similarly, an E. coli strain

expressing the genes for β-glucosidase from Thermobifida fusca and cellulase from Bacillus sp. was used

for the cellulose-based production of polyhydroxybutyrate (Table 1; Gao et al., 2015).

Since natural cellulose degrading microorganisms often possess minicellulosomes, C. glutamicum has

been engineered to produce the scaffolding protein CpbA from Clostridium cellulovorans and the

chimeric C. thermocellum endoglucanase (Table 2). CbpA contains nine cohesin domains, which bind to

the dockerin domains of cellulosomal enzyme subunits. The recombinant strain hydrolyzed

carboxymethyl cellulose (Hyeon, Jeon, Whang, & Han, 2011). Furthermore, a cellulase complex

containing two cellulolytic enzymes, endoglucanase CelE and β-glucosidase BglA from C. thermocellum,

was displayed on the cell surface of C. glutamicum (Table 2) by anchoring to the mechanosensitive

channel Msc (S. J. Kim, Hyeon, Jeon, Choi, & Han, 2014). C. glutamicum has also been metabolically

engineered to use other cellulosic compounds such as D-cellobiose (Table 2), which required

overexpression of the endogenous genes bglF317A and bglA, encoding a variant of

phosphoenolpyruvate:carbohydrate PTS β-glucoside-specific enzyme IIBAC component and phospho-β-

glucosidase, respectively (Sasaki, Jojima, Inui, & Yukawa, 2008). Alternatively, β-glucosidade from the

cellulose degrading Saccharophagus degradans (Sde1394) was displayed on the cell surface display to

allow for direct production of L-lysine from cellobiose (Adachi et al., 2013). Access to the polysaccharide

β-glucan has been achieved for C. glutamicum by biosynthesis and secretion of endoglucanase from C.

thermocellum based on the torA signal sequence from E. coli. Glutamate production from barely β-

glucan assisted by addition of β-glucosidase from Aspergillus oryzae was achieved (Tsuchidate et al.,

2011). Furthermore, C. glutamicum strains producing lysine and co-expressing endoglucanase genes

from Xanthomonas campestris XCC3521 and XCC2387 and β-glucosidase gene from S. degradans
Sde1394 were constructed (Table 2). These strains also efficiently saccharified pretreated biomass like

sugarcane tops and rice straw to reducing sugars (Anusree, Wendisch, & Nampoothiri, 2016).

S. cerevisiae was engineered to use various substrates including the cellulosic carboxymethylcellulose,

hydroxyethylcellulose, laminarin, barley glucan and cellobiose (Van Rensburg, Van Zyl, & Pretorius,

1998). S. cerevisae produces glucanases, but lacks the multicomponent cellulase complexes that

hydrolyze cellulose-rich biomass. A cellulase gene cassette encoding the Butyrivibrio fibrisolvens endo-β-

1,4-glucanase End1p, the Phanerochaete chrysosporium cellobiohydrolase Cbh1p, the Ruminococcus

flavefaciens cellodextrinase Cel1p and the Endomyces fibuliger cellobiase Bgl1p were produced by

recombinant S. cerevisae (Table 5). These enzymes represent the four main groups of cellulases required

for the complete degradation of crystalline cellulose (Van Rensburg et al., 1998). -Glucosidase

displaying S. cerevisiae cells showed improved degradation of cellulose and improved ethanol

production as consequence of expressing the genes for endoglucanase from Trichoderma reesei and

cellobiohydrolase from Talaromyces emersonii (Z. Liu et al., 2015).

2.3 Xylan

Xylans are xylose-containing polysaccharides in hemicellulose. To utilize hemicellulose economically,

it is desirable to develop bioprocesses in which all steps from biomass degradation to the production

of target products occur in a single bioreactor (Yim, Choi, Lee, & Jeong, 2016). B. subtilis can utilize

xylans due to its ability to secrete glycoside hydrolase GH11 and GH30 endoxylanases XynA and XynC,

respectively (Rhee et al., 2014), with internal xylose processing fine regulated by XylR (Gu et al.,

2010), see Table 4.

A three-module C. glutamicum strain was constructed to allow this bacterium to utilize xylans: I)

xylulokinase (xylB) and xylose isomerase (xylA) from E. coli for xylose utilization (Kawaguchi et al.,

2006; Majewski and Domach, 1990; Sasaki et al., 2008); II) xylose uptake by XylE from E. coli
MG0155; III: degradation of xylan by endoxylanase (XlnA) from Streptomyces coelicolor A3 and

xylosidase (XynB) from Bacillus pumilus (Yim et al., 2016) as shown in Table 2.

Y. lipolytica degraded xylans when endowed with endoxylanase XynII from T. harzianum or XlnD from

A. niger (Wang et al., 2014), see Table 5. In Pichia stipitis Xyn2 (β-Xylanase) from Trichoderma reesei,

Xyn-C (β-Xylanase) from Aspergillus kawachii, and XlnD (β-xylosidase) from Aspergillus niger were

expressed(Den Haan & Van Zyl, 2003) as shown in Table 5.

2.4 Chitin and Chitosan

Chitin is a linear homopolymer of N-acetylglucosamine (GlcNAc) and chitosan a copolymer of GlcNAc

and glucosamine (GlcN). Pure homopolymeric chitin is rarely found in nature and the varying degree

of deacetylation yields a continuum of structures between chitin and chitosan. Only chitosan is

readily soluble in aqueous acidic solutions (Roberts, 1992). Chitin/chitosan belong to the most

abundant polymers found in Nature. Large amounts of chitin-containing waste arise in the shrimp

industry which represent a major source of surface pollution in coastal areas. Although purified chitin

and chitosan preparations find various applications, chitin-containing waste may be considered an

under-utilized source of carbon, energy and nitrogen. Chitin utilization by Serratia marcescens is well-

studied and this property may pertain to its lifestyle as fungal pathogen or symbiont since fungal cell

walls may contain chitin. S. marcescens possesses processive exo-chitinases ChiA and ChiB, endo-

chitinase ChiC and chitin-active lytic polysaccharide monooxygenase CBP21. E. coli strains growing

with the GlcNAc-containing disaccharide chitobiose can be selected easily since ‘decryptifying’

mutations lead to transcription of the cryptic cel operon chbBCARFG (Keyhani & Roseman, 1997).

2.5 Alginate

Alginate is lucrative for the worldwide market because it is used in various industries such as food,

textile, printing and chemical industries (Wong, Preston, & Schiller, 2000) and it has the potential to be a
substrate for the production of biofuels (Beer, Boyd, Peters, & Posewitz, 2009). The biopolymer alginate

is composed of β-D-mannuronate and α-L-guluronate. Three types of alginate are known: poly-β-D-

mannuronate (polyM), poly-α-L-guluronate (polyG) and heteropolymeric (polyMG) alginate (Gacesa,

1988). Alginate can be biosynthesized by bacteria and algae with the bacterial alginate containing more

M units and the algal containing more G units (F. Thomas et al., 2013).

Different prokaryotic organisms like Sphingomonas sp. A1 (Hashimoto, Miyake, Momma, Kawai, &

Murata, 2000; Yonemoto, Murata, Kimura, Yamaguchi, & Okayama, 1991), Stenotrophomonas

maltophilia KJ-2 (Shin, Lee, Park, Kim, & Lee, 2015) and Vibrio splendidus 12B01 (Badur et al., 2015) are

able to use alginate as carbon and energy source, but these microorganisms are currently not employed

in biotechnology.

Genes for the degradation of alginate to pyruvate and D-glyceraldehyde-3-phosphate (GAP) were cloned

in a plasmid and used to transform E. coli ATCC8739 (Table 1) (Wargacki et al., 2012). The

depolymerization of alginate into oligoalginate via endolytic β-elimination was catalyzed by the alginate

lyase (SM0524 aly EC: 4.2.2.3) from Pseudoalteromonas sp. SM0524 (Wong et al., 2000). The genes for

the following reactions for degradation of oligoalginate to glycolytic intermediates were taken from

Vibrio splendidus 12B01. Oligoalginate (3-5 mers) was transported in the periplasm with the aid of

porins (KdgMN). The periplasmic alginate lyases (AlyABCD) shortened the oligoalginates (to 2-4 mers).

