Kechun Zhang

Expanding metabolism for biosynthesis of nonnatural alcohols

Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.

Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine

The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a nonnatural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammonium-assimilating enzyme glutamate dehydrogenase. The specificity constant kcat/Km of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase IlvE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, ΔrhtA), 5.4 g/L L-homoalanine was produced from 30 g/L glucose (0.18 g/g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates.

Engineering cooperativity in biomotor-protein assemblies

A biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.

Scalable production of mechanically tunable block polymers from sugar

Significance In recent years there has been extensive research toward the development of sustainable polymeric materials. However, environmentally benign, bioderived polymers still represent a woefully small fraction of plastics and elastomers on the market today. To displace the widely useful oil-based polymers that currently dominate the industry, a bioderived synthetic polymer must be both cost and performance competitive. In this paper we address this challenge by combining the efficient bioproduction of β-methyl-δ-valerolactone with controlled polymerization techniques to produce economically viable block polymer materials with mechanical properties akin to commercially available thermoplastics and elastomers. Development of sustainable and biodegradable materials is essential for future growth of the chemical industry. For a renewable product to be commercially competitive, it must be economically viable on an industrial scale and possess properties akin or superior to existing petroleum-derived analogs. Few biobased polymers have met this formidable challenge. To address this challenge, we describe an efficient biobased route to the branched lactone, β-methyl-δ-valerolactone (βMδVL), which can be transformed into a rubbery (i.e., low glass transition temperature) polymer. We further demonstrate that block copolymerization of βMδVL and lactide leads to a new class of high-performance polyesters with tunable mechanical properties. Key features of this work include the creation of a total biosynthetic route to produce βMδVL, an efficient semisynthetic approach that employs high-yielding chemical reactions to transform mevalonate to βMδVL, and the use of controlled polymerization techniques to produce well-defined PLA–PβMδVL–PLA triblock polymers, where PLA stands for poly(lactide). This comprehensive strategy offers an economically viable approach to sustainable plastics and elastomers for a broad range of applications.

A Synthetic Metabolic Pathway for Production of the Platform Chemical Isobutyric Acid

