Pathway and protein engineering approaches to produce novel and commodity small molecules

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Pathway and protein engineering approaches to produce novel and commodity small molecules Namita Bhan1,2, Peng Xu1,2 and Mattheos AG Koffas1,2 Nature has provided us the basis of designing the most integrated and efficacious production platforms. Cell factories via their millions of years of evolution have nearly perfected each of their production systems. We have been trying to imitate, utilize and tweak this system to our advantage by using slightly overlapping and greatly interdependent approaches such as metabolic engineering and systems biology to make nature work for us in an efficient and robust way, without producing toxic waste and/or unnecessary side products. Systems biology, metabolic engineering and ‘omics’ technologies have paved the way for protein and pathway engineering. To this end we will talk about the recent advances in production of novel pharmaceutical and commodity small molecules by designing novel proteins and pathways. Addresses 1 Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, United States Corresponding author: Koffas, Mattheos AG ([email protected])

Current Opinion in Biotechnology 2013, 24:1137–1143 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Ajikumar Parayil and Federico Gago For a complete overview see the Issue and the Editorial Available online 14th March 2013 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.02.019

Introduction Living organisms have been tuned by millions of years of evolution to efficiently utilize renewable sources of energy to produce a variety of chemicals, many of which have found extensive human applications. The emergence of recombinant DNA technologies and metabolic engineering have allowed the expression of entire metabolic pathways in heterologous hosts and the fine tuning of the resulting recombinant organisms in order to produce such important chemicals at high titers and high yields. In recent years, the engineering of biological systems has expanded in other directions, beyond making the production of natural compounds economically viable. Metabolic engineers are becoming more and more interested www.sciencedirect.com

in creating novel metabolic pathways by mixing and matching biosynthetic enzymes from different sources or altering the biochemical properties of enzymes in order to generate novel molecules. Commodity chemicals such as building blocks for biopolymers, aroma chemicals, etc. are of significant interest for biological engineering [1] (Figure 1). Extensive work has been done on small molecules such as n-butanol, isobutanol and higher alcohols, which are proposed to be the ideal replacement for the conventional fossil-based fuels [2]. In addition, pharmaceutically important small molecules have been diversified using tailoring enzymes, fusion proteins and protein engineering techniques [3]. The different approaches involve in vitro or in vivo production systems. While in vitro production platforms enjoy the benefit of not needing to streamline all the other processes of the cell to enhance yield, assembly of several different enzymes and cascading reactions can be a daunting task. In addition, at an industrial level, optimization of conditions for in vitro enzymatic reactions is more difficult than that for whole cell systems. On the other hand, in vivo production platforms suffer from difficulty in channeling intermediates intrinsic to the heterologous host into the pathway of interest as well as the potential toxic effects of pathway intermediates or the end product. The recent advances in understanding cellular metabolism have significantly facilitated the progress of pathway engineering for production of novel and commodity chemicals. In addition, development of more efficient cloning techniques has made it possible to express entire pathways heterologously in genetically tractable organisms, usually bacteria and yeast [4–6]. Metabolomics has also played a pivotal role in the synthetic microbiology of secondary metabolism. It has helped to identify bottlenecks for improving yields and novel compounds in microbes, and the discovery of entirely new pathways, with the assistance of metabolic modeling and genome mining [7,8].

In vitro approaches In vitro enzymatic reactions have the distinct advantage of producing regio-selective compounds usually at high purity. The conversion rate and specificity of the enzymes can be tweaked by modifying the catalytically important residues of the enzymes involved [9–11]. Certain robust enzymes such as polyketide synthase (PKS) type 1 are difficult to functionally express in vitro. Recently however Ma et al. have reported the isolation of functional lovastatin nonoketide synthase (LovB) by altering the Current Opinion in Biotechnology 2013, 24:1137–1143

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Figure 1

S Metabolic Engineering Systems Biology ‘Omics’ Techniques

S Protein Engineering

E1*

In vitro assays

E2 Modular Approach

Tunable Control

E3 T

P

P

Current Opinion in Biotechnology

Schematic representation of strategies to generate novel pharmaceuticals and commodity chemicals. Metabolic engineering, systems biology and ‘omics’ techniques provide the basis for protein and pathway engineering. The boxes in blue represent in vivo approaches. Green box represents in vitro and in vivo approaches. Tailoring enzymes can be used to decorate the small molecules through glycosylation, acylation, alkylation, etc. Notations: E1*: engineered protein; E2, E3: enzymes of engineered pathway; T: tailoring enzymes.

