Metal Reduction and Protein Secretion Genes Required for Iodate Reduction by Shewanella oneidensis.

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO3-) as an anaerobic terminal electron acceptor. Microbial IO3- electron transport pathways are postulated to terminate with nitrate (NO3-) reductase, which reduces IO3- as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO3- reductase is not involved in IO3- reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO3- reduction by Shewanella oneidensis with lactate, formate, or H2 as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO3- reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO3- reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO3- reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane ß-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO3- reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO3- reduction activity. These findings indicate that IO3- reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO3- reductase to the S. oneidensis outer membrane.IMPORTANCE Microbial iodate (IO3-) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO3- reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO3- reduction by S. oneidensis On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO3- reduction by S. oneidensis These findings indicate that the IO3- electron transport pathway terminates with an as yet unidentified IO3- reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO3.

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.

Combinatorial synthetic pathway fine-tuning and comparative transcriptomics for metabolic engineering of Raoultella ornithinolytica BF60 to efficiently synthesize 2,5-furandicarboxylic acid.

The compound 5-hydroxymethylfurfural (HMF) has attracted much attention due to its versatility as an important bio-based platform chemical. Here, we engineered Raoultella ornithinolytica BF60 as a whole-cell biocatalyst for a highly efficient synthesis of 2,5-furandicarboxylic acid (FDCA) from HMF. Specifically, various expression cassettes of key genes, such as hmfH (gene encoding HMF/furfural oxidoreductase [HmfH]) and hmfo (gene encoding HMF oxidase), were designed and constructed for fine-tuning FDCA synthesis from HMF. The FDCA titer reached 108.9 mM with a yield of 73% when 150 mM HMF was used as the substrate. This yield was 16% higher than that without balancing key gene expression in FDCA synthetic pathways. Additionally, to strengthen HmfH expression at the translational level, ribosomal binding site (RBS) sequences, which were computationally designed using the RBS calculator, were assembled into HmfH expression cassettes. The HmfH expression in the presence of these sequences enhanced FDCA titer to 139.6 mM with a yield of 93%. Next, previously unknown candidate genes, such as aldR, dkgA, akR, AdhP1, and AdhP2, which encode enzymes that catalyze the reactions leading to the formation of the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol) from HMF, were identified by RNA-sequencing-based transcriptomics. Combinatorial deletion of these five candidate genes led to an 88% reduction in HMF alcohol formation and 12% enhancement in FDCA production (175.6 mM). Finally, FDCA synthesis was further improved by the substrate pulse-feeding strategy, and 221.5 mM FDCA with an 88.6% yield was obtained. The combinatorial synthetic pathway fine-tuning and comparative transcriptomics approach may be useful for improving the biocatalysis efficiency of other industrially useful compounds.

Comparative genomics and transcriptomics analysis-guided metabolic engineering of Propionibacterium acidipropionici for improved propionic acid production.

Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31 ± 0.84 g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools.

Pgas, a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger.

The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only.IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.

Metabolic Engineering of Raoultella ornithinolytica BF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural.

2,5-Furandicarboxylic acid (FDCA) is an important renewable biotechnological building block because it serves as an environmentally friendly substitute for terephthalic acid in the production of polyesters. Currently, FDCA is produced mainly via chemical oxidation, which can cause severe environmental pollution. In this study, we developed an environmentally friendly process for the production of FDCA from 5-hydroxymethyl furfural (5-HMF) using a newly isolated strain, Raoultella ornithinolytica BF60. First, R. ornithinolytica BF60 was identified by screening and was isolated. Its maximal FDCA titer was 7.9 g/liter, and the maximal molar conversion ratio of 5-HMF to FDCA was 51.0% (mol/mol) under optimal conditions (100 mM 5-HMF, 45 g/liter whole-cell biocatalyst, 30°C, and 50 mM phosphate buffer [pH 8.0]). Next, dcaD, encoding dicarboxylic acid decarboxylase, was mutated to block FDCA degradation to furoic acid, thus increasing FDCA production to 9.2 g/liter. Subsequently, aldR, encoding aldehyde reductase, was mutated to prevent the catabolism of 5-HMF to HMF alcohol, further increasing the FDCA titer, to 11.3 g/liter. Finally, the gene encoding aldehyde dehydrogenase 1 was overexpressed. The FDCA titer increased to 13.9 g/liter, 1.7 times that of the wild-type strain, and the molar conversion ratio increased to 89.0%. IMPORTANCE: In this work, we developed an ecofriendly bioprocess for green production of FDCA in engineered R. ornithinolytica This report provides a starting point for further metabolic engineering aimed at a process for industrial production of FDCA using R. ornithinolytica.

Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects.

