Whole-cell Biocatalysis Research Articles

Stabilization and scale-up of photosynthesis-driven ω-hydroxylation of nonanoic acid methyl ester by two-liquid phase whole-cell biocatalysis.

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O2 via photosynthetic water oxidation. Thus far, research on photosynthesis-driven bioprocesses mainly focuses on strain development and the proof of principle in small-scale biocatalytic reaction setups. This study investigates the long-term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω-hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity-independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two-liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7-fold increased initial specific activities (5.6 ± 0.1 U/gCDW ) and 7.6-fold increased specific yields on biomass (3.8 ± 0.1 mmolH-NAME /gCDW ) as compared with single aqueous phase biotransformations. Finally, the whole-cell biotransformation system was successfully scaled from glass tubes to a stirred-tank photobioreactor. This is the first study reporting the application of the two-liquid phase concept for efficient phototrophic whole-cell biocatalysis.

The new way to synthesize ethyl 1-butyryloxy-1-phenylmethane(P-phenyl)phosphinate and whole-cell biocatalysis by Escherichia coli and Pseudomonas fluorescens

A new compound, ethyl 1-carboxy-1-hydroxy-1-phenylmethane(P-phenyl)phosphinate, was synthesized and the configuration of its diastereoisomers was described using NMR spectroscopy. Acylation with butyryl chloride gave an unexpected product, ethyl butyryloxy(phenyl)methane(P-phenyl)phosphinate, which was then hydrolyzed using two bacterial species as biocatalysts. Good results were achieved for biocatalytic hydrolysis performed by Escherichia coli cells, giving optically active products with 84% enantiomeric excess, and enantioselectivity of 25.8 for one pair of enantiomers. Better results were obtained when the biocatalytic reaction was carrying out for a longer period and the conversion degree reached 71%, the enantiomeric excess of unreacted substrate was >99% and enantioselectivity increased to 32.1. In all cases, isomers bearing α–carbon atom of S configuration were hydrolyzed preferentially.

Multi-level engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the production of C9 chemicals from oleic acid

Whole-cell biotransformation is one of the promising alternative approaches to microbial fermentation for producing high-value chemicals. Baeyer–Villiger monooxygenase (BVMO)-based Escherichia coli biocatalysts have been engineered to produce industrially relevant C9 chemicals, such as n-nonanoic acid and 9-hydroxynonanoic acid, from a renewable long-chain fatty acid. The key enzyme in the biotransformation pathway (i.e., BVMO from Pseudomonans putida KT2440) was first engineered, using structure modeling-based design, to improve oxidative and thermal stabilities. Using a stable and tunable plasmid (STAPL) system, E. coli host cells were engineered to have increased plasmid stability and homogeneity of the recombinant E. coli population, as well as to optimize the level of BVMO expression. Multi-level engineering of the key enzyme in host cells, allowed recombinant E. coli expressing a fatty acid double-bond hydratase, a long-chain secondary alcohol dehydrogenase, and the engineered BVMO from P. putida KT2440 (i.e., E6BVMO_C302L/M340L), to ultimately produce C9 chemicals (i.e., n-nonanoic acid and 9-hydroxynonanoic acid) from oleic acid, with a yield of up to 6 mmoL/g dry cells. This yield was 2.4-fold greater than the yield in the control strain before engineering. Therefore, this study will contribute to the development of improved processes for the biosynthesis of industrially relevant medium chain fatty acids via whole-cell biocatalysis.