The symporter ToaABC imported these into the cytoplasm, where they were degraded to unsaturated

monosaccharides by exolytic oligoalginate lyase OalABC. The monosaccharides rearranged non-

enzymatically to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). DEH reductase converted DEH to 2-

keto-3-deoxygluconate (KDG), which entered the Entner-Doudoroff pathway (Ochiai, Yamasaki, Mikami,

Hashimoto, & Murata, 2006; Takase, Ochiai, Mikami, Hashimoto, & Murata, 2010). The Entner-

Doudoroff enzymes 2-keto-3-deoxygluconate kinase (kdgK; EC: 2.7.1.45) and 2-dehydro-3-deoxy-

phosphogluconate aldolase (eda, EC: 4.1.2.14) cleaved KDG after phosphorylation to KDPG (2-dehydro-

3-deoxy-D-gluconate-6-phosphate) to yield pyruvate and GAP (Conway, 1992) (Table 1).

The yeast Yarrowia lipolytica hydrolyzed alginate to oligoalginate when the alginate lyase gene (alyVI, EC

4.2.2.11) from Vibrio sp. QY101 was expressed. It became apparent that the alginate lyase AlyVI

preferred alginate with poly-M-blocks (G. Liu et al., 2009)(Table 5).

2.6 Polypectate

Cell walls of land plants contain pectins, which are methyl ester of polygalaturonic acids (polypectate),

as major polysaccharides (Sriamornsak, 2003; Willats, McCartney, Mackie, & Knox, 2001).The

biopolymer pectin can be readily extracted from citrus peel or apple pomace (May, 1990) and is used in

the food, beverage and pharmaceutical industries due to its gelling properties (Sriamornsak, 2003).

Plants protect themselves from tissue-invading phytopathogenic fungi by the cell wall protein

polygalacturonase-inhibiting protein that inhibits fungal polygalacturonases (Spadoni et al., 2006). Some

bacteria like Erwinia carotovora and Erwinia chrysanthemi use pectin as energy and carbon source

(Abbott & Boraston, 2008). Yersinia enterocolitica, Yersinia pseudotuberculosis and Klebsiella

pneumoniae on the other hand only show a weak degradation activity for polygalacturonic acids (Starr,

Chatterjee, Starr, & Buchanan, 1977).

Hydrolysis of polygalacturonic acids by S. cerevisiae was achieved by expression of heterologous genes

for the degradation of polygalacturonic acids (Van Rensburg et al., 1998). A combination of a cellobiase

(BGL1; Endomyces fibuliger Y646), endo-β-1,4-glucanase (END1; EC 3.2.1.4; Butyrivibrio fibrisolvens),

cellobiohydrolase (CBH1; EC 3.2.1.91; Phanerochaete chrysosporium) and cellodextrinase (CEL1;

Ruminococcus flavefaciens) was necessary to enable access of S. cerevisiae to polygalacturonic acids

(Van Rensburg et al., 1998) (Table 5).

Some microorganisms, like E. coli, Agrobacterium tumefaciens (Richard & Hilditch, 2009) and B. subtilis

(Yoon, Moon, Iranpour, Lanza, & Prather, 2009) degrade naturally only the major component of

polypectate, the galaturonic acid. C. glutamicum was engineered to utilize glucuronic acid and

galacturonic acid based on the respective genes from E. coli (Hadiati, Krahn, Lindner, & Wendisch, 2014).
2.7 Cyanophycin

Cyanophycin (CGP) is a natural biopolymer synthesized by most cyanobacteria as transient nitrogen

storage compound (Mackerras, de Chazal, & Smith, 1990). CGP accumulates in cytoplasm as insoluble

membraneless inclusions. CGP consists of a poly(aspartic acid) backbone with arginine side chains. Per

aspartic acid in the backbone one arginine is linked with its α-amino group to the β-carboxyl group of

each aspartic acid to form multi-L-arginyl-poly(L-aspartic acid)(Simon & Weathers, 1976). The

poly(aspartic acid) backbone is an efficient substance for a substitute for non-biodegradable

polyacrylates or as an additive for paper, paint and oil industries (Joentgen, Müller, Mitschker, &

Schmidt, 2005; Schwanborn & Joentgen, 1998).

Different bacteria such as Pseudomonas anguilliseptica BI, Bacillus megaterium BAC19 and

Pseudomonas alcaligenes DIP1 are able to utilize CGP as sole carbon and energy source (Obst,

Oppermann-Sanio, Luftmann, & Steinbüchel, 2002; Obst, Sallam, Luftmann, & Steinbüchel, 2004). The

peptidase cyanophycinase hydrolyzes the polymer to a dipeptide consisting of aspartic acid and arginine

(Richter, Hejazi, Kraft, Ziegler, & Lockau, 1999). This property has been used to develop a process for

producing Asp-Arg dipeptides by degradation of cyanophycin (Sallam et al., 2011). Recombinant E. coli

strains degraded cyanophycin when the gene for the extracellular cyanophycinase (CphEal ; EC:3.4.15.6)

from Pseudomonas alcaligenes DIP1 was expressed (Sallam et al., 2011)(Table 1).

3. Di- and monosaccharides

Since maltose and cellobiose are generated during degradation of starch and cellulose, respectively, the

metabolic engineering strategies for access to these disaccharides are detailed in the section on the

respective polymers.
3.1 Lactose

Whey is a by-product of the dairy and cheese industries and contains lactose as major carbohydrate.

This disaccharide consists of galactose and glucose. Use of whey as an abundant and renewable raw

material for microbial fermentations is restricted since it is often too dilute.

E. coli can utilize lactose as sole carbon source, while B. subtilis cannot. This is surprising given the

fact that genes which encode the proteins necessary for the degradation of lactose and xylose are

present in B. subtilis and that these genes are expressed under inducing conditions. For xylose it has

been shown that this sugar is not transported into the cell (Krispin & Allmansberger, 1998). The

metabolic engineering of S. cerevisiae cells for conversion of lactose to ethanol involved expressing K.

lactis genes (lac4 and lac12) under control of other yeast promoters (Guimarães, Teixeira, &

Domingues, 2010) as shown in Table 5. C. glutamicum that lacks the capacity for lactose catabolism

(Guimarães et al., 2010) has been engineered by heterologous expression of the lacZYA operon from

E. coli which enabled growth on lactose as sole carbon source (Brabetz, Liebl, & Schleifer, 1991). This

was the first evidence that a transport protein, i.e. lactose permease, from the Gram-negative E. coli

functioned in Gram-positive mycolic acid-containing C. glutamicum (Brabetz et al., 1991). Expression

of lacZYA, however, only allowed utilizing of the glucose monomer liberated from lactose by -

galactosidase, while the galactose monomer accumulated in the culture medium (Brabetz et al.,

1991). Full utilization of the disaccharide lactose became possible when genes for galactose utilization

were expressed in addition (Table 2). A strain expressing the genes for lactose permease and β-

galactosidase from Lactobacillus delbrueckii subsp. bulgaricus and the genes for aldose-1-epimerase,

galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-epimerase from Lactococcus

lactis subsp. cremoris grew with lactose sole carbon source and utilized both monomers of the

disaccharide (Barrett et al., 2004). Whey-based L-lysine production was characterized by low yields

and productivities (Barrett et al., 2004). The lacZYA operon of E. coli was also integrated in the

chromosome of P. fluorescens and P. putida yielding lac+ recombinants (Hansen, Sorensen, & Jensen,
1997) (Table 3). Ethanol-producing S. cerevisiae utilized lactose when the K. lactis genes (lac4 and

lac12) were expressed under control of yeast promoters (Domingues, Guimarães, & Oliveira, 2010)

(Table 5). The wild type strain of Pichia pastoris cannot grow on medium containing lactose as sole C

source because it lacks β-galactosidase (Larsen et al., 2013).

Nevertheless, a P. pastoris strain secreting β-D-galactosidase into a whey permeate, which was

supplemented with L-arabinose isomerase from Arthrobacter sp. 22c, hydrolysed lactose, utilized the

glucose monomer to 90% and converted the galactose moiety to 30% to tagatose (Wanarska & Kur,

2012) (Table 5).

3.2 Galactose

Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins. E. coli

can grow with galactose as sole carbon source, but is has been engineered to circumvent catabolite

repression. Thus, a synthetic operon galETKM under control of a strong constitutive promoter was

expressed (Lim, Seo, & Jung, 2013) see Table 1. Similarly, glucose repression of the Leloir pathway

genes for galactose utilization of Y. lipolytica was avoided by their overexpression. This strain was

used to produce citric acid and lipids from galactose, an important step for the usage of food-based

substrates in industrial applications (Lazarev, Puskarz, & Breaker, 2003)(Lazar, Gamboa-Melendez, Le

Coq, Neuveglise, & Nicaud, 2015) as shown in Table 5. B. subtilis cannot grow with galactose because

it cannot be taken up into the cell. This might have evolved since already very small concentrations of

UDP-galactose seem to be toxic for B. subtilis, a phenomenon still not understood (Krispin &

Allmansberger, 1998). For C. glutamicum, growth with galactose became possible by heterologous

expression of the galETKM operon from Lactococcus lactis subsp. cremoris MG1363 (Barrett et al.,

2004) as shown in Table 2.