Society currently relies on fossil-based resources for energy and chemical feedstocks. Due to the depletion of oil reserves, there is a growing interest in exploring alternatives to petroleum-based products. Biosynthesis is a promising approach that enables the sustainable production of fuels or chemicals from renewable carbon sources. A great challenge is that many useful chemicals are not accessible by natural biological systems. Therefore it is necessary to design or evolve novel metabolic pathways for the production of non-natural metabolites. Here, we report the development of a biosynthetic route to isobutyric acid. Isobutyric acid (1) is a useful platform chemical. It can be converted into methacrylic acid (2) by catalytic oxidative dehydrogenation. The methyl ester of 2 (i.e. , methyl methacrylate) is produced in a quantity of 2.2 million tonnes per year for the synthesis of poly(methyl methacrylate). 1 can also be used to manufacture sucrose acetate isobutyrate (3), an emulsifier that is used in printing inks, automotive paints, and beverage additives with a market size of 100 000 tonnes annually. Another application of 1 is the synthesis of 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (4) (Texanol) or diisobutyrate (TXIB). TXIB is a non-phthalate plasticizer and Texanol is the most widely used coalescent (produced ca. 50 000 tonnes per year). More applications of 1 include the preparation of isopropyl ketones, such as isobutyrone (5), by decarboxylative coupling. The current manufacturing process of 1 is acid-catalyzed Koch carbonylation of propylene (Scheme 1 a). There are two major concerns for this chemical process: (i) propylene is produced by cracking larger hydrocarbon molecules from non-renewable resources such as petroleum and natural gas, the long-term sustainable supply of which is not guaranteed; and (ii) the usage of carbon monoxide and hydrogen fluoride may cause environmental damage. Such problems could be addressed by replacing chemical synthesis with microbial biosynthesis. While there are some bacteria that can overproduce butyric acid, no natural organism has been discovered to produce a significant amount of isobutyric acid. Retrosynthetic analysis suggests that acids can be prepared by the oxidation of alkanes, alcohols, or aldehydes. Because isobutyraldehyde (10) is a metabolic intermediate in the Ehrlich pathway, we can develop a synthetic metabolic pathway that is composed of a natural metabolic route for generating 10 from glucose and a nonnatural step for oxidizing 10 into 1 (Scheme 1 b). In this pathway, glucose is metabolized to pyruvate (6) through glycolysis. 6 is then converted into 2-ketovaline (9) by valine biosynthetic enzymes AlsS, IlvC and IlvD. 9 can be decarboxylated into 10 by Ehrlich pathway enzyme 2-ketoacid decarboxylase (KIVD) from Lactococcus lactis. The key question for the synthetic pathway is whether or not we can identify an enzyme that could effectively catalyze the conversion of 10 into 1. We speculated that we might discover enzymes capable of catalyzing the oxidation of isobutyraldehyde, despite the fact that these enzymes’ natural functions do not include isobutyric acid biosynthesis. We chose seven aldehyde dehydrogenases as possible candidate enzymes: acetaldehyde dehydrogenase AldB, 3-hydroxypropionaldehyde dehydrogenase AldH, succinate semialdehyde dehydrogenase GabD, phenylacetaldehyde dehydrogenase PadA, g-aminobutyraldehyde dehydrogenase YdcW from E. coli, and a-ketoglutaric semialdehyde dehydrogenase from Burkholderia ambifaria KDHba or from Pseudomonas putida KT2440 KDHpp. [18] These enzymes share little homology and cover a wide range of aldehyde substrates. They (designated X) were individually cloned after KIVD to build an expression cassette kivd-x on a high-copy plasmid (Figure 1 a). Another operon on a medium-copy plasmid in the transcriptional order ilvD-alsS (Figure 1 a) was also constructed to drive the carbon flux towards 2-ketovaline (ilvC was not cloned because the chromosomal copy could be overexpressed upon induction by its substrate acetolactate). The cloned plasmids were transformed into wild-type E. coli strain BW25113. Shake flask fermentation was performed at 30 8C for 48 h. Cultures were grown in M9 minimal medium, containing 40 g L 1 glucose as carbon source, and 0.1 mm IPTG Scheme 1. a) Chemical synthesis of isobutyric acid from petrochemical feedstock, and its representative applications. b) Design of a metabolic pathway for biosynthesis of isobutyric acid from the renewable carbon source glucose. One critical step is to identify an enzyme “X” that can efficiently convert isobutyraldehyde 10 into isobutyric acid.

A Bio-Catalytic Approach to Aliphatic Ketones

Depleting oil reserves and growing environmental concerns have necessitated the development of sustainable processes to fuels and chemicals. Here we have developed a general metabolic platform in E. coli to biosynthesize carboxylic acids. By engineering selectivity of 2-ketoacid decarboxylases and screening for promiscuous aldehyde dehydrogenases, synthetic pathways were constructed to produce both C5 and C6 acids. In particular, the production of isovaleric acid reached 32 g/L (0.22 g/g glucose yield), which is 58% of the theoretical yield. Furthermore, we have developed solid base catalysts to efficiently ketonize the bio-derived carboxylic acids such as isovaleric acid and isocaproic acid into high volume industrial ketones: methyl isobutyl ketone (MIBK, yield 84%), diisobutyl ketone (DIBK, yield 66%) and methyl isoamyl ketone (MIAK, yield 81%). This hybrid “Bio-Catalytic conversion” approach provides a general strategy to manufacture aliphatic ketones, and represents an alternate route to expanding the repertoire of renewable chemicals.

Engineering nonphosphorylative metabolism to generate lignocellulose-derived products

Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.

Engineered biosynthesis of medium-chain esters in Escherichia coli.

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36g/L of IBAc was produced after 72h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.

Generation of Surface-Bound Multicomponent Protein Gradients

Spatial control of bioactive ligands is achieved by integrating microfluidics and protein engineering. The proteins of interest are mixed in a gradient generator and immobilized on artificial polypeptide scaffolds through the strong association of heterodimeric ZE/ZR leucine zipper pairs. Protein densities and gradient shapes are easily controlled and varied in this method.

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

ABSTRACT Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA. The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask). IMPORTANCE Metabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals.