Construction Of Recombinant Strains Research Articles

Construction of recombinant Pichia pastoris strains for ammonia reduction by the gdhA and glnA regulatory genes in laying hens

Ammonia emissions have become an important environmental challenge for the livestock industry. Probiotics are often used as additives to reduce ammonia, and the ammonia reduction efficiency of common probiotics is approximately 20–40%. In this study, we constructed a gdhA recombinant Pichia pastoris strain, glnA recombinant Pichia pastoris strain and gdhA-glnA Pichia pastoris recombinant strain using the gdhA and glnA genes, which have the potential function of reducing ammonia emissions. The results of in vitro fermentation showed that compared with the control, wild-type Pichia pastoris and pPICZA strains, the gdhA, glnA and gdhA-glnA recombinant strains significantly reduced ammonia emissions in laying hens (P < 0.05), with emission reduction efficiencies of 63.95%, 65.68% and 74.04%, respectively. The reason may be that the recombinant Pichia pastoris strains can convert ammonium nitrogen into amino acids for self-growth through ammonia assimilation, and reduce the pH, uric acid and urea content in the intestinal tract of livestock and poultry, and urease activity. Therefore, the construction of recombinant strains can provide technical support for reducing ammonia pollution in the livestock industry.

Construction of an ultra-strong PtacM promoter via engineering the core-element spacer and 5′ untranslated region for versatile applications in Corynebacterium glutamicum

As one of the most important synthetic biology elements in transcriptional regulation, promoters play irreplaceable roles in metabolic engineering. For the industrial microorganism Corynebacterium glutamicum, both the construction of a promoter library with gradient strength and the creation of ultra-strong promoters are essential for the production of target enzymes and compounds. In this work, the spacer sequence (both length and base) between the −35 and −10 regions, and the 5′-terminal untranslated region (5′UTR) were particularly highlighted to investigate their contributions to promoter strength. We constructed a series of artificially induced promoters based on the classical tac promoter using C. glutamicum ATCC13032 as the host. Here, we explored the effect of sequence length between the −35 and −10 regions on the strength of the tac promoter, and found that the mutant with 15 nt spacer length (PtacL15) was transcriptionally stronger than the classic Ptac (16 nt); subsequently, based on PtacL15, we explored the effect of the nucleotide sequence in the spacer region on transcriptional strength, and screened the strongest PtacL15m-110 (GAACAGGCTTTATCT), and PtacL15m-87 (AGTCGCTAAGACTCA); finally, we investigated the effect of the length of the 5′-terminal untranslated region (5′UTR) and screened out the optimal PtacM4 mutant with a 5′UTR length of 32 nt. Based on our new findings on the optimal spacer length (15 nt), nucleotide sequence (AGTCGCTAAGACTCA), and 5′UTR (truncated 32 nt), an ultra-strong PtacM, whose transcriptional strength was about 3.25 times that of the original Ptac, was obtained. We anticipate that these promoters with gradient transcriptional strength and the ultra-strong PtacM will play an important role in the construction of recombinant strains and industrial production.

Рекомбинантные штаммы-продуценты термостабильной липазы из Thermomyces lanuginosus и их применение в гетерогенном биокатализе, в том числе, в процессах низкотемпературного синтеза сложных эфиров

Abstract-This work was devoted to the construction of recombinant strains Escherichia coli BL21 (DE3) and Pichia pastoris X33, producing a 1,3-specific thermostable lipase from Thermomyces lanuginosus. The sequences of two lipase genes were optimized for expression in bacteria and methylotrophic yeasts, then synthesized and cloned into the corresponding expression vectors. As a result of genetic engineering manipulations, E. coli and P. pastoris strains were constructed that efficiently produced recombinant lipase from T. lanuginosus, which accumulated in the cytoplasm in an amount of 30-40% of the total cellular protein. Recombinant P. pastoris clones secreted lipase into the nutrient medium at a concentration of at least 1 g/L. Lipases produced by the recombinant clones, designated as rE.coli/lip and rPichia/lip, respectively, contained a six-histidine sequence (-His6) in the C-terminal region. The resulting lipases were immobilized on/in solid inorganic supports in order to develop heterogeneous biocatalysts (HB) for the enzymatic conversion of triglycerides and fatty acids. The rPichia/lip enzyme was adsorbed on mesoporous silica and macroporous carbon aerogel. The properties of the prepared HB, their enzymatic activity, substrate specificity and operational stability were studied in the reaction of esterification of fatty acids with aliphatic alcohols in organic solvents at 20 ± 2°C. It was found that immobilized lipases had a relatively wide substrate specificity, as well as high operational stability, and the prepared HB almost completely retained their high esterifying activity for several tens of reaction cycles. Key words: Escherichia coli, Pichia pastoris, recombinant strains-producers, Thermomyces lanuginosus lipase gene, immobilization, biocatalysts, esterification The authors are grateful to V. L. Kuznetsov for the provided samples of carbon aerogel and A. V. Ryabchenko for gene-engineering manipulation aimed at obtaining the recombinant rE. coli strain, a producer of the rE.coli/lip enzyme. The work was carried out under the Project on Fundamental Research within the framework of a state assignment to the Institute for Catalysis "Catalysts and Processes of Renewable Raw Material Conversion" (no. 0239-2021-0005).