3.3 L-Levoglucan
L-Levoglucosan is a pyrolytic sugar present in bio-oil or wood pyrolysate liquor. Levoglucosan is not

abundant in Nature, although studies have shown that several microorganisms are capable of utilizing

it. However, expression of levoglucosan kinase from Lipomyces starkeyi was required to enable

growth of E. coli KO11 (Layton, Ajjarapu, Choi, & Jarboe, 2011), C. glutamicum (E.-M. Kim, Um, Bott, &

Woo, 2015), and Pseudomonas putida KT2440 (Linger, Hobdey, Franden, Fulk, & Beckham, 2016)

(Tables 1-3). The yeasts P. stipitis and S. cerevisiae can utilize levoglucosan for growth but with a

lower efficiency compared to glucose (Prosen, Radlein, Piskorz, Scott, & Legge, 1993). In conclusion,

natural and engineered microorganisms are able to utilize levoglucosan present in wood pyrolysate

liquor but inhibition due to toxic pyrolysis products, therefore post-pyrolysis clean-up is needed.

3.4 Xylose

Xylose is present e.g. in hemicelluloses. P. putida S12 has been engineered to utilize xylose (Table 3)

as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from E. coli. The initial

yield on xylose was low (0.09 g CDW/g), due to oxidation of xylose to xylonate by endogenous glucose

dehydrogenase (Gcd), which was then deleted (Meijnen, De Winde, & Ruijssenaars, 2008).

C. glutamicum has also been engineered for growth on D-xylose (Gopinath, Meiswinkel, Wendisch, &

Nampoothiri, 2011; Kang et al., 2014; Hideo Kawaguchi et al., 2006; Meiswinkel, Gopinath, Lindner,

Nampoothiri, & Wendisch, 2013) by heterologously expressing xylose isomerase gene xylA from E.

coli. This was sufficient to convert D-xylose to D-xylulose, which is then phosphorylated by

endogenous xylulokinase to yield xylulose-5-phosphate, an intermediate of the pentose phosphate

pathway (Table 2). Faster growth and amino acid production from xylose resulted from

overexpression of the xylose isomerase gene from Xanthomonas campestris in combination with

overexpression of the endogenous xylulokinase gene xylB (Meiswinkel, Gopinath, et al., 2013). C.

glutamicum ATCC 13032 converted xylose as well as arabinose to the non-fermentable sugar alcohol

xylitol (Table 2), when genes for NAD(P)H-dependent xylose reductase (xr) from Rhodotorula

mucilaginosa, L-arabinose isomerase (araA) from E. coli, D-psicose 3 epimerase (dpe) from
Agrobacterium tumefaciens, and L-xylulose reductase (lxr) from Mycobacterium smegmatis were

expressed (Kiran et al., 2016). Xylose conversion to xylitol was enhanced by heterologous expression

of arabinose transporter gene araT from B. licheniformis leading to 6.7 ± 0.4 g/L of xylitol in batch

fermentations with a specific productivity of 0.28 ± 0.05 g/g cdw/h (Kiran et al., 2016).

However, D-xylose assimilation by this so-called isomerase pathway has the drawback that a

significant fraction of the D-xylose-derived carbon is lost as CO2 while xylose assimilation by the

Weimberg pathway yields α-ketoglutarate without loss of CO2. Expression of the respective genes

from Caulobacter crescentus in C. glutamicum (Table 2) led to conversion of xylose to α-

ketoglutarate, the immediate precursor of L-glutamate without carbon loss (Radek et al., 2014).

3.5 Arabinose

Arabinose is a pentose sugar present in hemicellulose. E. coli can utilize arabinose, but its utilization is

subject to glucose repression. Also, due to repression by AraC arabinose is utilized prior to xylose

when both pentoses are present in mixtures such as hemicellulosic hydrolysates (Desai & Rao, 2010).

B. subtilis is able to use arabinose (Sa-Nogueira, Nogueira, Soares, & de Lencastre, 1997).

Its ara operon consists of nine genes where araA, araB and araD encode for L-arabinose isomerase, L-

ribulokinase and L-ribulose-5-phosphate 4-epimerase, respectively, while araL, araM, araN, araP,

araQ and abfA are not essential for arabinose utilization. P. pastoris is a natural utilizer of L-arabinose

(Knoshaug, Franden, Stambuk, Zhang, & Singh, 2009). In C. glutamicum, arabinose utilization became

possible by heterologous expression of the araBAD operon of E. coli (see Table 3), which encodes

arabinose isomerase AraA, ribulokinase AraB, and ribulose 5-phosphate 4-epimerase AraD (Hideo

Kawaguchi, Sasaki, Vertès, Inui, & Yukawa, 2008; Schneider, Niermann, & Wendisch, 2011). The

arabinose uptake system from C. glutamicum strain ATCC 31831 was not only useful to accelerate

import and catabolism of arabinose, but also utilization of xylose (Sasaki, Jojima, Kawaguchi, Inui, &

Yukawa, 2009). P. putida S12 was engineered to utilize xylose as a substrate by expressing xylose
isomerase (XylA) and xylulokinase (XylB) from E. coli (Table 3). A fast xylose-utilizing mutant

metabolized L-arabinose as efficient as D-xylose due to deletion of gcd (glucose dehydrogenase) has

been described (Meijnen et al., 2008).

A challenge emerging from the use of lignocellulosics for the production of ethanol by the yeast S.

cerevisiae is efficient fermentation of D-xylose and L-arabinose, as these sugars cannot be used by

natural S. cerevisiae strains (Hahn-Hägerdal, Karhumaa, Jeppsson, & Gorwa-Grauslund, 2007). AraT

from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana mediate the

uptake and utilization of L-arabinose (Subtil & Boles, 2011). A S. cerevisiae strain was metabolically

engineered to obtain both xylose and arabinose utilization. For this, the Piromyces XylA, S. cerevisiae

XKS1, and Lactobacillus plantarum araA, araB, and araD genes, as well as the endogenous genes of

the pentose phosphate pathway (rpe1, rki1, tkl1, and tal1) were overexpressed (H Wouter Wisselink

et al., 2007)(Qiao et al., 2015)(Qi et al., 2015) together with endogenous overexpression of galactose

permease (gal2) as shown in Table 5. The first engineered S. cerevisiae strain was further engineered

leading to a strain capable of fermenting mixtures of glucose, xylose, and arabinose with a high

ethanol yield (0.43 g g−1 of total sugar) without formation of the side products xylitol and arabinitol (H

W Wisselink, Toirkens, Wu, Pronk, & van Maris, 2009).

3.6 Aminosugars

N-Acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN) are monosaccharide derivatives of glucose.

Both amino sugars are widely distributed in Nature and can be derived from chitin. They are

synthesized in all organisms, including bacteria, yeasts, fungi, plants and animals (Hamid et al., 2013;

L. Liu et al., 2013). GlcNAc is present in the bacterial cell wall component murein and in hyaluronic

acid, the major component of the extracellular matrix in connective, epithelial and neural tissues

(Chen, Shen, & Liu, 2010). E. coli grows slower with GlcN than with GlcNAc as a sole source of carbon.

This could be overcome by derepression of the nag operon (Álvarez-Añorve, Calcagno, & Plumbridge,
2005). Genes for uptake and metabolism of GlcNAc (nagP and nagAB) are present in all bacilli except

for Anoxybacillus flavithermus (Gaugue, Oberto, Putzer, & Plumbridge, 2013). P. putida can efficiently

utilize glucosamine involving the enzymes encoded by nagQ, nagA, nagB2, pstI, nagE. C. glutamicum

strains have been constructed to enable access to glucosamine and to GlcNAc (Matano et al., 2014; A

Uhde et al., 2013) (Table 1). In C. glutamicum, glucosamine uptake is imported by the glucose specific

permease PtsG. Fast growth with glucosamine as sole source of carbon and nitrogen required

overexpression/derepression of the endogenous glucosamine-6-phosphate deaminase gene nagB (A

Uhde et al., 2013). To enable growth of C. glutamicum with GlcNAc the GlcNAc -specific PTS gene

nagE from the related C. glycinophilum had to be expressed in addition to overexpression of

endogenous nagB and endogenous nagA which encodes cytoplasmic GlcNAc -6-phosphate

deacetylase (Matano et al., 2014). Glucosamine-based as well as GlcNAc -based L-lysine production

was reported (Matano et al., 2014; A Uhde et al., 2013). Deletion of nanR, coding for the

transcriptional regulator of the sialic acid utilization operon and of nagA and nagB, was efficient to

allow for fast utilization of the amino sugars (Uhde et al., 2016).