Cost-practical of glycolic acid bioproduction by immobilized whole-cell catalysis accompanied with compressed oxygen supplied to enhance mass transfer

Bioprocess for Glycolic acid (GA) production from ethylene glycol by whole-cell catalysis of Gluconobacter oxydans is restrained by various biological impediments and high production costs. In this study, these limitations were subsided through the implementation of immobilized whole-cell bio-catalysis combined with increased oxygen supply. Results indicated that this strategy noticeably enhanced mass transfer efficiency, and prolonged cell life that significantly reduced the cost of biomass. Ultimately, with immobilized whole-cell catalysis in air-open and oxygen-open bioreactor, 41.3 and 66.9 g/L of GA was obtained within 48 h, with an increment of 62.0%. Additionally, in oxygen-compressed bioreactor, 63.3 g/L of GA was accumulated with the yield of 97.2%. Subsequently, 605.7 g of GA was produced after 10 rounds of recovery experiments. Although there was a slight decrease in GA production compared with pure-oxygen supply, production cost was reduced with limited oxygen supply. This strategy commendably demonstrated cost-practical bioprocess for GA production.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems

Enzyme-mediated synthesis of pharmaceutical compounds is a ‘green’ alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell–mediated biocatalysis by the halophilic archaeon H. volcanii.

Engineering a Microbial Consortium Based Whole-Cell System for Efficient Production of Glutarate From L-Lysine.

Glutarate is an important C5 platform chemical produced during the catabolism of L-lysine through 5-aminovalerate (5-AMV) pathway. Here, we first established a whole-cell biocatalysis system for the glutarate production from L-lysine with the engineered Escherichia coli (E. coli) that co-expressed DavAB and GabDT. However, the accumulation of intermediate 5-AMV was identified as one important factor limiting glutarate production. Meanwhile, the negative interaction of co-expressing DavAB and GabDT in a single cell was also confirmed. Here, we solved these problems through engineering a microbial consortium composed of two engineered E. coli strains, BL21-22AB and BL21-YDT, as the whole-cell biocatalysts, each of which contains a part of the glutarate pathway. After the optimization of bioconversion conditions, including temperature, metal ion additives, pH, and cell ratio, 17.2 g/L glutarate was obtained from 20 g/L L-lysine with a yield of 95.1%, which was improved by 19.2% compared with that in a single cell. Little accumulation of 5-AMV was detected. Even at the high substrate concentration, the reduced 5-AMV accumulation and increased glutarate production were achieved. This synthetic consortium produced 43.8 g/L glutarate via a fed-batch strategy, the highest titer reported to date.

Enhanced N-acetyl-D-neuraminic production from glycerol and N-acetyl-D-glucosamine by metabolically engineered Escherichia coli with a two-stage pH-shift control strategy.

Typical N-acetyl-D-neuraminic acid (Neu5Ac) production uses N-acetyl-D-glucosamine (GlcNAc) and excess pyruvate as substrates in the enzymatic or whole-cell biocatalysis process. In a previous study, a Neu5Ac-producing biocatalytic process via engineered Escherichia coli SA-05/pDTrc-AB/pCDF-pck-ppsA was constructed without exogenous pyruvate. In this study, glycerol was found to be a good energy source compared with glucose for the catalytic system with resting cells, and Neu5Ac production increased to 13.97 ± 0.27gL-1. In addition, a two-stage pH shift strategy was carried out, and the Neu5Ac yield was improved to 14.61 ± 0.31gL-1. The GlcNAc concentration for Neu5Ac production was optimized. Finally, an integrated strategy was developed for Neu5Ac production, and the Neu5Ac yield reached as high as 18.17 ± 0.27g L-1. These results provide a new biocatalysis technology for Neu5Ac production without exogenous pyruvate.

Enantiocomplementary decarboxylative hydroxylation combining photocatalysis and whole-cell biocatalysis in a one-pot cascade process

The first demonstration of photochemo-enzymatic whole-cell one-pot enantiocomplementary decarboxylative hydroxylation.

Molecular optimization of autotransporter-based tyrosinase surface display

Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia coli surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.

Biodegradation of Microplastic Derived from Poly(ethylene terephthalate) with Bacterial Whole-Cell Biocatalysts.

At present, the pollution of microplastic directly threatens ecology, food safety and even human health. Polyethylene terephthalate (PET) is one of the most common of microplastics. In this study, the micro-size PET particles were employed as analog of microplastic. The engineered strain, which can growth with PET as sole carbon source, was used as biocatalyst for biodegradation of PET particles. A combinatorial processing based on whole-cell biocatalysts was constructed for biodegradation of PET. Compared with enzymes, the products can be used by strain growth and do not accumulated in culture solution. Thus, feedback inhibition of products can be avoided. When PET was treated with the alkaline strain under high pH conditions, the product concentration was higher and the size of PET particles decreased dramatically than that of the biocatalyst under neutral conditions. This shows that the method of combined processing of alkali and organisms is more efficient for biodegradation of PET. The novel approach of combinatorial processing of PET based on whole-cell biocatalysis provides an attractive avenue for the biodegradation of micplastics.

Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols.

Technologies enabling new enzyme discovery and efficient protein engineering have spurred intense interest in the development of biocatalytic reactions. In recent years, whole-cell biocatalysis has received attention as a simple, efficient, and scalable biocatalytic reaction platform. Inspired by these developments, we have established a whole-cell protocol for oxidative dearomatization of phenols using the flavin-dependent monooxygenase, TropB. This approach provides a scalable biocatalytic platform for accessing gram-scale quantities of chiral synthetic building blocks.

Efficient biosynthesis of heterodimeric C3-aryl pyrroloindoline alkaloids

Many natural products contain the hexahydropyrrolo[2, 3-b]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C3-aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon–carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C3-aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli, we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.

Biocatalytic synthesis of acylated derivatives of troxerutin: their bioavailability and antioxidant properties in vitro

BackgroundFlavonoid glycosides have many beneficial effects on health, but these bioactivities tend to decrease after oral administration owing to their poor lipophilicity. In this study, a facile whole-cell-based method was developed for selective preparation of monoester or diester of troxerutin, a flavonoid derivative. In addition, the bioavailabilities and antioxidant properties of troxerutin and its acylated derivatives were also investigated in cells.ResultsPseudomonas aeruginosa and Pseudomonas stutzeri cells showed high catalytic efficiency (substrate conversion > 90%) and different preferences for troxerutin, resulting in the production of its monoester (TME) and diester (TDE), respectively. The logP values of troxerutin, TME, and TDE were − 2.04 ± 0.10, − 0.75 ± 0.08, and 1.51 ± 0.05 and their Papp values were 0.34 × 10−6 ± 0.05, 0.99 × 10−6 ± 0.12, and 1.54 × 10−6 ± 0.17 cm/s, respectively. The results of hydroxyl radical, ABTS, and ORAC assays indicated that the antiradical activities of acylated derivatives did not exceed that of troxerutin, but showed higher inhibition effects upon 2,2′-azobis(2-amidinopropane) dihydrochloride-induced erythrocyte hemolysis than that of troxerutin (P < 0.05).ConclusionA facile and efficient whole-cell biocatalysis method was developed to synthesize troxerutin-acylated derivatives, markedly enhancing the bioavailability and antioxidant activities of troxerutin in cells. Additionally, the mechanism underlying the observed difference in the antioxidant activities of troxerutin and its esters was ascribed to both their free radical scavenging abilities and distribution on the cell membrane surface.

Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering

α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain Escherichia coli YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an Abies grandis geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between A. grandis GPPSD90G/L175P and Pinus taeda Pt1Q457L. In an effort to improve the production, an E. coli-E. coli modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of A. grandis GPPSD90G/L175P and P. taeda Pt1Q457L. Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain E. coli YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best E. coli-E. coli co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The E. coli-E. coli modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L.

Recent Technology Development for the Biosynthesis of Human Milk Oligosaccharide.

Human milk oligosaccharide (HMO) is a third most abundant component in breast milk. HMOs are molecules naturally tailored to the need of an infant. They protect infants from diseases, foster healthy gastrointestinal systems, reinforce fledgling immune function, and promote early brain development. Supplement HMOs to infant formula, which lack this critical element, would substantially improve the function of formula. Overwhelming evidence also indicate that HMOs can be used for the treatment of arthritis and related autoimmune disease, and inhibition of bacteria adhesion or as potential prebiotics. The prospect of using HMO in these applications has stimulated worldwide interest in developing synthesis technology for these valuable products. As the quantities extracted from human milk are limited, and chemical synthesis methods are time-consuming, costly, and complex, biotechnological approach, involving either enzyme catalysis or metabolically engineered bacteria is preferred. In this review, we highlight the most recent advances in the synthesis technologies, as disclosed in patents and patent applications, and analyze these technologies against those reported in literatures. Significant progress has been made over the past decade, especially in whole-cell biocatalysis for smaller HMOs. Significant challenges remain for larger and more complex HMOs.