Construction of Recombinant Strains for 3-hydroxypropionaldehyde Biosynthesis and Comparison of Glycerol Dehydratase Gene Expression in Hosts

3-hydroxypropionaldehyde(3-HPA) is an important chemical product,which could be transformed from glycerol by glycerol dehydratase.In order to acquire an engineering strain to produce 3-HPA,gdrB encoding glycerol dehydratase reactivating factor small-subunit was amplified and employed to construct the plasmid pEtac-dhaB-gdrA-gdrB on the basis of the plasmid pEtac-dhaB-gdrA,which contained glycerol dehydratase reactivating factor large-subunit.The common Escherichia coli tac promoter was used for expression of pEtac-dhaB-gdrA-gdrB in different hosts E.coli BL21,DH5a,JM109.The positive clones1were induced with IPTG,and the complete cDNA was obtained by RT-PCR using the total RNA as template.It was found that the gene of glycerol dehydratase could be transcribed well in all 3 hosts.The results of SDSPAGE,enzyme analysis and the fermentation of 3-HPA indicated that the expression of glycerol dehydratase in different hosts were not the same.The specific enzyme activities of recombinant strains with different hosts(E.coli BL21,DH5a,JM109) were 4.7(±0.44),3.5(±0.95) and 8.1(±0.66) U/mg,respectively,and the 3-HPA productions by shake flask fermentation were 0.012(±0.0044),0.014(±0.003) and 0.375(±0.018) g L-1,respectively.The recombinant E.coli JM109/pEtac-dhaB-gdrA-gdrB had the best ability in glycerol dehydratase expression and 3-HPA production.Compared with Klebsiella pneumoniae,the byproducts of recombinant E.coli were much less,which was good for separation and purification.This study provides a new route for the biosynthesis of 3-HPA.

Proteomic Analysis ofBacillus thuringiensisStrain 4.0718 at Different Growth Phases

The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains.

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF CHITINASE GENE chiA FROM LOCAL ISOLATE OF Bacillus licheniformis MS1

Detection of chitin-degrading bacteria from natural sources such as rhizosphere soil is useful for the isolation of bacteria that produce antifungal or other novel compounds. A high correlation between chitinolysis and production of bioactive compounds has been reported (Chen et al., 1991; Pisano et al., 1992; Hoster et al., 2005). Microorganisms, which secret a complex of mycolytic enzymes, are considered to be used as bio- logical control agents of plant diseases (Helisto et al., 2001; Chang et al., 2003; Hoster et al., 2005). Chitin hydrolysis is performed by a major pathway composed of three separate enzymes to break down polymeric chitin to chitin oligosaccharides, diacetylchitobiose and N- actylglucosamine, separately or synergistically and consecutively in the degradation of chitin to free N-actylglucosamine. Endochitinase (E.C. 3.2.1.14) is a poly (1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolases, which produce multimers of β-N-acetylglucosamine by random hydrolysis of β-1-4-linkages in chitin and chitodextrins while Exochitinase (E.C. 3.2.1.52) β-1,4-N-acetylhexosaminidase, is catalyzing the sequential release of soluble dimers starting at the non-reducing end of the polymer. Chitobiases (E.C. 3.2.1.30) or β-N-acetylglucosaminidase, helps the hydrolysis of chitobiose into monomers of N-actylglucosamine (Souza et al., 2003). It is though that chitin hydrolysis occurs either by a sequential or synergetic action of endochitinase, exochitinase, and chitobiases (Tanaka et al., 2003). While exoand endo-chitinases are able to depolymerize chitin alone. The presence of both activities significantly increases the efficiency of chitinolytic system (Howard et al., 2003). Recently, the cloning and expression of the chitinase gene and its introduction into the biologically susceptible species or the construction of recombinant strains with new capacities have been recommended to be one of the interesting areas of chitinase studies and applications. Chitinase genes have been cloned and characterized from many microorganisms (Ueda et al., 2003; Hobel et al., 2005; Yano et al., 2005; Yong et al., 2006). Some of which were either transformed into plants and/or bacterial strains to increase their ability to control phytopathogens (Koby et al., 1994; Punja, 2001) or were high level of expression in Escherichia coli cells to enhance the activity of Bacillus thuringiensis to control pests (Regev et al., 1996; Sampson and Gooday, 1998). Chitinase has received increased attention because of their potential application in the biological control of plants-pathogenic fungi and pests, as well as in the bioconversion of shellfish chitin wastes (Chang et al., 2003; Hoster et al., 2005). In our previous study, local isolate of Bacillus licheniformis MS1 isolated from agricultural fields, Giza, Egypt, was found to be one of the most producing a large amount of chitinase enzyme (Kamil et al., 2007). For this reason this isolate was selected in the present study for cloning, sequencing and molecular analysis of its chitinase gene (chiA).