While Yarrowia lipolytica is able to grow with amino sugars (Groenewald et al., 2014), in S. cerevisiae,

four C. albicans genes (nag3 or its nag4 paralog, nag5, nag2, and nag1) have been expressed. These

genes code for GlcNAc permease, GlcNAc kinase, GlcNAc-6-phosphate deacetylase, and glucosamine-

6-phosphate deaminase, respectively (Wendland, Schaub, & Walther, 2009)(Table 5).

3.7 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)

The monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) is an intermediate of alginate

degradation (Preiss & Ashwell, 1962). DEH is not fermented by many microorganisms (D. M. Wang et al.,

2014), but can easily be generated from brown macro algae (Takeda, Yoneyama, Kawai, Hashimoto, &

Murata, 2011; Wargacki et al., 2012). Naturally DEH degrading microorganisms such as Sphingomonas

sp. Strain A1 (Takase, Mikami, Kawai, Murata, & Hashimoto, 2014) or Flavobacterium sp. strain UMI-01
(Inoue, Nishiyama, Mochizuki, & Ojima, 2015) possess either NADPH-dependent (Takase et al., 2010) or

NADH-dependent DEH reductase (Takase et al., 2014). A pathway for degrading DEH was established in

S. cerevisiae and the heterologously expressed genes were codon optimized for S. cerevisiae except for

the DEH reductase from Agrobacterium tumefaciens C58 (Enquist-Newman et al., 2014). Besides the

DEH reductase gene, the genes for the DEH transporter (Ac_DHT1) from Asteromyces cruciatus ATCC

26324, 2-dehydro-3-deoxygluconokinase (kdgK; EC: 2.7.1.45) from E. coli and 2-dehydro-3-deoxy-

phosphogluconate aldolase (KdgpA; EC: 4.1.2.14) from Vibrio splendidus 12B01 were required (Enquist-

Newman et al., 2014) (Table 5).

4. Aromatic compounds

Aromatic compounds can be classified as organic molecules that consist of at least one aromatic ring, a

benzene ring in particular. Aromatic compounds can be separated into polycyclic aromatic hydrocarbons

(PAHs), heterocyclic, and substituted aromatics. Bacteria that are capable of degradation of aromatic

compounds are of high interest with regard to bioremedation and biodegradation of organic pollutants

(Seo, Keum, & Li, 2009).

4.1 Organophosphates

Organophosphates like parathion and paraoxon are components of pesticides and chemical warfare

agents functioning as toxic cholinesterase inhibitors (DeFrank, 1991; Murray, Rathbone, & Ray, 2005).

Groundwater contamination with pesticides is a major problem (Beceiro-González et al., 2007).

Therefore, biodegradation is an alternative to chemical and physical methods for decontamination

leading to complete degradation of organophosphates (Russell, Berberich, Drevon, & Koepsel, 2003).

P. putida was genetically engineered for the use of parathion as well as paraoxon as sole carbon and

energy source (De La Peña Mattozzi, Tehara, Hong, & Keasling, 2006; Walker & Keasling, 2002). The first
step is the hydrolysis of the organophosphate by heterologously produced organophosphate hydrolase

(EC 3.1.8.1) from Flavobacterium sp. ATCC 27551. Parathion is hydrolysed to p-nitrophenol and diethyl

thiophosphate (DETP), while paraoxon is hydrolysed to p-nitrophenol and diethyl phosphate (DEP). The

additional expression of the operon for transformation of p-nitrophenol into β-ketoadipate from

Pseudomonas sp. ENV2030 (Table 3) enabled P. putida to convert p-nitrophenol via hydroquinone into

β-ketoadipate, which is then further catabolized in the β-ketoadipate pathway (De La Peña Mattozzi et

al., 2006; Walker & Keasling, 2002). DEP can be used as phosphorus source by P. putida when pde

(phosphodiesterase, EC 3.1.3.-) from Delftia acidovorans and phoA (alkaline phosphatase, EC 3.1.3.1)

from P. aeruginosa HN854 were heterologously expressed (De La Peña Mattozzi et al., 2006).

4.2 2,4-dinitrotoluene

2,4-dinitrotoluene is a toxic agent used in the explosives and polyurethane industry. As for the

organophosphates, there is a high demand for mineralization of this compound with regard to

bioremediation.

P. fluorescens was capable of using 2,4-dinitrotoluene as sole source of carbon and energy when the

plasmid pJS1 from Burkholderia sp. strain 2,4-dinitrotoluene, harboring the dnt genes (Table 3) for

degradation of 2,4-dinitrotoluene, was introduced (Monti, Smania, Fabro, Alvarez, & Argarana, 2005).

Since the plasmid was not stable in P. fluorescens, the dnt genes were introduced into the chromosome

leading to the ability of using 2,4-dinitrotoluene as sole nitrogen source, but not as sole carbon and

energy source (Monti et al., 2005).

5. Glycerol
Glycerol is a stoichiometric by-product of the biodiesel process and, thus, potentially available in large

quantities (Khanna et al., 2012). However, volatility of crude glycerol prices is high and technical

qualities of glycerol contain impurities that may be growth inhibitory (Meiswinkel et al., 2013a).

Many microorganisms can use glycerol as source of carbon and energy. Glycerol is a natural growth

substrate for S. cerevisiae (Swinnen, Ho, Klein, & Nevoigt, 2016), Y. lipolytica (Dobrowolski, Mituła,

Rymowicz, & Mirończuk, 2016), E. coli (Cheng et al., 2014), B. subtilis (Holmberg, Beijer, Rutberg, &

Rutberg, 1990), P. putida (Nikel, Romero-Campero, Zeidman, Goñi-Moreno, & de Lorenzo, 2015), but

not for C. glutamicum (Rittmann et al., 2008).

Isolates of S. cerevisiae CEN.PK113-1A growing faster with glycerol carried mutations in glycerol kinase

GUT1, ubiquitin-protein ligase subunit E3 (UBR2), and phosphorelay intermediate osmosensor and

regulator SSK1 (Swinnen et al., 2016) Additional introduction of glycerol facilitator FPS1 from

Cyberlindnera jadinii improved growth with glycerol (Swinnen et al., 2016). Fast growing mutants of E.

coli (carrying mutations in glpK and in RNA polymerase β′ subunit gene rpoC) were isolated (Cheng et al.,

2014). In P. putida, the prolonged lag phase observed with glycerol, but not with other carbon

substrates, was avoided by deletion of the repressor gene glpR (Nikel et al., 2015). Although the C.

glutamicum genome encodes glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD), it

cannot grow with glycerol since expression of glpK and glpD is too low to sustain growth (Meiswinkel et

al., 2013a). Overexpression of endogenous glpK with glpD or of the respective E. coli genes enabled fast

growth with glycerol. Additional expression of the glycerol facilitator gene glpF from E. coli accelerated

growth (Rittmann et al., 2008). Deletion of glycerol 3-phosphatase gene gpp, which is responsible for

formation of glycerol as by-product by C. glutamicum was not required (Lindner et al., 2012). Notably,

the recombinant C. glutamicum strains co-utilized glycerol with glucose (Rittmann et al., 2008).

Recombinant C. glutamicum strains were used for glycerol-based production of L-glutamate, L-lysine

and succinic acid (Rittmann et al., 2008; Litsanov et al., 2013). Production of the amino acids L-

glutamate, L-lysine, L-ornithine and L-arginine and the diamine putrescine from crude glycerol obtained
from various biodiesel manufacturers has been described for recombinant C. glutamicum strains

(Meiswinkel et al., 2013a).