TCA cycle-powered synthesis of fucosylated oligosaccharides.

Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progress, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, given the stunning diversity and many varied applications of glycans, broadening the range of glycans accessible via microbial synthesis is of paramount importance. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular "power house," the TCA cycle, to provide the cellular energy for synthesis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis by-products. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding and pH control in the synthesis stage. The system could achieve several grams per liter (3-4 g/L) within a 24-h period in shaker flask cultivation for two targets, fucosyllactose and fucosyllactulose, demonstrating efficiency of the biocatalyst developed and its applicability to both natural and non-natural targets. To the best of our knowledge, this is the first use of TCA cycle intermediates as the energy source for oligosaccharide synthesis and the first description of successful synthesis of fucosyllactulose with titers in several grams per liter.

Production of D-Glyceric Acid by a Two-step Culture Strategy Based on Whole-cell Biocatalysis of Acetobacter tropicalis

Production of D-Glyceric Acid by a Two-step Culture Strategy Based on Whole-cell Biocatalysis of Acetobacter tropicalis

Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis.

(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.

Enhancement of α,ω-Dicarboxylic Acid Production by the Expression of Xylose Reductase for Refactoring Redox Cofactor Regeneration.

The production of α,ω-dicarboxylic acids (DCAs) by whole-cell biocatalysis is often limited by cofactor regeneration. Here, ω-oxidation pathway genes (monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase) were coexpressed with a xylose reductase (XR) gene to regenerate cofactors in an engineered Escherichia coli strain that cometabolizes glucose and xylose. The resulting strain exhibited a 180% increase in DCA production compared with the control strain without XR, and produced xylitol in the presence of xylose. Expression of monooxygenase and XR without other ω-oxidation pathway genes resulted in an additional increase in tetradecanedioic acid concentration and a substrate conversion of 95%, which was 198% higher than that associated with the control strain. The expression of XR helped the system to regenerate and balance the cofactors thereby achieving maximum substrate conversion efficiency. It could serve as an efficient platform for the industrial production of α,ω-DCAs.

Enzyme and microbial technology for synthesis of bioactive oligosaccharides: an update.

Oligosaccharides, in either free or bound forms, play crucial roles in a wide range of biological processes. Increasing appreciation of their roles in cellular communication, interaction, pathogenesis, and prebiotic functions has stimulated tremendous interests in their synthesis. Pure and structurally defined oligosaccharides are essential for fundamental studies. On the other hand, for those with near term medical and nutraceutical applications, their large-scale synthesis is necessary. Unfortunately, oligosaccharides are notoriously difficult in their synthesis, and their enormous diverse structures leave a vast gap between what have been synthesized in laboratory and those present in various biological systems. While enzymes and microbes are nature's catalysts for oligosaccharides, their effective use is not without challenges. Using examples of galactose-containing oligosaccharides, this review analyzes the pros and cons of these two forms of biocatalysts and provides an updated view on the status of biocatalysis in this important field. Over the past few years, a large number of novel galactosidases were discovered and/or engineered for improved synthesis via transglycosylation. The use of salvage pathway for regeneration of uridine diphosphate (UDP)-galactose has made the use of Leloir glycosyltransferases simpler and more efficient. The recent success of large-scale synthesis of 2' fucosyllactose heralded the power of whole-cell biocatalysis as a scalable technology. While it still lags behind enzyme catalysis in terms of the number of oligosaccharides synthesized, an acceleration in the use of this form of biocatalyst is expected as rapid advances in synthetic biology have made the engineering of whole cell biocatalysts less arduous and less time consuming.