Metabolic engineering of D-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid.

Clostridium beijerinckii is an attractive butanol-producing microbe for its advantage in co-fermenting hexose and pentose sugars. However, this Clostridium strain exhibits undesired efficiency in utilizing D-xylose, one of the major building blocks contained in lignocellulosic materials. Here, we reported a useful metabolic engineering strategy to improve D-xylose consumption by C. beijerinckii. Gene cbei2385, encoding a putative D-xylose repressor XylR, was first disrupted in the C. beijerinckii NCIMB 8052, resulting in a significant increase in D-xylose consumption. A D-xylose proton-symporter (encoded by gene cbei0109) was identified and then overexpressed to further optimize D-xylose utilization, yielding an engineered strain 8052xylR-xylT(ptb) (xylR inactivation plus xylT overexpression driven by ptb promoter). We investigated the strain 8052xylR-xylT(ptb) in fermenting xylose mother liquid, an abundant by-product from industrial-scale xylose preparation from corncob and rich in D-xylose, finally achieving a 35% higher Acetone, Butanol and Ethanol (ABE) solvent titer (16.91 g/L) and a 38% higher yield (0.29 g/g) over those of the wild-type strain. The strategy used in this study enables C. beijerinckii more suitable for butanol production from lignocellulosic materials.

Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018.

BACKGROUND: Clostridium acetobutylicum, a gram-positive and spore-forming anaerobe, is a major strain for the fermentative production of acetone, butanol and ethanol. But a previously isolated hyper-butanol producing strain C. acetobutylicum EA 2018 does not produce spores and has greater capability of solvent production, especially for butanol, than the type strain C. acetobutylicum ATCC 824. RESULTS: Complete genome of C. acetobutylicum EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, spo0A and adhEII have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose. CONCLUSIONS: Comparative analysis of C. acetobutylicum hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.

Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose.

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

Removal of L-alanine from the production of L-2-aminobutyric acid by introduction of alanine racemase and D-amino acid oxidase.

L-2-Aminobutyric acid can be synthesized in a transamination reaction from L-threonine and L-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product L-alanine was produced simultaneously. A small amount of L-alanine increased the complexity of the L-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the L-alanine concentration partially. To eliminate the remnant L-alanine, alanine racemase of Bacillus subtilis in combination with D-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the L-2-aminobutyric acid synthesis. L-Alanine could be completely removed by the action of alanine racemase of B. subtilis and D-amino acid oxidase of R. gracilis; thereby, high-purity L-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between L-alanine and L-2-aminobutyric acid, and selectively catalyzed L-alanine to D-alanine reversibly. D-Amino acid oxidase then catalyzed D-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product L-alanine in the production of other neutral amino acids such as L-tertiary leucine and L-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.

Gradually accumulating beneficial mutations to improve the thermostability of N-carbamoyl-D-amino acid amidohydrolase by step-wise evolution.

To further enhance repeated batch reactions with immobilized N-carbamoyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T (50) (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met(31), Asn(93), Gln(207), Asn(242), Glu(266), Thr(271), Ala(273)) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T(50) by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k(cat) and K(m) values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of D-p-hydroxyphenylglycine.

Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum.

D-xylose utilization is a key issue for lignocellulosic biomass fermentation, and a major problem in this process is carbon catabolite repression (CCR). In this investigation, solvent-producing bacterium Clostridium acetobutylicum ATCC 824 was metabolically engineered to eliminate D-glucose repression of d-xylose utilization. The ccpA gene, encoding the pleiotropic regulator CcpA, was experimentally characterized and then disrupted. Under pH-controlled conditions, the ccpA-disrupted mutant (824ccpA) can use a mixture of D-xylose and D-glucose simultaneously without CCR. Moreover, this engineered strain produced acetone, butanol and ethanol (ABE) at a maximal titer of 4.94, 12.05 and 1.04 g/L, respectively, which was close to the solvent level of maize- or molasses-based fermentation by wild type C. acetobutylicum. Molar balance analysis for improved process of mixed sugars utilization also revealed less acid accumulation and more butanol yield by the engineered strain as compared to the wild type. This study offers a genetic modification strategy for improving simultaneous utilization of mixed sugars by Clostridium, which is essential for commercial exploitation of lignocellulose for the production of solvents and biofuels.

Coupled bioconversion for preparation of N-acetyl-D: -neuraminic acid using immobilized N-acetyl-D: -glucosamine-2-epimerase and N-acetyl-D: -neuraminic acid lyase.

