Probing cytoplasmic peroxide metabolism in Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0-250 µM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases.

Iodate Reduction by Shewanella oneidensis Requires Genes Encoding an Extracellular Dimethylsulfoxide Reductase.

Microbial iodate (IO3 -) reduction is a major component of the iodine biogeochemical reaction network in anaerobic marine basins and radioactive iodine-contaminated subsurface environments. Alternative iodine remediation technologies include microbial reduction of IO3 - to iodide (I-) and microbial methylation of I- to volatile gases. The metal reduction pathway is required for anaerobic IO3 - respiration by the gammaproteobacterium Shewanella oneidensis. However, the terminal IO3 - reductase and additional enzymes involved in the S. oneidensis IO3 - electron transport chain have not yet been identified. In this study, gene deletion mutants deficient in four extracellular electron conduits (EECs; ΔmtrA, ΔmtrA-ΔmtrDEF, ΔmtrA-ΔdmsEF, ΔmtrA-ΔSO4360) and DMSO reductase (ΔdmsB) of S. oneidensis were constructed and examined for anaerobic IO3 - reduction activity with either 20 mM lactate or formate as an electron donor. IO3 - reduction rate experiments were conducted under anaerobic conditions in defined minimal medium amended with 250 µM IO3 - as anaerobic electron acceptor. Only the ΔmtrA mutant displayed a severe deficiency in IO3 - reduction activity with lactate as the electron donor, which suggested that the EEC-associated decaheme cytochrome was required for lactate-dependent IO3 - reduction. The ΔmtrA-ΔdmsEF triple mutant displayed a severe deficiency in IO3 - reduction activity with formate as the electron donor, whereas ΔmtrA-ΔmtrDEF and ΔmtrA-ΔSO4360 retained moderate IO3 - reduction activity, which suggested that the EEC-associated dimethylsulfoxide (DMSO) reductase membrane-spanning protein DmsE, but not MtrA, was required for formate-dependent IO3 - reduction. Furthermore, gene deletion mutant ΔdmsB (deficient in the extracellular terminal DMSO reductase protein DmsB) and wild-type cells grown with tungsten replacing molybdenum (a required co-factor for DmsA catalytic activity) in defined growth medium were unable to reduce IO3 - with either lactate or formate as the electron donor, which indicated that the DmsAB complex functions as an extracellular IO3 - terminal reductase for both electron donors. Results of this study provide complementary genetic and phenotypic evidence that the extracellular DMSO reductase complex DmsAB of S. oneidensis displays broad substrate specificity and reduces IO3 - as an alternate terminal electron acceptor.

Resistance of perfluorooctanoic acid to degradation by the microbially driven Fenton reaction.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) have received recent heightened attention as emerging contaminants. Due to widespread application in household products and aqueous film-forming foams, PFAS are globally distributed in the environment, and bioaccumulate in the blood and tissues of mammals including humans. The microbially driven Fenton reaction, a hybrid biotic/abiotic hydroxyl radical (HO•)-generating system, previously degraded a wide variety of persistant organic pollutants. In the present study, the microbially driven Fenton reaction was employed to attempt degradation of PFOA. Batch cultures of the facultatively anaerobic bacteria Shewanella oneidensis were amended with PFOA and Fe(III)-citrate. Under aerobic conditions, S. oneidensis reduced oxygen to hydrogen peroxide (H2O2), while under anaerobic conditions, S. oneidensis reduced Fe(III) to Fe(II). During aerobic-to-anaerobic transition periods, Fe(II) and H2O2 interacted chemically via the Fenton reaction to produce HO• radicals, which in turn interacted with PFOA. Batch reactors were cycled between aerobic and anaerobic phases for four cycles, residual PFOA was extracted via liquid-liquid extraction and analyzed by liquid chromatography combined with tandem mass spectrometry. Unlike degradation of other organic pollutants, PFOA concentrations remained unchanged, which indicated that PFOA was resistant to degradation by the microbially-driven Fenton reaction. Similar to abiotic (purely chemical) Fenton reaction systems, these results most likely reflect the inability of HO• radicals to oxidatively degrade PFOA.

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.

Combinatorial Fine-Tuning of Phospholipase D Expression by Bacillus subtilis WB600 for the Production of Phosphatidylserine.

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.

Biocatalytic Production of Glucosamine from N-Acetylglucosamine by Diacetylchitobiose Deacetylase.