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.

Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α-keto acids.

Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α-keto acid production-from the corresponding l-amino acids-catalyzed by FAD-dependent l-amino acid deaminase (l-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis. In the resulting E. coli strain PHCF7, strong overexpression of pma, ribC, and ribF and moderate overexpression of ribH yielded a 90% increase in phenylpyruvic acid (PPA) titer: 19.4 ± 1.1 g · L-1 . Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH2 /FAD using FDH for FADH2 /FAD regeneration and NOX for NAD+ /NADH regeneration. The resulting E. coli strain PHCF7-FDH-NOX yielded the highest PPA production: 31.4 ± 1.1 g · L-1 . Finally, this whole-cell system was adapted to production of other α-keto acids including α-ketoglutaric acid, α-ketoisocaproate, and keto-γ-methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH2 /FAD regeneration for production of α-keto acids. Notably, the strategy reported herein may be generally applicable to other flavin-dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. 2017;114: 1928-1936. © 2017 Wiley Periodicals, Inc.

Microbial response to environmental stresses: from fundamental mechanisms to practical applications.

Environmental stresses are usually active during the process of microbial fermentation and have significant influence on microbial physiology. Microorganisms have developed a series of strategies to resist environmental stresses. For instance, they maintain the integrity and fluidity of cell membranes by modulating their structure and composition, and the permeability and activities of transporters are adjusted to control nutrient transport and ion exchange. Certain transcription factors are activated to enhance gene expression, and specific signal transduction pathways are induced to adapt to environmental changes. Besides, microbial cells also have well-established repair mechanisms that protect their macromolecules against damages inflicted by environmental stresses. Oxidative, hyperosmotic, thermal, acid, and organic solvent stresses are significant in microbial fermentation. In this review, we summarize the modus operandi by which these stresses act on cellular components, as well as the corresponding resistance mechanisms developed by microorganisms. Then, we discuss the applications of these stress resistance mechanisms on the production of industrially important chemicals. Finally, we prospect the application of systems biology and synthetic biology in the identification of resistant mechanisms and improvement of metabolic robustness of microorganisms in environmental stresses.

Biological production of L-malate: recent advances and future prospects.

As intermediates in the TCA cycle, L-malate and its derivatives have been widely applied in the food, pharmaceutical, agriculture, and bio-based material industries. In recent years, biological routes have been regarded as very promising approaches as cost-effective ways to L-malate production from low-priced raw materials. In this mini-review, we provide a comprehensive overview of current developments of L-malate production using both biocatalysis and microbial fermentation. Biocatalysis is enzymatic transformation of fumarate to L-malate, here, the source of enzymes, catalytic conditions, and enzymatic molecular modification may be concluded. For microbial fermentation, the types of microorganisms, genetic characteristics, biosynthetic pathways, metabolic engineering strategies, fermentation substrates, and optimization of cultivation conditions have been discussed and compared. Furthermore, the combination of enzyme and metabolic engineering has also been summarized. In future, we also expect that novel biological approaches using industrially relevant strains and renewable raw materials can overcome the technical challenges involved in cost-efficient L-malate production.

Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis.

UNLABELLED: Shewanella oneidensis is unable to metabolize the sugar xylose as a carbon and energy source. In the present study, an otherwise silent xylose catabolic pathway was activated in S. oneidensis by following an adaptive evolution strategy. Genome-wide scans indicated that the S. oneidensis genome encoded two proteins similar to the xylose oxido-reductase pathway enzymes xylose reductase (SO_0900) and xylulokinase (SO_4230), and purified SO_0900 and SO_4230 displayed xylose reductase and xylulokinase activities, respectively. The S. oneidensis genome was missing, however, an Escherichia coli XylE-like xylose transporter. After 12 monthly transfers in minimal growth medium containing successively higher xylose concentrations, an S. oneidensis mutant (termed strain XM1) was isolated for the acquired ability to grow aerobically on xylose as a carbon and energy source. Whole-genome sequencing indicated that strain XM1 contained a mutation in an unknown membrane protein (SO_1396) resulting in a glutamine-to-histidine conversion at amino acid position 207. Homology modeling demonstrated that the Q207H mutation in SO_1396 was located at the homologous xylose docking site in XylE. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. IMPORTANCE: The activation of an otherwise silent xylose metabolic system in Shewanella oneidensis is a powerful example of how accidental mutations allow microorganisms to adaptively evolve. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass.

Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii.