N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.

Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio.

A possible way to improve the economic efficacy of acetone-butanol-ethanol fermentation is to increase the butanol ratio by eliminating the production of other by-products, such as acetone. The acetoacetate decarboxylase gene (adc) in the hyperbutanol-producing industrial strain Clostridium acetobutylicum EA 2018 was disrupted using TargeTron technology. The butanol ratio increased from 70% to 80.05%, with acetone production reduced to approximately 0.21 g/L in the adc-disrupted mutant (2018adc). pH control was a critical factor in the improvement of cell growth and solvent production in strain 2018adc. The regulation of electron flow by the addition of methyl viologen altered the carbon flux from acetic acid production to butanol production in strain 2018adc, which resulted in an increased butanol ratio of 82% and a corresponding improvement in the overall yield of butanol from 57% to 70.8%. This study presents a general method of blocking acetone production by Clostridium and demonstrates the industrial potential of strain 2018adc.

Improving the thermostability of N-carbamyl-D-amino acid amidohydrolase by error-prone PCR.

To facilitate the easier production of D-amino acids using N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 degrees C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of D-amino acids.

Ammonium acetate enhances solvent production by Clostridium acetobutylicum EA 2018 using cassava as a fermentation medium.

Cassava, due to its high starch content and low cost, is a promising candidate substrate for large-scale fermentation processes aimed at producing the solvents acetone, butanol and ethanol (ABE). However, the solvent yield from the fermentation of cassava reaches only 60% of that achieved by fermenting corn. We have found that the addition of ammonium acetate (CH(3)COONH(4)) to the cassava medium significantly promotes solvent production from cassava fermented by Clostridium acetobutylicum EA 2018, a mutant with a high butanol ratio. When cassava medium was supplemented with 30 mM ammonium acetate, the acetone, butanol and total solvent production reached 5.0, 13.0 and 19.4 g/l, respectively, after 48 h of fermentation. This level of solvent production is comparable to that obtained from corn medium. Both ammonium (NH(4) (+)) and acetate (CH(3)COO(-)) were required for increased solvent synthesis. We also demonstrated substantially increased acetic and butyric acid accumulation during the acidogenesis phase as well as greater acid re-assimilation during the solventogenesis period in ammonium acetate-supplemented cassava medium. Reverse transcription-polymerase chain reaction analysis indicated that the transcription of several genes encoding enzymes related to acidogenesis and solventogenesis in C. acetobutylicum EA 2018 were enhanced by the addition of ammonium acetate to the cassava medium.

Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli.

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of beta-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Construction of recombinant Escherichia coli D11/pMSTO and its use in enzymatic preparation of 7-aminocephalosporanic acid in one pot.

The main drawback in the industrial production of 7-aminocephalosporanic acid is the accumulation of intermediate (AKA-7-ACA) and destruction of substrate (cephalosporin C) catalyzed by catalase and beta-lactamase. To overcome the adverse effect of these enzymes on the conversion process, Escherichia coli D11 with mutation of katG, katE and ampC genes was constructed by P1 phage transduction, which enabled it not to produce catalase and beta-lactamase, respectively. At the same time, recA mutation in D11 increased the stability of foreign plasmid. With D11 used as host, both d-amino acid oxidase and GL-7-ACA acylase were cloned and expressed by the recombinant plasmids of pMSS or pMSTO, and the production of two enzymes could be increased by addition of 1.0% glucose. Cells of recombinant strain D11/pMSTO could directly convert cephalosporin C into 7-aminocephalosporanic acid at 25 degrees C, with the yield of more than 74%. The data suggested that the constructed D11/pMSTO could be an alternative catalyst for production of 7-aminocephalosporanic acid in one pot.

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin.

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes a previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.

Current status and prospects of industrial bio-production of n-butanol in China.

n-Butanol is an important bulk chemical. Commercial fermentative production of n-butanol has been applied more than 100 years ago but is currently more costly than production from propylene and syngas. Renewed interest in biobutanol as a biofuel has spurred technological advances to the fermentation process. This article reviewed the recent status including the commercialization, pilot production and R&D activities of n-butanol fermentation in China. Long-term bio-production of n-butanol as a next generation biofuel and biochemical from biomass waste and steel mill off-gas needs technology breakthroughs and more environmental concerns from policymakers.

