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Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d-xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry.

In this study, a combined optimization strategy, based on co-expression of molecular chaperones and supplementation of osmolytes, was used to reduce the formation of inclusion bodies and enhance the expression of the soluble form of 4-α-glucanotransferase. The 4-α-glucanotransferase yield was enhanced by co-expression with pGro7 and supplementation of trimetlylamine oxide. Subsequently, the effects of process conditions (temperature, inducer concentration, and arabinose concentration) on cell growth and 4-α-glucanotransferase production were also investigated in shake flasks. In addition, a modified high-cell-density fermentation approach was proposed and applied in 3-L fermentor supplied with l -arabinose and trimetlylamine oxide, which achieved a dry cell weight of 65.92 g·L−1. Through this cultivation approach at 28 °C, the activity of 4-α-glucanotransferase reached 332.5 U·g−1 dry cell weight, which was 24.6-fold greater than the initial activity in shake flask cultivation. This combined strategy is expected to provide an efficient and economical approach to overproduction of aggregation prone proteins on a large scale.

2′-Fucosyllactose (2′-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2′-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP- L -fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2′-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP- L -fucose. Besides, 2′-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2′-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2′-FL production in E. coli.

O-Succinyl- l -homoserine (OSH) is an important platform chemical in production of C4 chemicals such as succinic acid, homoserine lactone, γ‑butyrolactone, and 1,4‑butanediol. The production of OSH through chemical method or the current engineering strain is difficult and not optimal, and thereby there remains a need to develop new engineering strategy. Here, we engineered an OSH overproducing Escherichia coli strain through deleting the degradation and competitive pathways, overexpressing thrA and metL to enhance the metabolic flux from l -asparate to l -homoserine. Additionally, increasing the precursor succinyl-CoA supply through simultaneously knocking out sucD and overexpressing sucA further increased the yield of OSH. The engineered strain OSH9/pTrc-metA11-yjeH with above strategies produced OSH at the concentration of 24.1 g/L (0.609 g/g glucose) in batch fermentation. To gain detailed insight into metabolism of the engineered strain, comparative metabolic profiling was performed between the engineered and wide-type strain. The metabolomics data deciphered that the carbon was directed toward the OSH biosynthesis resulting in less flexibility of the genetically modified strain than the wide-type strain.

The possibility of using active dry microbial preparations in biotechnological processes is essential for the development of new modern industrial technologies. In this study, we show the possibility of obtaining such preparations of the genetically engineered yeast strain Ogataea (Hansenula) polymorpha with glutathione overproduction. Special pre-treatment involving the gradual rehydration of dry cells in water vapour led to the restoration/reactivation of almost 100% of dehydrated cells. Furthermore, dry cells do not lose their viability during storage at room temperatures. Application of dry cells as the inoculum provides the same levels of glutathione synthesis as that of a native yeast culture.

The por1 gene encoding one of the mitochondrial porin channels in C. utilis CCTCC M 209298 was disrupted using a homologous recombination method. The co-production of S-adenosylmethionine (SAM) and glutathione (GSH) in the mutant C. utilis Δpor1 increased by 34.9% and 25.1%, respectively, during batch and fed-batch fermentation, relative to the parental strain. The average oxygen consumption rate, activities of key enzymes involved in SAM and GSH biosynthesis, levels of intracellular cofactors such as NADH and ATP, and carbon fluxes of key metabolites were compared between the parental strain and the Δpor1 mutant. The disruption of por1 gene increased the rate of mitochondrial respiration, increased the activities of both methionine adenosyltransferase and γ-glutamylcysteine synthetase, and enhanced the supply of energy and substrates for SAM and GSH biosynthesis, all of which favored the overproduction of SAM and GSH in the Δpor1 mutant.

Most expression systems used for the over production of many enzymes employ carbon catabolite repressible promoters and hence must use sugar free, rich complex media. Use of expression systems to overcome carbon repression opens an avenue for exploiting cheap carbon sources for the production of recombinant enzymes. A self-inducible, catabolite repression free and above all a glucose-activated expression system has been developed using an industrially suitable thermophilic alpha-amylase as a model. The alpha-amylase gene of Bacillus licheniformis MSG without the 5' cre operator produced unimpeded glucose-enhanced expression when fused to the phosphate starvation-inducible strong pst promoter with optimum translation signals in a protease deficient Bacillus subtilis. A combination of high glucose with limited phosphate permitted sufficient biomass and fast transition to quiescent phase by phosphate starvation permitting 1250-fold induction for 70 h. A ~300-fold high productivity (9070 U mL(-1)) and 131-fold increase in specific expression in 2% glucose and a 100-fold high yield in 4% molasses were obtained compared to the production by the parent strain. The yield was 18.5-fold higher than that from the native promoter in an isogenic clone. This strategy of catabolite enhanced enzyme expression uncoupled from biomass formation can be applied for cost effective high production of proteins using starch or molasses.

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L(-1). The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. cerevisiae.

Pleiotropic drug resistance 1 (Pdr1p) protein is a key transcription factor in multidrug resistance. Specifically, R821S point mutation in the PDR1 gene in Saccharomyces cerevisiae diploid KK-211 strain plays an important role in tolerance to hydrophobic organic solvents, though the molecular mechanisms underlying organic solvent tolerance are unclear. In KK-211, several ABC transporters and cell wall proteins were upregulated. PDR1 R821S mutant derived from the laboratory haploid strain MT8-1 confirmatively tolerated hydrophobic organic solvents, and this study is the first to reveal its tolerance of the hydrophilic organic solvent, dimethyl sulfoxide (DMSO). To identify the genes involved in the organic solvent tolerance, we focused on the upregulated 4 ABC transporters and 6 cell wall proteins in KK-211, and demonstrated 2 ABC transporters, Pdr10p and Snq2p, and the 4 cell wall proteins, Wsc3p, Pry3p, Pir1p, and Ynl190wp were responsible for hydrophobic organic solvent (n-decane and n-undecane) tolerance. Tolerance to the hydrophilic organic solvent, DMSO, was facilitated by the overproduction of 2 ABC transporters, Snq2p and Yor1p and, 3 cell wall proteins, Wsc3p, Pry3p, and Ynl190wp. Our results suggest that overexpression of genes encoding proteins effective for tolerance to specific organic solvents would enable enhanced tolerances for practical use.

In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).

By combining a homologous recombinant gene expression system and optimization of the culture conditions, hyper overproduction of Pleurtous ostreatus MnP2 was achieved. Genetically modified P. ostreatus strains with the recombinant mnp2 sequence under the control of sdi1 expression signals, were subjected to agitated culture using media supplemented with wheat bran or its hot-water extract. The best result, whereby 7300 U/l of MnP was produced by a recombinant strain TM2-18, indicated that more than 30-fold overproduction of the recombinant MnP2 compared to the previous result was achieved. On the other hand, no MnP activity was detected for the wild-type strain under the same conditions. Accumulation of the recombinant, but not endogenous, mnp2 transcripts was demonstrated in reverse-transcription PCR experiments. These results indicated that the recombinant MnP2 was exclusively expressed by the recombinant strain. Purified recombinant MnP2 showed almost identical properties to native MnP2 in electrophoresis, spectroscopic and kinetic analyses, including determination of Km and Vmax values for Mn(II), H2O2 and veratryl alcohol. Moreover, the recombinant MnP2 directly oxidized a high-molecularweight substrate RNase A in the absence of redox mediators, as does native MnP2. The homologous overproduction system will provide a plat form for exclusive production of mutant or variant peroxidases with a desired property in basidiomycete.

The obligate methylotroph Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine could secrete L-lysine into the medium, but also maintained a high concentration of intracellular L-lysine. To improve the yield from excretion, we attempted to introduce an L-lysine/L-arginine exporter (LysE) from Corynebacterium glutamicum 2256 into M. methylotrophus. We were unable to stably transform M. methylotrophus with a plasmid expressing the wild type lysE gene, but happened to obtain a transformant carrying a spontaneously mutated lysE gene (designated lysE24) which could induce L-lysine production even in the wild type strain. The transformant also possessed increased tolerance to S-(2-aminoethyl)-L-cysteine (an L-lysine analog). lysE24 has a single-base insertion mutation in the middle of the lysE gene, and its product is presumably quite different in structure from wild-type LysE. When lysE24 was introduced into an L-lysine producer of M. methylotrophus carrying dapA24, the level of intracellular L-lysine fell. During fermentation, M. methylotrophus carrying both lysE24 and dapA24 produced 10-fold more L-lysine (11.3 gl(-1) in jar-fermentation) than the parent producer carrying only dapA24 or lysE24. These results show the importance of the factor (lysE24) involved in the excretion of L-lysine on its overproduction in M. methylotrophus.