expression host (Saccharomyces cerevisiae instead of Aspergillus) and formed the nonaketide, dihydromonacolin L acid, by coupling LovB and enoyl reductase (LovC) in vitro [12]. Further fusion of different iterative enzymes like PKSs and nonribosomal peptide synthases (NRPS), which act on similar substrates has resulted in in vitro synthesis of the siderophore yersiniabactin [13], tetramic acid-containing macrolactams (antifungal agents) [14], and cyclopiazonic acid intermediates (inhibitors of Ca2+-ATPase) [15]. Type II PKSs are analogous to the bacterial fatty acid synthases (FAS), and catalyze the formation of bacterial aromatic natural products such as actinorhodin, frenolicin and tetracenomycin. Khosla et al. have extensively worked on in vivo expression of type II PKSs. They have also recently reported in vitro analysis of the hedamycin PKS, which has shed some light on the functionality of type II PKSs [16]. Diversification of products of type III PKSs is a classic example of protein engineering. Unlike their type I counterparts, type III PKSs are relatively small proteins with a single polypeptide chain. Site-directed engineering of the active-site architecture of these enzymes has been used to produce unnatural polyketide scaffolds [17]. Abe et al. have recently reported producing a novel polyketide-alkaloid scaffold [18]. A precursor-directed Current Opinion in Biotechnology 2013, 24:1137–1143

approach was applied, where malonyl-CoA synthetase (MCS) in conjunction with chalcone synthase (CHS) was expressed in vitro and synthetic starter molecules were used as substrates and malonate as extender. Kwon et al. have also showed how the immobilization of the enzymes helps to improve the yields by a great extent (30% in this case), which can be an important factor when trying to reconstitute an entire pathway in vitro [19,20] (Figure 2). Tailoring enzymes are nature’s own way of diversifying the secondary metabolites by decorating the molecules produced with different chemical moieties such as sugar and alkyl groups and halogen molecules. In a recent study the DesVII, a glycosyltransferase from S. venezuelae was coupled with an auxiliary protein, DesVIII, to form a number of unnatural macrolides [21]. In addition, more than 50 novel O-glycosides, S-glycosides and N-glycosides of oleandomycin were produced using the wild-type glycosyltransferase OleD and a triple mutant of the same enzyme (A242V/S132F/P67T) [22]. Various geranyl and dimethylallyl aromatic polyketides were synthesized in vitro, after expression and purification of Fur7, a prenyltransferase (PT) from Escherichia coli [23]. Terpenoids constitute another class of highly structurally diverse molecules. The substrate flexibility of the terpenoid pathway enzymes is relatively limited and as a result diversification of products is achieved by altering chain length, branching and cyclization. A recent successful attempt was made to form chlorinated analogs of GGPP by supplying chlorinated isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as substrates for farnesyl pyrophosphatase (FPPase) [24]. In addition, chimeric enzymes were prepared by fusing catalytic fragments of FFPase and chrysanthemyl pyrophosphatase (CPPase), so that the resulting enzyme could catalyze chain branching and cyclobutanation reaction [25]. Moreover, a novel triterpenoid (13aH)-isomalabarica14(27),17E,21-trien-3b-ol was created by mutating lanosterol synthase [26]. Promiscuity of various sesquiterpene synthases from Coprinus cinereus was tested for different FPP stereoisomers [27].

In vivo approaches Construction of new pathways by assembling enzymes from different unrelated organisms or from different pathways from the same organism has enabled the construction of systems that can result in a wide variety of nonnatural or diversified natural products. Tseng and Prather have recently addressed the issue of removing the bottlenecks for different modules in a synthetically designed pathway based on the butanol biosynthetic pathway [28]. They achieved this by creating a bypass for each module by developing a CoA addition/removal tool kit. All the intermediates of the pathway are CoA derivatives, and are thus trapped inside the cell. The use www.sciencedirect.com

Bioengineering of novel & commodity small molecules Bhan, Xu and Koffas 1139

Figure 2

(a)

Type I PKS KS

MAT

DH

MT

ER0

KR

ACP

CON

+

ER

/

M1cG

Coupling of domains (b) Type III PKS

(c)

Tailoring enzymes

Altering catalytically important residues

H PT

Cy c

Gly GT

P

PO

E Re

Ox CT

Current Opinion in Biotechnology

Schematic representation of different techniques that have been employed to produce novel compounds in vitro. (a) Coupling different domains to study the function of type I polymerases, which can be an important approach to diversify their products. (b) Detailed understanding of catalytically important residues has helped to alter the substrate specificity and the products formed by type III PKS [17,20]. (c) Tailoring enzymes that can be used to alter products. Notations: Product (P); halogenase (H); cyclase (CYC); oxygenase/oxidase (Ox); reductase (Re); glycosyltransferase (GT); epimerase (E); peroxidase (PO); prenyltransferase (PT) and carbamoyltransferase (CT).