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dacph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

Synergistic Rewiring of Carbon Metabolism and Redox Metabolism in Cytoplasm and Mitochondria of Aspergillus oryzae for Increased l-Malate Production.

l-Malate is an important platform chemical that has extensive applications in the food, feed, and wine industries. Here, we synergistically engineered the carbon metabolism and redox metabolism in the cytosol and mitochondria of a previously engineered Aspergillus oryzae to further improve the l-malate titer and decrease the byproduct succinate concentration. First, the accumulation of the intermediate pyruvate was eliminated by overexpressing a pyruvate carboxylase from Rhizopus oryzae in the cytosol and mitochondria of A. oryzae, and consequently, the l-malate titer increased 7.5%. Then, malate synthesis via glyoxylate bypass in the mitochondria was enhanced, and citrate synthase in the oxidative TCA cycle was downregulated by RNAi, enhancing the l-malate titer by 10.7%. Next, the exchange of byproducts (succinate and fumarate) between the cytosol and mitochondria was regulated by the expression of a dicarboxylate carrier Sfc1p from Saccharomyces cerevisiae in the mitochondria, which increased l-malate titer 3.5% and decreased succinate concentration 36.8%. Finally, an NADH oxidase from Lactococcus lactis was overexpressed to decrease the NADH/NAD+ ratio, and the engineered A. oryzae strain produced 117.2 g/L l-malate and 3.8 g/L succinate, with an l-malate yield of 0.9 g/g corn starch and a productivity of 1.17 g/L/h. Our results showed that synergistic engineering of the carbon and redox metabolisms in the cytosol and mitochondria of A. oryzae effectively increased the l-malate titer, while simultaneously decreasing the concentration of the byproduct succinate. The strategies used in our work may be useful for the metabolic engineering of fungi to produce other industrially important chemicals.

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.

Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60.

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes-one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)-were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%.

Metabolic engineering of carbon overflow metabolism of Bacillus subtilis for improved N-acetyl-glucosamine production.

Bacillus subtilis is widely used as cell factories for the production of important industrial biochemicals. Although many studies have demonstrated the effects of organic acidic byproducts, such as acetate, on microbial fermentation, little is known about the effects of blocking the neutral byproduct overflow, such as acetoin, on bioproduction. In this study, we focused on the influences of modulating overflow metabolism on the production of N-acetyl-d-glucosamine (GlcNAc) in engineered B. subtilis. We found that acetoin overflow competes with GlcNAc production, and blocking acetoin overflow increased GlcNAc titer and yield by 1.38- and 1.39-fold, reaching 48.9 g/L and 0.32 g GlcNAc/g glucose, respectively. Further blocking acetate overflow inhibited cell growth and GlcNAc production may be induced by inhibiting glucose uptake. Taken together, our results show that blocking acetoin overflow is a promising strategy for enhancing GlcNAc production. The strategies developed in this work may be useful for engineering strains of B. subtilis for producing other important biochemicals.

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.

Efficient expression of cyclodextrin glycosyltransferase from Geobacillus stearothermophilus in Escherichia coli by promoter engineering and downstream box evolution.

Cyclodextrin glycosyltransferase (CGTase) catalyzes hydrolysis, cyclization, coupling, and disproportionation reactions and is widely used in the starch processing industry. In this work, the expression of CGTase from Geobacillus stearothermophilus in Escherichia coli BL21 (DE3) was significantly improved by promoter engineering and downstream box evolution. Firstly, the effects of the promoter type (PT7, Ptrp, PlacUV5, and the hybrid promoters PtacI and PtacII) and spacer sequence on the expression of CGTase were examined. PtacI demonstrated the highest rate of transcriptional activity, which was 4.4-, 7.1-, 3.3-, and 1.5-fold greater than that of PT7, Ptrp, PlacUV5, and PtacII, respectively. The spacer sequence of the promoter was optimized using a degenerate base library, and the GC content of the spacer was found to be inversely proportional to CGTase expression. In addition, CGTase expression was higher when TG:CA and TA:TA dimers were present in the spacer sequence. Under the control of the PtacI promoter with an optimized spacer sequence, extracellular CGTase activity reached 170.6 U/mL, which was seven times higher than that of the original strain (25.2 U/mL). Directed evolution of the downstream box sequence was then performed by randomization of the sequence using degenerate codons, similarly as for the optimization of the spacer sequence. After optimizing the downstream box sequence, CGTase activity increased from 170.6 to 214 U/mL. The results obtained here indicate that in addition to promoter type, the spacer sequence of the promoter and the downstream box are important target elements for improved gene expression.

