Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.     

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Expression and stability of a recombinant plasmid in Zymomonas mobilis and Escherichia coli

A recombinant plasmid was constructed by ligating EcoRI digests of the plasmid cloning vector pBR325 and pZMO2, one of the natural plasmids of Zymomonas mobilis ATCC 10988. This vector, named pDS212 (total size 7.9 kb), which was able to transform Escherichia coli efficiently, was also transferred to Z. mobilis hosts by mobilization during conjugation using the helper plasmid pRK2013. pDS212 was inherited stably in both E. coli and Z. mobilis hosts and could be recovered intact from them. Markers of pBR325 and pRK2013 were also transferred in Z. mobilis but at very low frequencies. Neither pBR325 nor pRK2013 could be recovered intact from the Z. mobilis hosts. It is proposed that expression and stability of pDS212 in Z. mobilis is due to the origin of replication of pZMO2 that it carries, and that it may be used for developing a gene transfer system in Z. mobilis.     

An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

Background  

Xylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR) in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR.     

Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.The biological conversion of lignocellulosic biomass to ethanol represents a potential major source of future domestic transportation fuels, but the current cost of converting biomass to fermentable sugars still needs to be reduced further (12). Most current strategies for ethanol production via biochemical conversion of lignocellulosic feedstocks utilize simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and cofermentation (SSCF) processes (8, 21, 22). The process configuration known as consolidated bioprocessing (CBP) (20) would alleviate the financial strain of producing saccharolytic enzyme cocktails by combining the necessary steps for ethanol production as the action of one microorganism.A particularly attractive microbial candidate for the development of a CBP microorganism is the Gram-negative fermentative bacterium Zymomonas mobilis. Z. mobilis has been studied for its exceptionally high ethanol production rate, yield, and tolerance to the toxicity of the final product (15-17, 20, 31-33, 35, 43). In addition, Z. mobilis has the ability to ferment sugars at low pH and has a naturally high tolerance to many of the inhibitory compounds found in hydrolysates derived from lignocellulosic biomass (45, 46) Furthermore, the use of the Entner-Doudoroff pathway (37) allows Z. mobilis to achieve the near-theoretical maximum ethanol yields during fermentation while achieving relatively low biomass formation. Accordingly, Z. mobilis has been used successfully in SSF and SSCF processes (14, 24, 36). Additionally, Z. mobilis has been successfully engineered to ferment the pentose (C₅) sugars, xylose (45) and arabinose (10).A necessary prerequisite to establishing Z. mobilis as a CBP host is the ability to achieve high levels of cellulolytic enzyme expression. However, there is not yet a strong consensus on how to achieve maximal heterologous protein expression in Z. mobilis. Multiple groups have attempted heterologous expression of numerous genes, including cellulolytic enzymes in Z. mobilis with various degrees of success (6, 7, 9, 19, 27, 42, 44). Unfortunately, there are no obvious correlations between the expression strategies employed compared to the results obtained. Intriguingly, however, when researchers used the tac promoter (P_tac) to drive expression of native Z. mobilis genes, they were able to express several genes to extremely high levels (2). The results from this study (2) suggest that while the potential to achieve high levels of heterologous cellulase expression in Z. mobilis certainly exists, the ability to do so on a consistent basis will need further investigation.While achieving high-level expression of cellulases is an important hurdle to overcome in the development of the CBP technology, it is imperative that these enzymes additionally be translocated to the extracellular medium in order to directly contact the lignocellulosic substrate. The most obvious means by which to achieve this translocation is by harnessing the host cell''s protein secretion apparatus. There is, however, in general, very little fundamental knowledge regarding the capacity of Z. mobilis to secrete proteins. There is only one account to our knowledge of fusing secretion signals native to Z. mobilis onto proteins from exogenous sources, where extracellular secretion of a recombinant β-glucosidase reached only 11% of the total amount of enzyme synthesized (43).We initially report the finding that several Z. mobilis strains natively produce an endogenous activity against carboxymethyl cellulose (CMC) and that this activity can be detected extracellularly. Together, these results suggest that Z. mobilis may be adept at producing and secreting cellulolytic enzymes, and as this attribute is essential for a CBP organism, Z. mobilis serves as an ideal candidate for further investigation.We next describe the expression of two cellulolytic enzymes (E1 and GH12) in both Escherichia coli and Z. mobilis. E1 (locus tag Acel_0614) and GH12 (locus tag Acel_0619) are both from the acidothermophile Acidothermus cellulolyticus and are representative of glycoside hydrolase families 5 and 12, respectively (10a, 38a). E1 is an endo-1,4-β-glucanase, and GH12 is an uncharacterized enzyme that has a very high sequence identity to the GH12 domain of GuxA (Acel_0615) from A. cellulolyticus. GuxA has activities against a wide variety of substrates, including carboxymethyl cellulose, arabinoxylan, xylan, and xyloglucan (W. Adney, unpublished results). While GH12 has yet to be fully characterized, we used homology modeling (3) to predict the enzyme class of GH12 and found that it strongly resembles an endo-1,4-β-glucanase. These enzymes were chosen because of their relatively low molecular weight, high stability, and activity over a broad temperature and pH range using only the catalytic domains (Adney, unpublished). We report the successful expression of both enzymes in Z. mobilis by addressing several variables related to gene expression. Additionally, the use of codon optimization was explored as a way of enhancing heterologous expression in Z. mobilis. After successfully demonstrating the intracellular expression of E1 and GH12 in Z. mobilis, we further show that Z. mobilis is capable of secreting these proteins extracellularly through the use of native secretion signals predicted to utilize two separate protein translocation pathways in Z. mobilis, the SecB-dependent and twin arginine translocation (TAT) pathways. This finding should prove valuable beyond the production of cellulases and could include all classes of recombinant proteins.     