of this tool kit provided the desired product stream or feed stream by either hydrolyzing metabolites within a module or activating exogenously supplied free acids once inside the cell. They reported successful production of propionate, trans-2-pentenoate, valerate, and pentanol. In another example Ajikumar et al. have used a modular approach to optimize the production of taxadiene. They divided the pathway into upstream methylerythritolphosphate (MEP) pathway forming isopentenyl pyrophosphate and a downstream terpenoid-forming pathway and balanced the two modules to reduce the amount of indole formed, which is considered to have inhibitory effects on taxadiene production [29]. Butanol and ethanol, along with their branched chain variants, have been identified as promising replacements for the fast depleting nonrenewable fossil fuels. Attempts to achieve the efficient production of these alternative sources of energy via microbial fermentations include alteration of the keto acid pathway and improvement of the tolerance of microorganisms to these alcohols (Figure 3). Along these lines, initial attempts included overexpression of 2-ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (AdhA) and the valine biosynthetic pathway to redirect the precursors of the aliphatic amino acids into synthesis of isobutanol [30]. Further optimization of isobutanol production in lab scale fermentors with in situ product removal increased the effective www.sciencedirect.com

production titer of isobutanol to 50 g/L [31]. Liao et al. have used norvaline, a valine analog, to select a mutant strain of E. coli to improve isobutanol production [32]. Engineered keto acid reductoisomerase (IIvC), which utilizes NADH as reducing power, has been used to produce isobutanol anaerobically with a yield almost close to the theoretical maximum [33]. Isobutanol production using glucose as the substrate has also been implemented in other microbes such as S. cerevisiae [34–36], Corynebacterium glutamicum [37,38], and Bacillus subtilis [39]. 2methyl-1-butanol and 3-methyl-1-butanol [40] are also produced from naturally occurring 2-keto acids, 2-keto-3methylvalerate and 2-keto-4-methylvalerate, respectively. Longer chain alcohols require longer 2-keto acid precursors, thus the 2-isopropylmalate synthase (IPMS; LeuA) and KivD have been engineered to accept larger substrates [41,42], leading to the production of heptanol and octanol. Previously, the n-butanol pathway has been heterologously expressed in different microbes such as E. coli [43,44], S. cerevisiae [45], Lactobacillus brevis [46], Pseudomonas putida and B. subtilis [47]. However, the titers obtained were low, due to the poor expression of the butyryl-CoA dehydrogenase electron transferring flavoprotein (Bcd/Etf) complex. This problem was overcome by expressing trans-2-enyol-CoA reductase (Ter) for the reduction of crotonyl-CoA using NADH without requirement of additional ferredoxin partners as compared to Bcd/Etf [48,49]. Dekishima et al. have used this E. coli Current Opinion in Biotechnology 2013, 24:1137–1143

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Figure 3

Glucose/Glycerol 1 Threonine Pathway

Phosphoenolpyruvate 1

Citramalate Pathway

Pyruvate Propionyl-CoA

IPMS Chain Elongation

2

Valine Pathway

2 3-Hydroxyvalerate

Acetyl-CoA

Malonyl-CoA

E1

3

3 Valeraldehyde FAS Pathway

CoA-dependent pathway E3

Fatty Acids

Butyraldehyde

Isobutyraldehyde E2 Pentanol Isobutanol

E2

β-Oxidation Pathway

Butanol Current Opinion in Biotechnology

Different approaches to pathway engineering for optimizing n-butanol, isobutanol and fatty acid ethyl esters (FAEEs) production in microbes. The boxes in pink represent the intermediates. Boxes in blue are the pathways altered or used for biofuel production. Boxes in green represent the approach used by Tseng and Prather for modular pathway generation [28]. Light green boxes represent the modules used by Xu et al. [52]. The red arrow indicates reversing the b-oxidation pathway. Notations: E1: 2-ketoacid decarboxylase; E2: alcohol dehydrogenase; E3: CoA-acylating alcohol dehydrogenase; IPMS: isopropylmalate synthesis.