6. Organic acids

6.1 Acetate and acetoin

Acetate is an intermediate of central carbon metabolism. Acetate may be excreted in overflow

metabolism, a trait usually avoided in industrial fermentation processes. Formation and accumulation of

acetate as a by-product in E. coli negatively affect product formation and growth in large-scale fed-batch

processes (Kabisch et al., 2013; Nakano, Rischke, Sato, & Märkl, 1997). Glyoxylate cycle-positive

bacteria, e.g. E. coli (Kornberg & Madsen, 1958), C. glutamicum (Reinscheid, Eikmanns, & Sahm, 1994) or

Bacillus licheniformis (Schroeter et al., 2011; Voigt et al., 2007) are able to grow with acetate as sole

carbon source. B. subtilis, however, does not possess the glyoxylate cycle and, thus, can use acetate,

acetoin and 2,3-butanediol, typical overflow metabolites of this bacterium, only to generate reduction

equivalents during their oxidation to carbon dioxide in the tricarboxylic acid cycle. Expression of an

operon encoding the key enzymes of the glyoxylate cycle, isocitrate lyase (aceB, EC 4.1.3.1) and malate

synthase (aceA, EC 2.3.3.9), from B. licheniformis enabled growth of B. subtilis with acetate (Table 4).

The aceBA operon also encoded a hypothetical protein (BL02643) which was shown to be essential for

the functional transfer of the cycle from B. licheniformis to B. subtilis. The engineered B. subtilis strain

showed improved production of recombinant α-amylase and a more solid growth behavior with excess

glucose (Kabisch et al., 2013).

6.2 Methylacetate
As alternative to use the C1 sources methanol and carbon monoxide, the methyl ester of acetic acid,

methylacetate, may be used as carbon source. Methylacetate can be formed from methanol through

carbonylation with CO in the presence of a metal-catalyst (C. M. Thomas & Süss-Fink, 2003).

To engineer C. glutamicum towards methylacetate utilization, mekB (homoserine O-acetyltransferase,

EC 2.3.1.31) from Pseudomonas veronii coding for an enzyme showing high esterase activity (Onaca,

Kieninger, Engesser, & Altenbuchner, 2007) was heterologously expressed (Choo, Um, Ok, & Min, 2016).

The engineered strain exhibited rapid and complete degradation of methylacetate yielding acetate and

methanol. Acetate is a native carbon and energy source of C. glutamicum, while methanol is dissimilated

to yield carbon dioxide.

6.3 Dicarboxylic acids

The dicarboxylic acids succinate, fumarate and malate are intermediates of the TCA cycle and they

usually serve as carbon sources. However, in C. glutamicum, overexpression of the endogenous genes

for dicarboxylic acid uptake had to be overexpressed to allow for growth (Teramoto, Shirai, Inui, &

Yukawa, 2008; Youn, Jolkver, Kramer, Marin, & Wendisch, 2008, 2009). For instance, growth with

succinic acid was only possible when dccT encoding a sodium dependent dicarboxylate transporter of

the DASS family or dctA encoding a proton dependent dicarboxylate uptake system was overexpressed

(Table 2).

7. C1 compounds

7.1 Methanol

Methanol has gained attention as alternative energy source. The feasibility of a “methanol economy”

was proposed before (Olah, Goeppert, & Prakash, 2009; Olah, 2013). Since methanol can be used as

non-food carbon source in biotechnological processes, interest to produce valuable products from
methanol has risen in the past decades (Brautaset et al., 2007; Marx et al., 2012; Naerdal,

Pfeifenschneider, Brautaset, & Wendisch, 2015; Sonntag et al., 2015). During utilization by

methylotrophic organisms, methanol is first oxidized to formaldehyde, which can be further oxidized to

CO2 through linear or cyclic detoxification pathways, or assimilated into the central carbon metabolism

by e.g. the ribulose monophosphate pathway (RuMP) or the serine cycle in bacteria, or the xylulose

monophosphate pathway (XuMP) in yeasts (Anthony, 1983). A number of yeasts are known to use

methanol as sole carbon and energy source in peroxisomes as different cell compartment to protect the

cell from toxic intermediates (Yurimoto, Oku, & Sakai, 2011). There is a strong and increasing knowledge

about methylotrophy in bacteria (Lidstrom & Stirling, 1990; Lidstrom, 1990; Šmejkalová, Erb, & Fuchs,

2010; Vorholt, 2002) e.g. about the Gram-negative serine cycle- and EMCP-utilizing Methylobacterium

extorquens (Ochsner, Sonntag, Buchhaupt, Schrader, & Vorholt, 2014; Vuilleumier et al., 2009) or the

Gram-positive RuMP-utilizing Bacillus methanolicus (Brautaset et al., 2007; Müller, Heggeset, Wendisch,

Vorholt, & Brautaset, 2015).

Transfer of dissimilatory oxidation of methanol is straightforward and often non-methylotrophs like E.

coli or C. glutamicum possess the respective enzymes (Gonzalez et al., 2006; Gutheil, Holmquist, &

Vallee, 1992; Herring & Blattner, 2004; Lessmeier, Hoefener, & Wendisch, 2013; Witthoff et al., 2013;

Witthoff, Eggeling, Bott, & Polen, 2012). Transfer of methylotrophy to non-methylotrophs has not yet

been successful (Lessmeier et al., 2015; Müller, Meyer, et al., 2015). However, when methanol was

provided as co-substrate, incorporation of methanol-carbon in central metabolites or secreted products

could be observed in engineered E. coli (Table 1) (Müller, Meyer, et al., 2015; Orita, Sakamoto, Kato,

Yurimoto, & Sakai, 2007), C. glutamicum (Table 2) (Lessmeier et al., 2015) and P. putida (Table 3)

(Koopman, De Winde, & Ruijssenaars, 2009). In E. coli, mdh2 (methanol dehydrogenase, EC 1.1.1.244)

and the gene for its activator protein act from B. methanolicus PB1 were expressed heterologously,

together with hps (3-hexulose-6-phosphate synthase, EC 4.1.2.43) and phi (6-phospho-3-

hexuloisomerase, EC 5.3.1.27) from B. methanolicus MGA3 (Table 1) (Müller, Meyer, et al., 2015).
 13
Labelling experiments with C methanol revealed up to 40% incorporation of methanol into central

metabolites. Labelling could not be increased by deletion of frmA (formaldehyde dehydrogenase, EC

1.1.1.284), which is essential for the endogenous glutathione-dependent, linear formaldehyde

detoxification pathway (Müller, Meyer, et al., 2015). In another approach, hps and phi from

Mycobacterium gastri MB19 were heterologously expressed as fusion protein in E. coli. The purified hps-

phi fusion showed approximately twofold activity during in-vitro assays, compared to an equimolar

mixture of both single enzymes. Strains expressing the fusion protein also exhibited improved

formaldehyde consumption and a better growth in comparison to the wild type, when formaldehyde

was present in the medium (Table 1) (Orita et al., 2007). To some extent, increased biomass

concentrations of P. putida S12 expressing the hps and phi genes from Bacillus brevis in chemostat

cultures feeding methanol as co-substrate were observed (Table 3) (Koopman et al., 2009). For co-

utilization of methanol in C. glutamicum, the linear detoxification pathway was removed by deleting ald

(aldehyde dehydrogenase, EC 1.2.1.3) and fadH (mycothiol-dependent formaldehydedehydrogenase, EC

1.1.1.306) (Lessmeier et al., 2013) and hxlA (3-hexulose-6-phosphate synthase, EC 4.1.2.43), hxlB (6-

phospho-3-hexulose isomerase, EC 5.3.1.27) from B. subtilis and mdh from B. methanolicus MGA3 were

expressed. The labelling of glucose-6-phosphate and fructose-6-phosphate from 13C-methanol indicated

operation of the RuMP in vivo (Table 2) (Lessmeier et al., 2015). Applying this strategy to a cadaverine

producing strain resulted in 13C-labelling of cadaverine from 13C-methanol. Thus, the non-natural carbon

source methanol was converted to the non-natural product cadaverine by recombinant C. glutamicum

to some extent, however, net biomass formation from methanol was not observed (Lessmeier et al.,

2015).

7.2 Formate

Various methanogenic archaea can use formate instead of hydrogen as electron donors. For instance,

Methanobacterium formicum can grow with formate as sole carbon and energy source producing
methane (Schauer & Ferry, 1980). A synthetic pathway for formate utilization was designed to overcome

challenges in heterologous expression of naturally occurring one-carbon assimilation pathways (Table 1)

(Siegel et al., 2015). Enzymes of the synthetic pathway were heterologously expressed in E. coli and

purified for in vitro characterization. First, formate is reduced to formaldehyde via formyl-CoA by

endogenous acetyl-CoA synthase (ecacs, EC 6.2.1.1) and aldehyde dehydrogenase (lmacdh, EC 1.2.1.10)

from Listeria monocytogenes. For condensation of three formaldehyde molecules to dihydroxyacetone

an enzyme named formolase was derived rationally from benzaldehyde lyase (bal, EC 4.1.2.38) from

Pseudomonas biovar I. Finally, dihydroxyacetone is phosphorylated to dihydroxyacetone phosphate, a

glycolytic intermediate, by dihydroxyacetone kinase (ydhak, EC 2.7.1.29) from S. cerevisiae. Whereas in

13
vitro labelling experiments with C-formate revealed formation of labelled DHAP when all

heterologously expressed enzymes were present, no growth with formate as sole carbon source was

achieved (Siegel et al., 2015). This may be due to low formolase activity or to the preferred formation of

glycoaldehyde from two molecules of formaldehyde rather than dihydroxyacetone from three

molecules of formaldehyde (Poust et al., 2015).