Tobacco suspension culture cell (Nicotiana tabacum, BY2) was transformed with an Aspergillus niger pectin methylesterase (PME; EC 3.1.1.11) cDNA under the control of cauliflower mosaic virus (CaMV) 35S promoter. The transformant indicated a significant rise of PME and the level of methanol in the transformant increased by 28.7% compared to the vector control transformant. This is the first report of methanol overproduction in plant cells by means of genetic engineering.

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l-1 in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP. © 2003 Elsevier Science B.V. All rights reserved.

a b s t r a c t Heterologous expression of secondary metabolite biosynthesis pathways in a surrogate host, e.g. Escherichia coli, has emerged in recent years as an effective way to produce complex natural products. The nonribosomal peptide (NRP) antibiotic valinomycin has been recombinantly produced in E. coli through reconstitution of its biosynthetic pathway from the native producer Streptomyces tsusimaensis. In this study, a discrete protein type II thioesterase (TEII) encoded in the valinomycin gene cluster was coex- pressed in the valinomycin producing E. coli strain. Valinomycin titers were significantly improved from 0.5 (without TEII coexpression) to 3.3 mg L −1 , which demonstrates the reconstitutive function of TEII involved in NRP biosynthesis. Based on a flask scale fed-batch cultivation system, repeated feeding of the glucose polymer during the cultivation further increased cell density and valinomycin titer up to 55 (OD600) and 13 mg L −1 , respectively. This indicates scalable high cell density cultivation in a bioreactor for overproduction of valinomycin will be a potential and feasible approach. In this work we present an in vivo example to show that TEII plays a positive role in heterologous valinomycin production. © 2014 Elsevier B.V. All rights reserved.

S-Adenosyl-L-methionine (SAM) is an important metabolite having prominent role in treating various diseases. Due to increasing demand of SAM, improvement in its production is essential. For this purpose, S-adenosyl-l-methionine synthetase gene (sam2) was overexpressed in the present study, and we studied the effect of coexpression of methionine permease (mup1) and adenylate kinase (adk1) genes. From the recombinant strains expressing individual genes, we observed that SAM2 synthetase is the primary limiting factor and its overexpression is essential to increase the SAM productivity. Coexpression of mup1 with sam2 did not enhance SAM production, while coexpression of adk1 with sam2 improved SAM production, clearly indicating that ATP is the primary limiting precursor in SAM production. However, coexpression of all three genes synergistically improved SAM productivity with better L-methionine (L-met) conversion efficiency in every stage, and it was 77% more compared to overexpressing sam2 alone. Sparging pure oxygen reduced cultivation time. Feeding nitrogen source and additional L-met during induction phase enhanced SAM yield by 38.4% and 55.1%, respectively. Moreover, building up biomass before induction resulted in 145% increase in specific yield and 83% higher L-met conversion efficiency. This is the first report on increasing both the precursors L-met and ATP availability through molecular strategies using microorganisms for the production of SAM.

The extracellular xylanase from Bacillus stearothermophilus T-6 is a thermostable alkaline tolerant enzyme that was found to bleach pulp optimally at pH 9 and 65°C, and was successfully used in a large-scale biobleaching mill trial. In an attempt to obtain a heavy atom derivative suitable for complete X-ray analysis, xylanase T-6 was labeled biosynthetically with seleno-methionine, resulting in a ‘built-in’ array of atoms with specific X-ray anomalous scattering signal. Optimization of growth conditions resulted in over 0.8 g of homogenous seleno-methionine xylanase T-6 per liter culture. The seleno-methionine enzyme was shown to be fully active and produced single crystals suitable for complete multiple wavelength anomalous diffraction (MAD) structural analysis.

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

An extended mathematical model for the biosynthesis of tryptophan (trp) in bacteria is proposed. The model considers both feedback inhibition of the biosynthetic enzymes and repression of the trp operon by tryptophan and explicitly takes into account the growth rate and the demand of tryptophan for protein synthesis. The intracellular concentration of tryptophan is found to drastically decline with the growth rate, particularly at low values of growth rate. This also applies to genetically modified strains with reduced strain levels of the repressor and/or alleviated feedback inhibition. At low growth rates the intracellular concentrations of tryptophan and the synthetic enzymes are also strongly affected by the strain level of repressor, feedback inhibition and the consumption of tryptophan for protein synthesis. It can be concluded that for an effective production of tryptophan the growth rate of cells should be kept as low as possible. Another important factor for the production of tryptophan is the stability and dynamics of the trp operon. Model analysis revealed that the intracellular concentration of tryptophan is determinant for the occurrence of oscillatory expression of the trp operon. Instability of tryptophan synthesis occurs at low intracellular tryptophan concentrations, irrespective of the growth rate. A reduction of the repressor level can reduce the instability region so that a stable operation with improved production is possible. In addition, a high demand of tryptophan for protein synthesis should be avoided which may lead to a periodic expression of the trp operon, even at low growth rate.