A rapid and specific method to screen environmental microorganisms for cephalosporin acylase activity.

Medically useful semisynthetic cephalosporin antibiotics are made from precursor 7-aminocephalosporanic acid (7-ACA). Cephalosporin acylase (CA), which catalyzes hydrolysis of both glutaryl-7-aminocephalosporanic acid (GL-7ACA) and cephalosporin C (CPC) to 7-ACA, is thus a very important enzyme for producing semisynthetic beta-lactam antibiotics. To facilitate the attempts of obtaining the microorganisms with higher CA activity from natural environments, a new and specific method for screening environmental microorganisms with cephalosporin acylase activity was developed. The core part of cephalosporin was replaced by 6-amino penicillinic acid (6-APA) to generate new substrates glutaryl-6-APA and adipoyl-6-APA for screening. Serratia marcescens that is sensitive to 6-APA and resistant to penicillin G, glutaryl-6-APA and adipoyl-6-APA was used as an indicator strain in an overlaid-agar screening system. A strain capable of producing cephalosporin acylase was selected from thousands of candidates by this method. Because of its specificity, simplicity and sensitivity, the method could be easily installed into a high-throughout system.

Expression of gene encoding GL-7ACA acylase in Escherichia coli.

Glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp.130 catalyzes hydrolysis of glutaryl 7-amino cephalosporanic acid to produce 7-amino cephalosporanic acid (7-ACA). 7-ACA is the starting material for the industrial production of most cephalosparonic derivatives. Six plasmids for expression of GL-7ACA acylase were constructed and these recombin ant plasmids presented different expression characteristics in Escherichia coli. The acylase gene from plasmid pKKCA1 was inserted into plasmid pMFT7-5 and the resulting plasmid pMFT7CA1 has higher expression in E.coli. The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7CA1 was near 5 u/g, so the over produced enzyme was easily purified by a single-step anion exchange column chromatography. The enzyme could be purified by immobilized ion affinity chromatography after fused by 6xHis in the N-terminal of its alpha-subunit. Because plasmid pSMLCA1 brings tc(R) and p15A origin, it is special useful plasmid in fermentation. Two secretory expression plasmids, pSUCA1S and pETCA1pelB, could secrete the acylase to periplasmic space of bacteria. The whole cells containing the secretory expression plasmid may be used for production of 7-ACA directly.

Cloning and Overexpression of the Mitomycin C Resistance Gene (mcr) from Streptoverticillium caespitosum.

Streptoverticillium caespitosum ATCC27422 is a major producer of an anti-cancer drug, mitomycin C. A 6.6 kb DNA fragment containing the mitomycin C resistance gene (mcr) was isolated from ATCC27422 by shotgun method in order to learn the molecular mechanism of mitomycin C resistance. By constructing a series of subclones from this 6.6 kb DNA fragment, the mitomycin C resistance gene was localized on a 3.1 kb DNA fragment. Sequence analysis revealed that the open reading frame of mcr gene was 1 347 bp in size, encoding 448 amino acids with ATG as initiation codon and TGA as termination codon. The mcr gene was specifically expressed under the control of T7 promoter in E.coli, and the resistance to mitomycin C in the transformant was over 100-fold higher than that in wild-type strain. The overexpression of mcr gene in E.coli is very helpful for the further research about the molecular mechanism of drug resistance.

Secretory Expression of GL-7-ACA Acylase in E. coli.

The gene of beta-subunit of GL-7-ACA acylase [7beta-(4-carboxybutanamido) cephalosporanic acid acylase] was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B. Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp. into pTrcCA1B. Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235. pTrcCA1S and pKKCA1S were allowed to transform TG1. These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria. As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24. And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.

Improvement of transformation and electroduction in avermectin high-producer, Streptomyces avermitilis.

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.

Economical challenges to microbial producers of butanol: feedstock, butanol ratio and titer.

Butanol is an important solvent and transport fuel additive, and can be produced by microbial fermentation. Attempts to generate a superior microbial producer of butanol have been made through different metabolic engineering strategies. However, to date, butanol bio-production is still not economically competitive compared to petrochemical-derived production because of its major drawbacks, such as, high cost of the feedstocks, low butanol concentration in the fermentation broth and the co-production of low-value by-products acetone and ethanol. Here we analyze the main bottlenecks in microbial butanol production and summarize relevant advances from recently reported studies. Further needs and directions for developing real industrially applicable strains in butanol production are also discussed.