6.3 CO2

Atmospheric carbon dioxide is an abundant, but rather inert carbon source (estimated 750 Gt of carbon

in the atmosphere), whose use as a fuel or a chemical feedstock would reduce emissions and

dependence on fossil fuels (Aresta & Dibenedetto, 2007). CO2 is fixed to some extent by many

carboxylases, e.g. aerobic production of succinate from glucose involves carboxylation of pyruvate or

PEP by anaplerotic carboxylase and three of the four carbon atoms of succinate are derived from

glucose and one from carbon dioxide (Litsanov, Brocker, & Bott, 2013; Wendisch, Bott, & Eikmanns,

2006). These reactions, however, do not allow for autotrophic growth with carbon dioxide. In Nature, six

autotrophic pathways for carbon dioxide fixation have been described to date (Bar-Even, Noor, & Milo,
2012; Ducat & Silver, 2013). The most common is the Calvin-Benson-Bassham (CBB) cycle, whose key

enzyme is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and which operates in plants,

algae and cyanobacteria. A functional CBB cycle in E. coli has recently been demonstrated by combining

rational metabolic rewiring, recombinant gene expression and adaptive laboratory evolution, although it

did not yet result in net carbon gain (Antonovsky et al., 2016). A synthetic operon encoding RuBisCO,

phosphoribulokinase and carbonic anhydrase was constructed to complete a carbon fixation cycle in E.

coli (Table 1). The reducing power and energy for operation of the CBB cycle was provided by oxidation

of pyruvate, which itself could not be used for sugar biosynthesis due to deletion of the genes encoding

gluconeogenetic phosphoglycerate mutase. Mutations in flux branchpoints observed in evolved strains

were shown to be essential for stable operation of the cycle (Table 1), however, while 13C-labeling from

CO2 could be demonstrated in sugar phosphates, biomass or product formation from CO2 was not

possible (Antonovsky et al., 2016).

The reductive citric acid pathway is used by members of the thermophilic bacterial phylum Aquificae,

Nitrospira and some proteobacteria and is the sole CO2 assimilation pathway in Chlorobium limicola, a

photosynthetic green sulfur bacterium (Buchanan & Arnon, 1990). Acetogens, mostly members of the

Clostridia, but also of the Firmicutes and Planctomycetes use the reductive acetyl-CoA pathway. The

dicarboxylate/4-hydroxybutyrate cycle is used by the thermophilic archaea Desulfurococcales and

Thermoproteales orders and the hyperthermophilic Ignicoccus hospitalis (Huber et al., 2008). The 3-

hydroxypropionate/4-hydroxybutyrate cycle is used by archaea of the order Sulfolobales and possibly

the Thaumarchaeota phylum (Bar-Even et al., 2012).

The alternative CO2 fixation pathway 3-hydroxypropionate (3-HPA) bicycle is used by the green

nonsulfur bacterium Chloroflexus aurantiacus (Holo & Sirevag, 1986; Mattozzi, Ziesack, Voges, Silver, &

Way, 2013; Zarzycki, Brecht, Müller, & Fuchs, 2009), and heterologous expression and functional activity

of six of its enzymes have been recently reported in the cyanobacterium Synechococcus elongates

PCC7942 (Shih, Zarzycki, Niyogi, & Kerfeld, 2014). This bicycle can be divided into four sub-pathways: it
starts with (1) propionyl-CoA synthesis from acetyl-CoA, followed by (2) succinate synthesis from

propionyl-CoA, (3) glyoxylate production and acetyl-CoA regeneration, and (4) formation of pyruvate

and regeneration of acetyl-CoA by assimilation of glyoxylate and propionyl-CoA. This bicycle is an

attractive target for engineering in E. coli as 3-HPA is a bioplastic precursor (Meng et al., 2012; Q. Wang,

Liu, Xian, Zhang, & Zhao, 2012; Zhou et al., 2011), in addition to its insensitivity to oxygen, high rate of

CO2 fixation and production of novel intermediates. Two subsets of the enzymes of the bicycle have

been successfully expressed heterologously in E. coli and activities were sufficient to complement host

mutations, however, autotrophic growth with carbon dioxide could not be achieved (Mattozzi et al.,

2013). In a different approach, E. coli has been engineered for the synthesis of carboxysomes (Bonacci et

al., 2012), which encapsulate RuBisCO and carbonic anhydrase and facilitate CO2 fixation (Frank,

Lawrence, Prentice, & Warren, 2013). Carboxysomes are bacterial microcompartments (BMCs),

metabolically active and functionally diverse structures with a proteinaceous shell. A significant number

of bacteria form BMCs, and specifically carboxysomes have been found in cyanobacteria and some

chemoautotrophs. Heterologous expression of the hnCB operon from H. neapolitanus has led to the

successful formation of carboxysomes and consequent CO2 fixation in E. coli (Table 1) (Bonacci et al.,

2012).

Conclusion

Feedstock flexibility is important for individual biotechnological processes, but it is central in

biorefineries. In analogy to petroleum refineries that produce about 100 different products from

petroleum (mostly fuels, but also chemical building blocks), biorefineries convert biomass to biofuels,

power, heat, and value-added chemicals. Thus, biorefineries combine physical, chemical and

biotechnological processes and depend on the type of biomass used. This dependence entails that the

biotechnological processes used in the biorefinery have to be flexible with respect to the carbon source

mixtures generated. In the future, metabolic engineering strategies have to be devised and realized to
allow optimal conversion of multiple carbon sources simultaneously under these variable carbon

substrate conditions. This development will take time until it reaches the efficiency of the petroleum

refineries, i.e. the almost complete utilization of all petrol oil components without non-utilized by-

products. Avoiding non-utilized by-products was and is pivotal for the economic success of the

petroleum refineries. Thus, a multitude of biotechnological processes in future biorefineries will have to

yield a multitude of biofuels and biochemicals without non-utilized residues.

Acknowledgements

This work was supported in part by ERASysAPP project MetApp and by Grant KF2969004SB4 from ZIM,

BMWi. LFB acknowledges support as fellow of the Ciência sem Fronteiras program of Brazil (Science

Without Borders program). All authors contributed to drafting the manuscript. VFW finalized the

manuscript. All authors read and approved the final manuscript. The authors declare no conflict of

interest.
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Figure Captions

Figure 1. Metabolic engineering strategies for access to non-native carbon sources. The utilization of a polymeric

carbon source by a bacterium is exemplarily shown. The polymeric carbon source may be hydrolyzed to

monomeric intermediates by secreted (2a) or cell wall-attached (2b) depolymerizing enzymes, which are imported

in to the cell (3). Alternatively, the polymeric carbon source is taken up into the cell (1) and then hydrolyzed to

monomeric intermediates by intracellular depolymerizing enzymes (2c). Finally, the intracellular monomeric

intermediates are introduced into the central metabolism. Additional modifications, for instance, prevention of the

uptake of competing substrates or prevention of carbon catabolite control, are not depicted, but described in the

text.
Tables

Table 1. Examples of E.coli strains engineered for access to non-native carbon sources