Propionic acid (PA) and its salts are widely used in the food, pharmaceutical, and chemical industries. Microbial production of PA by propionibacteria is a typical product-inhibited process, and acid resistance is crucial in the improvement of PA titers and productivity. We previously identified two key acid resistance elements-the arginine deaminase and glutamate decarboxylase systems-that protect propionibacteria against PA stress by maintaining intracellular pH homeostasis. In this study, we attempted to improve the acid resistance and PA production of Propionibacterium jensenii ATCC 4868 by engineering these elements. Specifically, five genes (arcA, arcC, gadB, gdh, and ybaS) encoding components of the arginine deaminase and glutamate decarboxylase systems were overexpressed in P. jensenii. The activities of the five enzymes in the engineered strains were 26.7-489.0% higher than those in wild-type P. jensenii. The growth rates of the engineered strains decreased, whereas specific PA production increased significantly compared with those of the wild-type strain. Among the overexpressed genes, gadB (encoding glutamate decarboxylase) increased PA resistance and yield most effectively; the PA resistance of P. jensenii-gadB was more than 10-fold higher than that of the wild-type strain, and the production titer, yield, and conversion ratio of PA reached 10.81 g/L, 5.92 g/g cells, and 0.56 g/g glycerol, representing increases of 22.0%, 23.8%, and 21.7%, respectively. We also investigated the effects of introducing these acid resistance elements on the transcript levels of related enzymes. The results showed that the expression of genes in the engineered pathways affected the expression of the other genes. Additionally, the intracellular pools of amino acids were altered as different genes were overexpressed, which may further contribute to the enhanced PA production. This study provides an effective strategy for improving PA production in propionibacteria; this strategy may be useful for the production of other organic acids. Biotechnol. Bioeng. 2016;113: 1294-1304. © 2015 Wiley Periodicals, Inc.

Metabolic engineering for amino-, oligo-, and polysugar production in microbes.

Amino-, oligo-, and polysugars are important for both medicinal and industrial applications. Microbial processes used in production of such sugars are not only carbon-intensive and energy-demanding processes but also have other distinct disadvantages such as low productivity, low yields, and by-product contamination. Therefore, metabolic engineering has emerged as an effective tool for developing engineered strains to deliver production strategies for many valuable sugars, which were previously difficult to manufacture by other means, in necessary amounts to support their applications. In this review, the recent strategies used for metabolic engineering are summarized and future prospects of this technique are discussed. We hope that this review will contribute to the development of functional and high-value sugar production by metabolic engineering strategies.

Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.

In our previous study, we produced phenylpyruvic acid (PPA) in one step from L-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase (L-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. In this study, metabolic engineering of the L-phenylalanine degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, L-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type L-AAD. Comparative kinetics analysis showed that compared with wild-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts.

Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose.

Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing ß-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

Improved production of propionic acid in Propionibacterium jensenii via combinational overexpression of glycerol dehydrogenase and malate dehydrogenase from Klebsiella pneumoniae.

Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA.

Toward metabolic engineering in the context of system biology and synthetic biology: advances and prospects.

Metabolic engineering facilitates the rational development of recombinant bacterial strains for metabolite overproduction. Building on enormous advances in system biology and synthetic biology, novel strategies have been established for multivariate optimization of metabolic networks in ensemble, spatial, and dynamic manners such as modular pathway engineering, compartmentalization metabolic engineering, and metabolic engineering guided by genome-scale metabolic models, in vitro reconstitution, and systems and synthetic biology. Herein, we summarize recent advances in novel metabolic engineering strategies. Combined with advancing kinetic models and synthetic biology tools, more efficient new strategies for improving cellular properties can be established and applied for industrially important biochemical production.

Engineering propionibacteria as versatile cell factories for the production of industrially important chemicals: advances, challenges, and prospects.

Propionibacteria are actinobacteria consisting of two principal groups: cutaneous and dairy. Cutaneous propionibacteria are considered primary pathogens to humans, whereas dairy propionibacteria are widely used in the food and pharmaceutical industries. Increasing attention has been focused on improving the performance of dairy propionibacteria for the production of industrially important chemicals, and significant advances have been made through strain engineering and process optimization in the production of flavor compounds, nutraceuticals, and antimicrobial compounds. In addition, genome sequencing of several propionibacteria species has been completed, deepening understanding of the metabolic and physiological features of these organisms. However, the metabolic engineering of propionibacteria still faces several challenges owing to the lack of efficient genome manipulation tools and the existence of various types of strong restriction-modification systems. The emergence of systems and synthetic biology provides new opportunities to overcome these bottlenecks. In this review, we first introduce the major species of propionibacteria and their properties and provide an overview of their functions and applications. We then discuss advances in the genome sequencing and metabolic engineering of these bacteria. Finally, we discuss systems and synthetic biology approaches for engineering propionibacteria as efficient and robust cell factories for the production of industrially important chemicals.

Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 µmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 µmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 µmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose.

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.