A semicontinuous bioprocess with simultaneous use of immobilized plant cells and in situ product extraction in a second liquid phase was developed to enhance the productivity of secondary metabolites and to overcome current limitations of plant cell cultures, such as slow metabolic rate, low product yield, and sensitivity to shear stress. Production of anthraquinones with Cruciata glabra was selected as a model system. The effects of cell immobilization and a second liquid phase using the triglyceride Miglyol were investigated in suspended and immobilized plant cells in batch operation as well as in semicontinuous processes. The concurrent use of cell immobilization and Miglyol improved the specific productivity of anthraquinones 5.5-fold. Continuous removal of metabolites from the solid phase by addition of a second liquid phase has been found to stimulate the biosynthetic capacity leading to overproduction and secretion of anthraquinones up to 58%. Semicontinuous process operations enabled a total 49-fold increase in specific productivity with selected ‘replacement rates’.

In this paper in vivo studies on the overall and cyanide-resistant respiration of Acremonium chrysogenum in connection with different oxygen transfer rates are discussed. Two parameters of the cyanide-resistant respiration, the activity and the capacity were examined separately during cultivation together with the possibly respiration-influencing factors like NADH/NAD content and ADP-limitation. It was finally concluded that the cyanide-resistant respiration seems to be independent of the amount and proportion of oxidized/reduced coenzymes and not limited by ADP or phosphate, but it is strongly affected by the oxygen available. It was also established that the examined path must have an important role in cephalosporin-C overproduction.

This paper compares three possible strategies for enhanced lipid overproduction in microalgae: the biochemical engineering (BE) approaches, the genetic engineering (GE) approaches, and the transcription factor engineering (TFE) approaches. The BE strategy relies on creating a physiological stress such as nutrient-starvation or high salinity to channel metabolic fluxes to lipid accumulation. The GE strategy exploits our understanding to the lipid metabolic pathway, especially the rate-limiting enzymes, to create a channelling of metabolites to lipid biosynthesis by overexpressing one or more key enzymes in recombinant microalgal strains. The TFE strategy is an emerging technology aiming at enhancing the production of a particular metabolite by means of overexpressing TFs regulating the metabolic pathways involved in the accumulation of target metabolites. Currently, BE approaches are the most established in microalgal lipid production. The TFE is a very promising strategy because it may avoid the inhibitive effects of the BE approaches and the limitation of "secondary bottlenecks" as commonly observed in the GE approaches. However, it is still a novel concept to be investigated systematically.

An alternative approach to the use of antibiotic selection markers for maintenance of recombinant plasmid vectors in Escherichia coli based on an aminoacid auxotrophy complementation has been developed. An E. coli M15-derivated glycine-auxotrophic strain of has been constructed by means of a PCR-based approach. This mutant strain contains a deletion in the glyA gene, which encodes for serine hydroxymethyl transferase, an enzyme involved in the main glycine biosynthesis pathway in E. coli. Also, we have constructed the complementation plasmid pQEalphabetarham derived from the commercially available expression vector pQE40 (QIAGEN) containing the glyA homologous gene under the control of the constitutive weak promoter P3. By using the E. coli M15DeltaglyA strain combined with the pQEalphabetarham plasmid, a successful complementation system was achieved, allowing transformants to grow on minimal media without glycine supplementation. The capability of the new system E. coli M15DeltaglyA/pQEalphabetarham for recombinant overproduction of rhamnulose 1-phosphate aldolase was evaluated in antibiotic free fed-batch cultures at controlled specific growth rate, obtaining high cell density cultures and high RhuA production and productivity levels comparable to those obtained with the conventional system. The new selection marker based on glycine-auxotrophy is a promising genetic tool, not only for recombinant protein production, but also for plasmid DNA production processes, where antibiotics can not be present in the medium formulation.

Although the enhancement of amino-acid synthesis by guanosine-3',5'-tetraphosphate (ppGpp) is well known, the effect of intracellular ppGpp levels on amino-acid overproduction in Escherichia coli has not been investigated. In this study, we demonstrate that overexpression of the relA gene, encoding ppGpp synthetase, increases the accumulation of amino acids, such as glutamate and lysine, in amino-acid-overproducing strains of E. coli. Elevation of intracellular ppGpp levels due to depletion of required amino acids also enhances glutamate overproduction. Moreover, the extent of overproduction is highly dependent on the intracellular ppGpp level. These results demonstrate that amino-acid overproduction in E. coli is closely connected to amino-acid auxotrophy via the accumulation of ppGpp.