Pr
Carbon source Sole carbon source Heterologous genes expressed Modifications of endogenous genes yie
(g/
Starch α-amylase (amyA, EC 3.2.1.1) and
+ glucoamylase (glaA, EC 3.2.1.3) genes
from Aspergillus tubingensis
+ amy1 (α-Amylase, EC 3.2.1.1) from Po
Penibacillus sp. T:
α-amylases (amyA, EC 3.2.1.1) from
+ Ita
B. amyloliquefaciens and S. bovis
AmyM (maltohexaose-forming
- exoamylase, EC 3.2.1.133) from
Corallococcus sp.(
α –amylase (amyA, EC 3.2.1.1) from
Pyrococcus furiosus
AmyM (maltohexaose-forming
- exoamylase, EC 3.2.1.133) from
Corallococcus sp.
α –amylase (amyA, EC 3.2.1.1) from
Pyrococcus furiosus
Cellulose cel-cd (cellulase, EC 3.2.1.4) from
Bacillus sp., tfu0937 (β-glucosidase,
EC 3.2.1.21) from Thermobifida fusca, Po
+
N-terminal 20 amino acid residues 0.1
(N20) of Cel-CD (cellulase, EC 3.2.1.4)
from Bacillus sp.
Alginate SM0524 aly (alginate lyase, EC
4.2.2.3) from Pseudoalteromonas sp.
SM0524; kdgN (Porin), toaC
(Symporter), toaA (Symporter), eda
(2-dehydro-3-deoxy-
phosphogluconate aldolase, EC
4.1.2.14), kdgk (2-dehydro-3-
deoxygluconokinase, EC 2.7.1.45),
oalB (oligoalginate lyase, EC 4.2.2.3), Eth
oalC (oligoalginate lyase, EC 4.2.2.3), 0.2
+
toaB (symporter), oalA (oligoalginate Sa
lyase, EC 4.2.2.3), dehR (DEH su
reductase, EC 1.1.1.126), alyA
(periplasmic alginate lyase, EC
4.2.2.3), alyB (periplasmic alginate
lyase, EC 4.2.2.3), alyC (periplasmic
alginate lyase, EC 4.2.2.3), kdgM
(Porin), alyD (periplasmic alginate
lyase, EC 4.2.2.3) from Vibrio
splendidus 12B01
Cyanophycin + cphEal (extracellular cyanophycinase,
 EC 3.4.15.6) from P. alcaligenes DIP1
Galactose galK (galactokinase, EC 2.7.1.6), galT
(UDP-glucose-1-P-uridylyltransferase,
+ EC 2.7.7.12), galE (UDP-galactose-4-
epimerase, EC 5.1.3.2), galM (aldose-
1-epimerase, EC 5.1.3.3) from E. coli
Levoglucosan EU751287.1 (levoglucosan kinase, EC
+ Eth
2.7.-.-) from Lipomyces starkeyi
Methanol mdh2 (methanol dehydrogenase, EC
1.1.1.244) from B. methanolicus PB1;
hps (3-hexulose-6-phosphate
- synthase, EC 4.1.2.43), phi (6-
phospho-3-hexuloisomerase, EC
5.3.1.27) operon from B.
methanolicus MGA3, act (Mdh
activator protein)
ΔfrmA (formaldehyde dehydrogenase,
EC 1.1.1.284 )
mdh2 (methanol dehydrogenase, EC
1.1.1.244) from B. methanolicus
- MGA3; hps (3-hexulose-6-phosphate
synthase, EC 4.1.2.43), phi (6-
phospho-3-hexuloisomerase, EC
5.3.1.27) operon from B.
methanolicus MGA3, act (Mdh
activator protein)
hps–phi (3-hexulose-6-phosphate
synthase, EC 4.1.2.43; 6-phospho-3-
- hexuloisomerase, EC 5.3.1.27) fusion
gene for formaldehyde consumption,
genes from M. gastri MB19

Formate lmacdh (aldehyde dehydrogenase, EC

1.2.1.10) from L. monocytogenes;
formolase rationally derived from bal
- (benzaldehyde lyase, EC 4.1.2.38)
from P. biovar I; ydhak
(dihydroxyacetone kinase, EC
2.7.1.29) from S. cerevisiae

CO2 hnCB operon (ribulose 1,5-

bisphosphate
carboxylase/oxygenase, EC 4.1.1.39;
- carbonate anhydrase, EC 4.2.1.1;
shell proteins CsoS1ABC and
CsoS4AB; shell-associated protein
CsoS2) from H. neapolitanus
 ΔgpmA and ΔgpmM
(phosphoglycerate mutase, EC
5.4.2.1); ΔpfkA and ΔpfkB (ATP-
dependent 6-
phosphofructokinase, EC
cbbM (RuBisCO, EC 4.1.1.39) from
2.7.1.11); Δzwf (glucose-6-
Rhodospirillum rubrum ATCC 11170, prkA
phosphate 1-dehydrogenase, EC
(phosphoribulokinase, EC 2.7.1.19) from
+ 1.1.1.49); ΔaceBAK (isocitrate
Synechococcus elongatus PCC 7942 and
lyase, EC 4.1.3.1; malate
Rru_A2056 (carbonic anhydrase, EC 4.2.1.1)
synthase, EC 2.3.3.9; and
from Rhodospirillum rubrum.
isocitrate dehydrogenase
kinase/phosphatase, EC
2.7.11.5); ΔmutS (methyl-
directed mismatch repair
protein, EG10625)
Table 2. Examples of C. glutamicum strains engineered for access to non-native carbon sources

Carbon source Sole carbon source Heterologous genes expressed Modifications of endogenous genes

amyA (α-amylase, EC 3.2.1.1) from

Starch -
S. griseus

amyA (α-amylase, EC 3.2.1.1) from

+
S. bovis

Carboymethylcellulos celE (chimeric endoglucanase E,

e consisting of endoglucanase E
catalytic backbone, EC 3.2.1.4 and
dockerin domain of endoglucanase
- B, EC 3.2.1.4) from C. cellulovorans,
minicbpA (scaffolding protein that
binds cellulosomal enzyme
subunits and to form a
minicellulosome)
Cellulose celE (endoglucanase, EC 3.2.1.4);
+ bglA (β-glucosidase, EC 3.2.1.21)
from C. thermocellum
Cellobiose Overexpression of bglA
(phospho-beta-glucosidase,
EC 3.2.1.86) and bglF
(phosphoenolpyruvate:carboh
-
ydrate phosphotransferase
system beta-glucoside-specific
enzyme IIBCA component)
from C. glutamicum
β-glucosidade (sde1394, EC
+ 3.2.1.21) from S. degradans

Endoglucanases (EC 3.2.1.4) from

X. campestris XCC3521 and
+ XCC2387, β-glucosidase (EC
3.2.1.21) from
S. degradans Sde1394
β-glucan celA (endoglucanase, EC 3.2.1.4)
- from C. thermocellum; torA (signal
sequence for protein secretion)
from E. coli K12
Glucosamine Overexpression of nagB
+ (glucosamine-6-phosphate
deaminase, EC 3.5.99.6)
Deletion of nanR (repressor of sialic
acid catabolism)
N-Acetyl-glucosamine nagE (N-acetyl-glucosamine- Overexpression of nagB
+ specific PTS, EC 2.7.1.69) from C. (glucosamine-6-phosphate
glycinophilum deaminase, EC 3.5.99.6) and nagA
 (N-acetylglucosamine-6-phosphate
deacetylase, EC 3.5.1.25)
nagE (N-acetyl-glucosamine-
Deletion of nanR (repressor of sialic
specific PTS, EC 2.7.1.69) from C.
acid catabolism)
glycinophilum
Levoglucosan EU751287.1 (levoglucosan kinase, EC 2.7.-
+
.-) from L. starkeyi
Xylose
xylA (xylose isomerase, EC 5.3.1.5) from X. Overexpression of xylB (xylulokinase,
+
campestris EC 2.7.1.17)

xylA (xylose isomerase, EC 5.3.1.5), xylB

(xylulokinase, EC 2.7.1.17) from E. coli
Arabinose

araA (arabinose isomerase, EC 5.3.1.3),

araB (ribulokinase, EC 2.7.1.16), and araD
+
(ribulose 5-phosphate 4-epimerase, EC
5.1.3.4 ) from E. coli

Galactose galK (galactokinase, EC 2.7.1.6), galT

(UDP-glucose-1-P-uridylyltransferase, EC
2.7.7.12), galE (UDP-galactose-4-
+
epimerase, EC 5.1.3.2) and galM (aldose-
1-epimerase, EC 5.1.3.3) from L. lactis
subsp. cremonis MG1363
Lactose lacY (lactose permease), lacZ (beta-
+ galactosidase, EC 3.2.1.23) from E.
coli
lacY (lactose permease), lacZ (beta-
galactosidase, EC 3.2.1.23) from L.
+
delbrueckii subsp. bulgaricus ATCC
11842
Glycerol glpD (glycerol 3-phosphate
dehydrogenase, EC 1.1.1.8); glpF
+
(aquaglyceroporin), glpK (glycerol
kinase, EC 2.7.1.30) from E. coli