Isolation of Noninhibitory Strains of Zymomonas mobilis

Wild-type Zymomonas mobilis strains inhibit the growth of Escherichia coli. We report the first isolation of noninhibitory strains, called Zymomonas inhibition negative (Zin⁻), after treatment with N-methyl-N′-nitro-N-nitrosoguanidine. A standardized soft-agar overlay procedure for detecting E. coli growth inhibition was also developed.     

Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli

Summary Levansucrase gene from Zymomonas mobilis was expressed efficiently in Escherichia coli and the overproduced recombinant levansucrase amounted to 40% of the total cell protein. Using E. coli lysate, levan was synthesized in a sucrose-based medium enzymatically with the conversion yields of up to 46% from fructose liberated in 25 hrs of incubation. More levan was formed at lower temperatures in the reaction mixture, whereas higher temperatures were favoured for the accumulation of free fructose or short chain oligosaccharides.     

Overexpression of extracellular sucrase (SacC) of Zymomonas mobilis in Escherichia coli

Abstract The extracellular sucrase (SacC) gene of Zymomonas mobilis was overexpressed in Escherichia coli BL21 using the T7 polymerase expression system. A low cell density induction method was designed to have maximum expression, and the conditions (IPTG concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing SacC protein.     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

Controlling Morphological Instability of Zymomonas mobilis Strains in Continuous Culture

[This corrects the article on p. 1900 in vol. 45.].     

Controlling Morphological Instability of Zymomonas mobilis Strains in Continuous Culture

Growth of Zymomonas mobilis ATCC 29191 and CP4 in a continuous stirred tank fermentor resulted in the selection of stable flocculating variants. Factors responsible for enhancing the system pressures selective for the morphological variants were identified. By incorporating some modifications into the design of the fermentor, it was possible to achieve steady-state operation of the chemostat with both wild-type and flocculating strains. Biochemical and microscopic studies were performed to elucidate the mechanism of flocculation in Z. mobilis.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

A simple method for the purification of over-expressed extracellular sucrase of Zymomonas mobilis from a recombinant Escherichia coli

The over-expressed extracellular sucrase (SacC) of Zymomonas mobilisfrom a recombinant Escherichia coli (pZSP62) carrying the sacC gene was purified partially by repeated cycles of freezing and thawing. This method separated the highly expressed recombinant protein from the bulk of endogenous E. coli proteins. The enzyme was further purified 14 fold with a 55% yield from the cellular extract of E. coli by hydroxyapatite chromatography. The purified enzyme had a Mr of 46 kDa by SDS-PAGE. Its km value for sucrose was 86 mM and was optimal at pH 5.0 and at 36°C.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.