strain to synthesize higher chain alcohols using a 3-ketothiolase (BktB) from Ralstonia eutropha by condensation of another acetyl-CoA to the butyryl-CoA. Machado et al. have used a selection platform to enhance higher alcohol production to produce hexanol and n-octanol [50]. Recently, pathway engineering strategies have been carried out in E. coli by reversing the b-oxidation pathway. This was achieved by mutating transcriptional regulators fadR, atoC, crp and by knockingout arcA. Upon further knockout of its fermentation pathways (DadhE, DfrdA, Dpta) [51] and overexpression of native thiolase (YqeF) and alcohol dehydrogenase (FucO), production of n-butanol reached 1.9 g/L. Recently, Xu et al. have divided the E. coli fatty acid synthesis pathway into three modules and identified conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Optimizing the transcription levels of these modules and refining protein translation efficiency led to the final fatty acid production of 8.6 g/L in a fed-batch bioreactor [52]. Biopolymers serve as economically important specialty chemicals, which can be either entirely produced in vivo Current Opinion in Biotechnology 2013, 24:1137–1143

or just the monomers produced in vivo and further modifications carried out in vitro [1]. In line with their de novo pathway engineering, the Prather group designed a pathway by combining enzymes from bacteria, fungi and mammalian sources to produce glucaric acid in E. coli [53]. Some of the other interesting specialty chemicals produced via pathway and protein engineering include 1,3-propanediol [54] and 1,4-butanediol [55]. Zhao et al. have constructed a synthetic pathway in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra for isoprene synthesis [56]. Previously, Leonard et al. have used a combination of protein and pathway engineering to optimize the terpenoid production in E. coli [57]. Several separate attempts have been made to generate poly(3-hydroxyalkanoates) (PHAs) molecules with prespecified chemistry and properties such as defined molar compositions of specific repeating units [58]. Differences in repeating unit composition influence the physical properties of PHAs [59,60]. However, Nomura et al. have shown very specific and regulated production of PHAs; repeating units between 4 and 12 carbons could be tailormade simply by using a fatty acid substrate of the same www.sciencedirect.com

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number of carbons [61]. This was achieved with the E. coli LSBJ strain, derived from the E. coli LS5218, which was developed to facilitate the constitutive expression of genes responsible for fatty acid utilization [62]. They further developed this strain, so that it could not oxidize fatty acids beyond enoly-CoA [63], thus the enoly-CoA intermediates of b-oxidation were utilized by transgenically expressed (R)-specific enoyl-CoA hydratase (PhaJ4) and PHA synthase (PhaC1) (STQk) to form PHAs. They also optimized the use of readily available carbon sources like glycerine and levulinic acid in varied ratios to form PHB to PHV homopolymers, with tunable 3-HB:3-HV contents [64]. Ultrahigh-molecular-weight poly[(R)-3hydroxybutyrate] was produced by rearranging the order of genes in the phaCAB operon [65]. It was found that both the molecular weight and accumulation levels were dependent on the order of the genes in the phaCAB operon relative to the promoter.

References and recommended reading

Diversification of natural products has been carried out by feeding a series of natural or nonnatural substrates depending on the substrate requirement of the enzymes involved [66]. In addition, successful expression of the tetracycline pathway in the ideal heterologous host along with pathway pairing led to the formation of several new tetracycline compounds and the discovery of a new set of tailoring enzymes [67]. Pathway engineering led to the production of quercetin-3-O-(6-deoxytalose), a novel quercetin glycoside as reported by Yoon et al. [68]. Extensive work has been done on in vivo engineering of PKSs; for an in depth review we would like the reader to refer to Khosla et al. [69].

Perspective As seen by the recent progress made in diversifying the production platform of heterologous hosts and in vitro expression systems, developing novel compounds of economical and pharmaceutical value is a rapidly evolving field. A better alignment of the data based fields (genomics, proteomics, metabolomics, etc.) will further assist in assembling enzymes from different pathways/organisms. The biggest obstacle in carrying out in vitro diversification is preserving the functionality of the proteins. At the same time, the bottleneck in the in vivo approach lies in streamlining the intrinsic pathways, so as to maximize yield. However, application of computational techniques for predicting optimal protein synthesis [70], novel pathways, retrosynthesis of metabolic compounds and mining of ‘omics’ data [71,72,73] represent promising approaches to overcome these limitations. All these advances along with improvement in expression systems and cloning techniques will help to pave the way to easier pathway and protein engineering [74].

Acknowledgements This work is supported by the startup funds and Biocatalysis Constellation funds awarded to M.A.G.K. by Rensselaer Polytechnic Institute. www.sciencedirect.com

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Current Opinion in Biotechnology 2013, 24:1137–1143