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.

Metabolic engineering of Aspergillus oryzae for efficient production of l-malate directly from corn starch.

l-Malate, an important chemical building block, has been widely applied in the food, pharmaceutical, and bio-based materials industries. In previous work, we engineered Aspergillus oryzae by rewiring the reductive tricarboxylic acid pathway to produce l-malate from glucose. To decrease the production cost, here, we further engineered A. oryzae to efficiently produce l-malate directly from corn starch with simultaneous liquefaction-saccharification and fermentation. First, a promoter PN5 was constructed by quintuple tandem of the 97-bp fragment containing the cis-element of the glucoamylase gene promoter (PglaA), and with the promoter PN5, the transcriptional level of glaA gene increased by 25-45%. Then, by co-overexpression of glaA, a-amylase (amyB) and a-glucosidase (agdA) genes with the promoter PN5, the l-malate titer increased from 55.5g/L to 72.0g/L with 100g/L corn starch in shake flask. In addition, to reduce the concentration of byproducts succinate and fumarate, a fumarase from Saccharomyces cerevisiae, which facilitated the transformation of fumarate to l-malate, was overexpressed. As a result, the concentration of succinate and fumarate decreased from 12.6 and 1.29g/L to 7.8 and 0.59g/L, and the l-malate titer increased from 72.0g/L to 78.5g/L. Finally, we found that the addition of glucose at the initial fermentation stage facilitated the cell growth and l-malate synthesis, and the l-malate titer further increased to 82.3g/L by co-fermentation of 30g/L glucose and 70g/L corn starch, with a productivity of 1.18g/L/h and a yield of 0.82g/g total carbon sources.

Combinatorial promoter engineering of glucokinase and phosphoglucoisomerase for improved N-acetylglucosamine production in Bacillus subtilis.

In previous work, a recombinant Bacillus subtilis strain was successfully constructed for microbial production of N-acetylglucosamine (GlcNAc). In this study, GlcNAc titer was further improved by combinatorial promoter engineering of key genes glck encoding glucokinase and pgi encoding phosphoglucoisomerase. First, three promoters including constitutive promoter P43, xylose inducible promoter PxylA, and isopropyl-ß-d-thiogalactoside inducible Pgrac were used to replace the native promoters of glcK and pgi, yielding 12 recombinant strains. It was found that when glcK and pgi were both under the control of promoter PxylA, the highest GlcNAc titer in 3-L fed-batch bioreactor reached 35.12g/L, which was 52.6% higher than that of the initial host. Next, the transcriptional levels of the related genes in glycolysis, GlcNAc synthesis pathway, peptidoglycan synthesis pathway, and pentose phosphate pathway were investigated by quantitative real-time PCR analysis. Fine-tuning upper GlcNAc synthesis pathway by combinatorial promoter substitution significantly enhanced GlcNAc production in engineered B. subtilis.

Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis.

N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-PxylA -glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 ± 0.25 to 9.6 ± 0.16 g L-1 ) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 ± 0.25 to 13.2 ± 0.31 g L-1 . Finally, the GlcNAc titer is further increased to 42.1 ± 1.1 g L-1 in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules.

Tuning the transcription and translation of L-amino acid deaminase in Escherichia coli improves α-ketoisocaproate production from L-leucine.

α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from l-leucine, using an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase (l-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of l-AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31.77%, 24.89%, and 30.20%, respectively, above that achieved with BL21/pet28a-lad. The highest KIC titers reached 76.47 g·L-1, 80.29 g·L-1, and 81.41 g·L-1, respectively. Additionally, the integration of these three engineering strategies achieved an even higher KIC production of 86.55 g·L-1 and a higher l-leucine conversion rate of 94.25%. The enzyme-engineering strategies proposed herein may be generally applicable to the construction of other biocatalysts.

Protein and metabolic engineering for the production of organic acids.

Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial organic acid production in the future.

Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate.

Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL-1 to 42.3gL-1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL-1. Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL-1 to 89.5gL-1. Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL-1. The final engineered A. oryzae strain produced 165gL-1 L-malate with a productivity of 1.38gL-1h-1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids.