Industrial strain improvement: rational screens and genetic recombination techniques

Industrial strain improvement is a largely empirical activity because the specific biochemical and genetic mechanisms involved are only poorly understood. Despite this, it is possible to reduce the empirical element by the application of basic biochemical and genetic principles known to operate in a wide spectrum of organisms. These principles may be applied in two ways: by devising more efficient screening procedures; or by using genetic recombination techniques. The latter approach has become even more powerful in recent years with the advent of in vitro DNA technology. The use of these approaches does not eliminate the need for titre testing to identify improved strains. If successful, however, they can greatly relieve the drudgery involved by reducing the numbers of isolates which need to be tested in this manner.     

A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2-deoxyglucose

Aims: The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods. Methods and Results: A new genetic variant was isolated from strain 9A02S1 and named S1M29. It was obtained by mutagenesis with H₂O₂ and two screening steps, which involved selection in Petri dishes using the medium supplemented with 2‐deoxyglucose and microfermentations in submerged culture. The mutant showed higher cellulase productivity than 9A02S1 based on the Filter Paper Activity assay and endoglucanase; the peak activities for these enzymes were reached significantly faster than for the parent strain. Conclusions: The mutant obtained after mutagenesis and selection could produce and secrete cellulase faster than the parent strain. Significance and Impact of the Study: Mutagenesis followed by selection is a useful tool for rapidly generating new cellulase‐producing phenotypes in fungi. Faster production and higher titers of cellulases in mutant strains contribute to reduce the production costs for enzymatic complexes that hydrolyse lignocellulose residues and form fermented syrups, thus contributing to the economic production of bioethanol.     

Creating a mutant luciferase resistant to HPV chemical inhibition by random mutagenesis and colony-level screening.

Firefly luciferase covers a wide range of applications. One common usage of the bioluminescence assay is the measurement of intracellular concentration of adenosine triphosphate (ATP) for cell viability. However, inhibition of the enzyme reaction by chemicals in the assay has so far limited the application of luciferase for high production volume (HPV) chemical testing. The objective of this research was to obtain a mutant luciferase with increased stability to inhibition by HPV chemicals, yet retaining specific activity comparable to, or better than, wild-type luciferase. The enzymatic properties of the wild-type luciferase were improved by random mutagenesis and colony-level screening. In this paper, the detailed process of creating mutant luciferases for testing the toxicity of HPV chemicals is described. As a result, two mutant luciferases were created, with different degrees of improved tolerance to inhibition by chloroform and other HPV chemicals.     

Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Aims:  The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants.
Methods and Results:  The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l⁻¹ of FaeB was selected (12-fold more than the A. niger overproducing strain).
Conclusions:  This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days.
Significance and Impact of the Study:  This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.     

Isolation and improvement of a thermotolerant Saccharomyces cerevisiae strain

The ambient temperature is a drawback in industrial ethanol production in Jaffna due to heat killing of yeast during fermentation. Thus a search was initiated for thermotolerant organisms suitable for fermentation in hot climates. The screening of the best wild-type organisms was undertaken as the first step. Thermotolerant strains were selected from environments where there are chances of organisms being exposed to high temperature. The samples were enriched and screened for thermotolerant organisms which survived at 45 °C for 15 h. Among the yeast strains selected from different sources, thermotolerant strains with the capacity to withstand 45 °C for 15 h were found in samples collected from the compost heap and distillery environments. Three colonies from the distillery environment were selected for further studies and named p1, p2 and p3. Exponential phase (18 h) cultures of p1, p2 and p3 were subjected to 15 temperature treatment cycles (at 50 °C each for 3 h) and thermally adapted strains pt1, pt2 and pt3 were obtained, showing 100, 30 and 20% viability at 50 °C for 30 min respectively. The initial round of thermal adaptation cycles increased the duration of 100% viability from 20 h (p1) to 68 h (pt1) when incubated at 40 °C. Very little benefit was obtained when pt1 was treated with u.v. and ethyl methanesulphonate. The selected strain was identified and designated as Saccharomyces cerevisiae S1. The ethanol produced from 100 g glucose l^–1 by S. cerevisiae S1 was 46 g l^–1 (36 h), 38 g l^–1 (48 h) and 26 g l^–1 (48 h) at 40, 43 and 45 °C respectively in rich nutrient medium.     

Cell-free synthesis and multifold screening of Candida antarctica lipase B (CalB) variants after combinatorial mutagenesis of hot spots

We have developed a strategy for rapid and combinatorial optimization of the hot spot residues of enzymes. After combinatorial randomization of target locations in the Candida antarctica lipase B (CalB) gene, the individual variant genes isolated in the E.coli cells were expressed in the cell-free protein synthesis system to analyze different parameters of the resulting CalB variants. The enzymatic assays for the hydrolysis of para-nitrophenyl-ester (pNP-ester) and triglyceride, synthesis of wax ester, and thermal stability of the variant enzymes were carried out simultaneously in 96-well microtiter plates. From the 1,000 variant genes tested in each assay, we were able to identify a series of the variant enzymes having markedly improved hydrolytic, synthetic activity, or thermal stability. The improved traits of the cell-free selected CalB variants were well reproduced when the corresponding genes were expressed in Pichia pastoris. Therefore, we expect that the proposed strategy of cell-free expression screening can serve as a viable option for rapid and precise tuning of enzyme molecules, not only for analytical purposes but also for industrial applications through large scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.     

微生物进化工程育种技术进展与展望

进化工程是一项全基因组水平的新兴技术,它通过模拟自然进化中的变异和选择过程,实现对微生物菌株优良性状的选育。近年来,进化工程在扩大底物利用范围,提高代谢物产量及提高微生物的耐受性等方面取得了显著进展。介绍了进化工程的概念、特点及发展过程,叙述了进化工程的原理和研究方法,重点总结了进化工程方法在菌株改进方面的研究进展,最后对该技术的发展趋势和潜在应用进行了展望。     

Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production

By combining induced mutation, using NTG and UV irradiation, and protoplasting of a wild type strain of Aspergillus oryzae ATCC 22788, a hyper-producing strain was obtained that accumulated 41 g kojic acid l(-1) in shake-flasks, which was 100-fold higher than that in the wild type strains. Similar production of kojic acid was obtained in 5 l stirred-tank fermentations.     

InsectDirectTM System: Rapid, High-level Protein Expression and Purification from Insect Cells

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells (Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirect^TM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases.     

Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis

Nannochloropsis oculata was subjected to N-methyl-N-nitrosourea-induced mutagenesis under the selection pressure of quizalofop, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity with the objective of generating genetically tractable mutants with altered fatty acid metabolism. Two mutants, QUIZ1 and QUIZ2, with stable resistance to quizalofop were isolated and partially characterized. The growth properties and morphology of the mutants appeared identical with the parent strain. However thermo-tolerance was observed in the mutants. Enhanced resistance to quizalofop suggested the presence of herbicide resistant isoforms of ACCase. In vitro assays for ACCase activity showed that ACCase in the wild strains was much more sensitive to quizalofop than the mutant strains. Gas chromatographic analysis of fatty acids revealed that the mutant strains were rich in polyunsaturated fatty acids (n– 3PUFAs), as well as total fatty acid contents; this was accompanied by a concomitant increase in triacylglycerol (TAG) followed by linoleic acid (18:2), arachidonic acid (20:4 n– 6) and EPA (20:5 n– 3). These results suggest that an increased substrate pool (malonyl-CoA) (due to increased specific activity of ACCase) in the mutant strains in vivo and in vitro may have led to the increased TAG accumulation. Random mutagenesis was shown to be a good tool to manipulate PUFAs and EPA in Nannochloropsis. The strains developed will be useful in understanding fatty acid metabolism using genetic and biochemical approaches and also for their direct use in mariculture.     

Production of phytase under solid-state fermentation using Rhizopus oryzae: novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (～34 kDa) showed optimal temperature of 45 °C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×10(6) M(-1) s(-1)). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.     

Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster

Several commercially improved strains of Penicillium chrysogenum have been shown to carry amplifications of the entire penicillin biosynthesis gene cluster. Analysis previously carried out using the strain BW 1890 has here been extended to the characterisation of other members of the SmithKline Beecham strain improvement series. We have determined the length of the amplicon to be 57.4 kb and shown a general increase in copy number and penicillin titre through the series. Sequence analyses of the promoter regions of the acvA, ipnA and aat genes in the high titre strain BW 1901, and comparisons with wild-type sequences have not identified any potentially titre-enhancing mutations. In addition, cDNA screening has failed to identify any further transcribed elements within the co-amplified region. The homogeneity of hybridisation patterns and the identification and analysis of a single copy revertant has shown that the amplification is of a direct tandem nature and we propose a model of chromatid misalignment and recombination as its mode of generation. Hybridisation analysis of penicillin non-producing mutants has indicated the loss, in all those investigated, of the entire penicillin biosynthesis gene cluster, similarities between the deletion junctions in these strains and comparison with previously published data indicating the presence of recombinogenic regions flanking the penicillin biosynthesis gene cluster. Received 05 November 1996/ Accepted in revised form 25 April 1997     

Immunological screening of standard cDNA libraries in pBR322 vectors: detection of human fibrinogen and prothrombin cDNA clones

The in situ immunological detection of antigens encoded by cDNA inserted into the PstI site of pBR322 plasmids was optimized. It was found that sensitivity of the detection was dramatically increased by in situ amplification of the recombinant plasmids on chloramphenicol-containing medium followed by a brief incubation without chloramphenicol during which protein synthesis resumes. In addition, several modifications of the previously described methods which permit total suppression of background and false positives are described. These techniques allowed easy detection of cDNA clones for human B beta- and gamma-fibrinogen and -prothrombin using a human liver double-stranded cDNA recombinant plasmid library in pBR322 vectors.     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

cDNA expression library screening and identification of two novel antigens: ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii antigens we constructed a lambdaTriplEx2 expression library of the parasite and screened this with pooled carp immune serum collected 6 weeks post-infection. Screening of the library not only revealed ribosomal proteins but identified ubiquitin and a homologue of the receptor for activated C kinase (RACK) as immunogenic proteins. Equivalents of all these proteins have been identified as immunogenic in expression library screenings of other Trypanosomatida, suggesting an evolutionary conservation of their immunogenicity. The possibility that ubiquitin and/or the homologue of RACK could represent protective antigens and be targets for the design of novel therapies is discussed.     

Induction of interleukin (IL)-1α and β gene expression in human keratinocytes exposed to repetitive strain: Their role in strain-induced keratinocyte proliferation and morphological change

Recent studies in our laboratory have demonstrated that mechanical strain alters many facets of keratinocyte biology including proliferation, protein synthesis, and morphology. IL-1 is known to play an important role in the autocrine regulation of these basic cellular properties under basal and stimulated conditions. However, it is not known whether IL-1 plays a role in strain-induced alteration of keratinocyte biology. Thus, the objective of this study was to test the hypothesis that cyclic strain stimulates IL-1 expression and that strain-induced changes in keratinocyte function is regulated by IL-1. To test this hypothesis, we examined the effect of cyclic strain (10% average deformation) on keratinocyte IL-1 gene expression and the effect of neutralizing antibodies of IL-1α and IL-1β on strain-induced changes in keratinocyte proliferation, morphology, and orientation. Northern blot analyses demonstrated that steady state levels of IL-1α and β mRNA were elevated by 4 h, peaked at 12 h of cyclic strain (IL-1α, 304 ± 14.2%; IL-1β, 212 ± 5.6% increase vs. static controls) and decreased gradually by 24 h. IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.01 μg/ml) significantly blocked strain-induced keratinocyte proliferation as well as the basal rate of proliferation. In contrast, IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.1 μg/ml) had no effect on strain-induced morphological changes such as elongation and alignment. We conclude that mechanical strain induces IL-1 mRNA expression in keratinocytes. The role of IL-1 in mediating strain-induced changes in keratinocyte biology remains to be determined but appears to be independent of morphological changes. J. Cell. Biochem. 69:95–103, 1998. © 1998 Wiley-Liss, Inc.