Development of Methodic Approach to Construction of Recombinant Strain, Producer of the Cholera Toxin B Subunit

Development of Methodic Approach to Construction of Recombinant Strain, Producer of the Cholera Toxin B Subunit

Creation of biocatalysts with prescribed properties

Oxidoreductases are widely used in different branches of industry (particularly, in pharmaceutical) and analytical biotechnology. To optimize the use and to reduce production cost of a target product, the properties of a biocatalyst should be modified to meet requirements of a specific process or method of analysis. Problem of creation of biocatalysts with prescribed properties includes complex set of R&D such as an engineering of enzyme properties, construction of recombinant strains overproducing the target protein, development of a large-scale downstream process and preparation of a working form of a biocatalysts. In the present work, the basic features and practical examples of such an over-all approach are considered for formate dehydrogenase and D-amino acid oxidase.

Microbial remediation of nitro-aromatic compounds: An overview

Nitro-aromatic compounds are produced by incomplete combustion of fossil fuel or nitration reactions and are used as chemical feedstock for synthesis of explosives, pesticides, herbicides, dyes, pharmaceuticals, etc. The indiscriminate use of nitro-aromatics in the past due to wide applications has resulted in inexorable environmental pollution. Hence, nitro-aromatics are recognized as recalcitrant and given Hazardous Rating-3. Although several conventional pump and treat clean up methods are currently in use for the removal of nitro-aromatics, none has proved to be sustainable. Recently, remediation by biological systems has attracted worldwide attention to decontaminate nitro-aromatics polluted sources. The incredible versatility inherited in microbes has rendered these compounds as a part of the biogeochemical cycle. Several microbes catalyze mineralization and/or non-specific transformation of nitro-aromatics either by aerobic or anaerobic processes. Aerobic degradation of nitro-aromatics applies mainly to mono-, dinitro-derivatives and to some extent to poly-nitro-aromatics through oxygenation by: (i) monooxygenase, (ii) dioxygenase catalyzed reactions, (iii) Meisenheimer complex formation, and (iv) partial reduction of aromatic ring. Under anaerobic conditions, nitro-aromatics are reduced to amino-aromatics to facilitate complete mineralization. The nitro-aromatic explosives from contaminated sediments are effectively degraded at field scale using in situ bioremediation strategies, while ex situ techniques using whole cell/enzyme(s) immobilized on a suitable matrix/support are gaining acceptance for decontamination of nitrophenolic pesticides from soils at high chemical loading rates. Presently, the qualitative and quantitative performance of biological approaches of remediation is undergoing improvement due to: (i) knowledge of catabolic pathways of degradation, (ii) optimization of various parameters for accelerated degradation, and (iii) design of microbe(s) through molecular biology tools, capable of detoxifying nitro-aromatic pollutants. Among them, degradative plasmids have provided a major handle in construction of recombinant strains. Although recombinants designed for high performance seem to provide a ray of hope, their true assessment under field conditions is required to address ecological considerations for sustainable bioremediation.

Biological production of semisynthetic opiates using genetically engineered bacteria.

Semisynthetic derivatives of morphine and related alkaloids are in widespread clinical use. Due to the complexity of these molecules, however, chemical transformations are difficult to achieve in high yields. We recently identified the powerful analgesic hydromorphone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Here we describe the construction of recombinant strains of Escherichia coli that express morphine dehydrogenase and morphinone reductase. These strains are capable of efficiently transforming the naturally occurring alkaloids morphine and codeine to hydromorphone and the antitussive hydrocodone, respectively. Our results demonstrate the potential for recombinant DNA technology to provide biological routes for the synthesis of known and novel semisynthetic opiate drugs.

Transformation of bacteria by electroporation

The introduction of DNA into bacteria by transformation is an essential step in the construction of recombinant strains. Recently, electroporation, or electropermeabilization, in which a brief high voltage electric discharge is used to render cells permeable to DNA, has revolutionized the transformation of bacteria. The technique is fast, simple, reproducible, frequently gives very high transformation frequency and appears to be applicable to a wide range of bacterial types previously thought untransformable. The technique can also offer advantages for transformable bacteria such as Escherichia coli.