Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh).

Toxic compounds, such as formic acid, furfural, and hydroxymethylfurfural (HMF) generated during pretreatment of corn stover (CS) at high temperature and low pH, inhibit growth of Zymomonas mobilis and lower the conversion efficiency of CS to biofuel and other products. The inhibition of toxic compounds is considered as one of the major technical barriers in the lignocellulose bioconversion. In order to detoxify and/or degrade these toxic compounds by the model ethanologenic strain Z. mobilis itself in situ the fermentation medium, we constructed a recombinant Z. mobilis ZM4 (pHW20a-fdh) strain that is capable of degrading toxic inhibitor, formate. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. The NADH regeneration reaction also improved both the energy efficiency and cell physiological activity of the recombinant organism, which were definitely confirmed by the improved cell growth, ethanol yield, and ethanol productivity during fermentation with CS hydrolysate.

Design and construction of improved new vectors for Zymomonas mobilis recombinants.

Zymomonas mobilis is a very important gram-negative bacterium having a potential application to simultaneous co-production of biofuel and other high value-added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis-acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram-negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tc(r) ). The utility of screening marker, lacZα with MCS, was confirmed by the blue-white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a-derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co-production of biofuel and high value added products.

Sorbitol production using recombinant Zymomonas mobilis strain.

A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose-fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml(-1), and 0.70 U mg(-1), respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn(2+) was found to be most effective by inhibiting the Entner-Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 gl(-1) and the sorbitol yield was increased to almost 100% from 89%. The Ca(2+) enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier.

Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds.

High cost of triacylglycerol lipid feedstock is the major barrier for commercial production of biodiesel. The fermentation of oleaginous yeasts for lipid production using lignocellulose biomass provides a practical option with high economic competitiveness. In this paper, the typical oleaginous yeast strains were screened under the pressure of lignocellulose degradation compounds for selection of the optimal strains tolerant to lignocellulose. The inhibitory effect of lignocellulose degradation products on the oleaginous yeast fermentation was carefully investigated. Preliminary screening was carried out in the minimum nutritious medium without adding any expensive complex ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute sulfuric acid. Seven typical lignocellulose degradation products formed in various pretreatment and hydrolysis processing were selected as the model inhibitors, including three organic acids, two furan compounds, and two phenol derivatives. The inhibition of the degradation compounds on the cell growth and lipid productivity of the selected oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors. Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose degradation compounds.

Carbon and energy balances of glucose fermentation with hydrogenproducing bacterium Citrobacter amalonaticus Y19.

For the newly isolated H2-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5- 9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (H2, ethanol, acetate, lactate, CO2, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal H2 production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.

Total biosynthesis of deoxynucleoside triphosphates using deoxynucleoside monophosphate kinases for PCR application.

Polymerase chain reaction (PCR) and other PCR applications for DNA synthesis require deoxynucleoside triphosphates (dNTP) as the essential precursors and substrates. Currently, the dNTP is commercially produced by a chemical method which is environmentally hazardous and costly due to its low yields in both the synthetic reaction and purification processes. In this study, a enzyme technology for the total integrated biosynthesis of all dNTP components is presented. The bioprocess technology developed and reported here involves two sequential enzymatic phosphorylation reactions coupled with the cofactor regeneration starting from deoxynucleoside monophosphates (dNMP) to deoxynucleoside diphosphates (dNDP) in the first reaction step and to dNTP in the second reaction step in the same bioreactor. The four genes encoding these deoxynucleoside monophosphate kinases were cloned into the recombinant E. coli and expressed using the recombinant E. coli strains. The reaction mechanisms and kinetics of the four kinase enzymes are studied and reported. The total enzymatic syntheses of the four dNTP products were carried out in four separate operations under the high substrate concentrations which emulate the practical application. The optimal process conditions were carefully investigated and complete conversion of dNMP to dNTP at high substrate concentration have been achieved. The purity and quality of dNTP products obtained from this work were analyzed and found to be at least equivalent or better than the commercially available dNTP products. The PCR application of dNTP products obtained from this work were also evaluated for isolating and amplifying genes of different sizes from different organisms. The PCR performance test also showed an equivalent quality as compared to the commercially available dNTP. The bioprocess technology developed and reported here for production of dNTP will provide economically competitive and environmentally friendly viable technology for the industry and research community as compared to the chemical technology currently in use.

Substrate specificity of nonribosomal peptide synthetase modules responsible for the biosynthesis of the oligopeptide moiety of cephabacin in Lysobacter lactamgenus.

Lysobacter lactamgenus produces cephabacins, a class of beta-lactam antibiotics which have an oligopeptide moiety attached to the cephem ring at the C-3 position. The nonribosomal peptide synthetase (NRPS) system, which comprises four distinct modules, is required for the biosynthesis of this short oligopeptide, when one takes the chemical structure of these antibiotics into consideration. The cpbI gene, which has been identified in a region upstream of the pcbAB gene, encodes the NRPS - polyketide synthase hybrid complex, where NRPS is composed of three modules, while the cpbK gene -- which has been reported as being upstream of cpbI-- comprises a single NRPS module. An in silico protein analysis was able to partially reveal the specificity of each module. The four recombinant adenylation (A) domains from each NRPS module were heterologously expressed in Escherichia coli and purified. Biochemical data from ATP-PPi exchange assays indicated that L-arginine was an effective substrate for the A1 domain, while the A2, A3 and A4 domains activated L-alanine. These findings are in an agreement with the known chemical structure of cephabacins, as well as with the anticipated substrate specificity of the NRPS modules in CpbI and CpbK, which are involved in the assembly of the tetrapeptide at the C-3 position.

Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates.

The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi-Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis-Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

Inhibitory effect of L-pyroglutamate on extremophiles: correlation with growth temperature and pH.

L-Pyroglutamate (PGA) is naturally occurring from L-glutamate solution with accelerated formation rate under high temperature and low pH. Even though PGA has been identified as a neurotoxic agent on brain cells, the effect of PGA on the growth of microorganisms is rarely known. Here various kinds of microorganisms differing in their optimal growth temperature, pH, phylogeny, and isolated biotope were investigated for the effect of PGA. We found that growth of thermoacidophiles, including both archaea and bacteria, was seriously inhibited by the presence of PGA, and the extent of the inhibitory effect was closely related with growth temperature and pH. Interestingly, only microbes that grow at high temperature and low pH are inhibited by PGA, while this compound may stimulate growth rates of organisms that live at neutral pH and low temperature.