Overexpression of glpD (glycerol 3-

phosphate dehydrogenase, EC
+
1.1.1.8), glpK (glycerol kinase, EC
2.7.1.30)
Dicarboxylic acids Overexpression of dccT (sodium
+ dependent dicarboxylate transporter
of the DASS family)
Overexpression of dctA (proton
+ dependent dicarboxylate uptake
system)
Methanol hxlA (3-hexulose-6-phosphate
(co-substrate) synthase, EC 4.1.2.43) hxlB (6-
Δald (aldehyde dehydrogenase, EC
phospho-3-hexulose isomerase, EC
1.2.1.3); ΔfadH (mycothiol-
- 5.3.1.27) from B. subtilis; mdh
dependent formaldehyde
(methandol dehydrogenase, EC
dehydrogenase, EC 1.1.1.306)
1.1.1.244) from B. methanolicus
MGA3
Table 3. Examples of Pseudomonas strains engineered for access to non-native carbon sources

Pr
Carbon source Sole carbon source Heterologous genes expressed Modifications of endogenous genes yie
(g/
Xylose xylA (α-ketoglutaric semialdehyde
dehydrogenase, EC 1.2.1.3), xylD (D-
+ xylonate dehydratase, EC 4.2.1.82), xylX
(2-keto-3-deoxy-D-xylonate dehydratase,
EC 5.3.3.-) from C. crescentus

xylA (xylose isomerase, EC 5.3.1.5), xylB Δgcd (glucose dehydrogenase, EC

+
(xylulokinase, EC 2.7.1.17) from E. coli 1.1.5.2)

Arabinose xylA (xylose isomerase, EC: 5.3.1.5), xylB

(xylulokinase, EC 2.7.1.17), araFGH Δgcd (glucose dehydrogenase, EC
+
(arabinose transporter, EC 3.6.3.17 from E. 1.1.5.2)
coli

Lactose lacY (lactose permease, EC 3.6.3.18), lacZ

+ (β-galactosidase, EC 3.2.1.23), lacA (β-
galactosidase, EC 3.2.1.23) from E. coli

Parathion opd (organophosphate hydrolase, EC

3.1.8.1) from Flavobacterium sp. ATCC
27551, pnpA (p-nitrophenol
monooxygenase, EC 1.14.13.166), pnpB
(benzoquinone reductase, EC 1.6.5.6),
pnpC (hydroquinone 1,2-dioxygenase, EC
+
1.13.11.66), pnpD (hydroxymuconic
semialdehyde dehydrogenase, EC
1.2.1.61), pnpE (maleylacetate reductase,
EC 1.3.1.32), pnpR, pnpS (LysR-type
regulatory proteins) from Pseudomonas
sp. ENV2030

Methanol hps (3-hexulose-6-phosphate synthase, EC

(co-substrate) - 4.1.2.43), phi (6-phospho-3-hexulose
isomerase, EC 5.3.1.27) from B. brevis

Paraoxon opd (organophosphate hydrolase, EC

3.1.8.1) from Flavobacterium sp. ATCC
27551, pnpA (p-nitrophenol
monooxygenase, EC 1.14.13.166), pnpB
(benzoquinone reductase, EC 1.6.5.6),
pnpC (hydroquinone 1,2-dioxygenase, EC
1.13.11.66), pnpD (hydroxymuconic
+
semialdehyde dehydrogenase, EC
1.2.1.61), pnpE (maleylacetate reductase,
EC 1.3.1.32), pnpR, pnpS (LysR-type
regulatory proteins) from Pseudomonas
sp. ENV2030, pde (phosphodiesterase, EC
3.1.3.-) from D. acidovorans , phoA
(alkaline phosphatase, EC 3.1.3.1) from P.
 aeruginosa HN854

dntA (2,4-DNT dioxygenase, EC 1.13.11.-),

dntB (4M5NC monooxygenase, EC
1.14.13.-), dntD (2,4,5-THT oxygenase, EC
2,4- 1.13.11.-), dntE (methylmalonate
+
dinitrotoluene semialdehyde dehydrogenase (EC
1.2.1.27), dntG (4-hydroxy-2-keto-5-
methyl-6-oxo-3-hexenoate hydrolase, EC
3.7.1.-) from Burkholderia sp. strain DNT
Table 4. Examples of B. subtilis strains engineered for access to non-native carbon sources

P
Carbon source Sole carbon source Heterologous genes expressed Modifications of endogenous genes y
(
Cellulose + bscel5 overexpression (endoglucanase,
L
EC 3.2.1.4)

+ β-glucosidase (EC 3.2.1.21) from 2

T. fusca Y

Acetate, acetoin aceBA operon from B.

- licheniformis (isocitrate lyase, EC
ΔamyE (α-amylase, EC 3.2.1.1)
4.1.3.1; malate synthase, EC
2.3.3.9; hypothetical protein
(BL02643))
Table 5. Examples of yeast strains engineered for access to non-native carbon sources

P
Carbon source Sole carbon source Heterologous genes expressed Modifications of endogenous genes y
(

S. cerevisiae
Starch amyA (α-amylase, EC 3.2.1.1) from
R. oryzae
glaA (glucoamylase EC 3.2.1.3) from
A. awamori
+ E
amyA (α-amylase, EC 3.2.1.1) and w
glaA (glucoamylase, EC 3.2.1.3) s
from A. tubingensis w
s
cel5a (endoglucanase EC 3.2.1.4)
E
from T. reesei, cel7
Cellulose + D
(cellobiohydrolase EC 3.2.1.91)
w
from T. emersonii
Carboxymethylce
llulose, bgl1 (cellobiase, EC 3.2.1.21) from
hydroxyethylcell E. fibuliger Y646, end1 (endo-β-1,4-
ulose, laminarin, glucanase, EC 3.2.1.4) from B.
barley glucan, fibrisolvens, cbh1
+
cellobiose, (cellobiohydrolase, EC 3.2.1.91)
polypectate, from P. chrysosporium, cel1
birchwood Xylan (cellodextrinase, EC 3.1.2.1.-) from
and methyl-β-D- R. flavefaciens
glucopyranoside
DEH (4-deoxy-L- dehR (DEH reductase, EC 1.1.1.126)
erythro-5- from different sources: (V.
hexoseulose splendidus 12B01, A. tumefaciens
urinate) C58 or Vibrio harveyi), kdgK (2-
dehydro-3-deoxygluconokinase, EC E
- 2.7.1.45) from E. coli, kdgpA (eda) (
(2-dehydro-3-deoxy- s
phosphogluconate aldolase, EC
4.1.2.14) from V. splendidus 12B01,
Ac_DHT1 (DEH transporter) from A.
cruciatus ATCC 26324
Xylan xlnD (β-xylosidase; EC 3.2.1.37)
+ from A. niger 90196, xyn2 (xylanase
II, EC 3.2.1.8 ) from T. reesei
Xylose E
xylA (xylose isomerase, EC 5.3.1.5)
+ X
from Piromyces sp. E2 (ATCC 76762)
G
Arabinose araT (arabinose transporter) from Overexpression of xks1
S. stipitis, stp2 (sugar transporting (xylulokinase, EC 2.7.1.17),gal2
+ protein 2) from A. thaliana (galactose permease, 2.A1.6), E
xylA (xylose isomerase, EC 5.3.1.5) rpE1 (D-ribulose-5-phosphate 3-
from Piromyces sp. E2, araA epimerase, EC 5.1.3.1),
 (arabinose isomerase, EC 5.3.1.3), rki1 (ribose-5-phosphate ketol-
araB (ribulokinase, EC 2.7.1.16), isomerase, EC 5.3.1.6),
and araD (ribulose 5-phosphate 4- tkl1 (transketolase, EC 2.2.1.1),
epimerase, EC 5.1.3.4) from S. tal1 (transaldolase, EC 2.2.1.2)
cerevisiae XKS1
Lactose lac4 (β-galactosidase, EC 3.2.1.23)
+ lac12 (β-galactosidase; EC 3.2.1.23)
K. lactis
Chitin chi42 (chitinase, EC 3.2.1.14) from
-
T. asperellum SH 16
Pichia pastoris
amyE (α-amylase, EC 3.2.1.1) from B.
-
subtilis
Starch
Thermostable α-amylase (EC 3.2.1.1) from
-
G. stearothermophilus
araA (L-arabinose isomerase, EC 5.3.1.4)
Lactose -
from Arthrobacter sp.22c

Pichia stipitis
Xylan + xyn2 (β-xylanase, EC 3.2.1.8) from T.
reesei, xynC (β-xylanase, EC 3.2.1.8) from
A. kawachii, xlnD (β-xylosidase, EC
3.2.1.37) from A. niger
Yarrowia
lipolytica
Alginate alyVI (alginate lyase, EC 4.2.2.3)
-
from Vibrio sp. QY101