Production of recombinant malaria proteins in the methylotrophic yeast Pichia pastoris has been difficult due to constraints in transcription, translation and/or post-translation controls. Use of codon-optimized genes has resolved many of the transcriptional controls; however, efforts to overcome translational and post-translational modifications involving disulfide bond formation and glycosylation have been mostly restricted to knocking-out putative N-linked glycosylation sites. We report now on the effect of overproduction of P. pastoris protein disulfide isomerase (PpPDI) and Plasmodium falciparum (PfPDI) on production of a disulfide-rich P. falciparum transmission-blocking vaccine candidate, Pfs25. Pfs25 is expressed in P. pastoris as two isoforms (A and B); the A form has been selected for Phase I human studies. Overproduction of PpPDI in the P. pastoris Pfs25 production clone markedly enhanced the expression level of Pfs25(A) and (B) by 3-fold, while overproduction of PfPDI increased the proportion of Pfs25(A) compared to (B). The resultant Pfs25 products were purified and fully characterized biochemically. In addition to differences in production levels, the mass spectra of PpPDI-Pfs25(A) compared to Pfs25(A) and PfPDI-Pfs25(A) were different due to the pattern and level of O-linked glycosylation. The overproduction of PpPDI or PfPDI provides new platforms for expression of disulfide-rich malaria proteins.

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).

A strategy of genetically manipulating carbon assimilation with respect to expression of the pac gene was employed for overproduction of recombinant penicillin acylase (PAC). Two expression plasmids of pCLL2902 and pCLL3201, which contain the pac coding region but differ in the pac regulatory region, were constructed for the production experiments. Expression of the pac gene was subjected to phenyl acetic acid (PAA-) induction and glucose catabolite repression for pCLL3201, whereas it was subjected to neither of the two transcriptional regulations for pCLL2902. The specific PAC activity for strains harboring pCLL2902 was significantly higher than that for strains harboring pCLL3201 due to an improved transcription efficiency. In addition, no inclusion bodies were observed upon production of PAC using the current expression systems. The results suggest that using the native pac promoter instead of a strong promoter such as tac for regulation is a feasible approach for production of PAC. The impact of the current expression systems is also significant from a process viewpoint since, using strains harboring pCLL2902, not only could glucose replace PAA as a carbon source of Escherichia coli cultures for production of PAC but also the volumetric PAC activity was highly improved.

The agarase gene from Streptomyces coelicolor has been cloned in the non-producer bacterium Streptomyces lividans under the control of its own set of promoters and under the control of a heterologous promoter that is functional only during exponential growth. The best level of overproduction was obtained when the strain containing the natural gene was cultivated in fed batch with mannitol as carbon source. The protein, with a relative molecular mass of 32 kDa, has been purified following an affinity purification method. Contaminating activities seem to be absent from the purified enzyme preparation that can be used to purify DNA from agarose gels.

Rhodococcus erythropolis is able to synthesize an anionic trehalose-2,2',3,4-tetraester during cultivation on n-alkanes. Preconditions for an overproduction are nitrogen limitation, temperature- and pH-shift. The optimum carbon source was technical grade n-C-10, which led to 0.35 g g-1 of glycolipid per n-alkane. Electron microscopical observations showed that n-C-14,15 (technical grade) grown cells contained numerous lipid inclusions in contrast to n-C-10 (technical grade) grown cells. Nocardia corynebacteroides synthesizes a novel pentasaccharide lipid and as size products small amounts of trehalose-corynomycolates. Optimum precursors for overproduction are n-alkanes from n-tetradecane to n-hexadecane with yields in the range of 0.17 g g-1 of glycolipid per carbon source.

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile− strain derived from strain EAPc7 using the Ile+ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA+ recombinant plasmid pYT471 (pBR322-aspA+), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA+-recombinant plasmid pYT471.

The aspartase and asparaginase genes of an industrial strain of Pseudomonas (PO7111) were cloned in the wide host range cosmid vector pJRD203 and identified by complementation of aspartase and asparaginase mutants. Subclones containing the asparaginase gene overproduced the enzyme 9-fold, but aspartase clones were highly unstable suggesting that aspartase overproduction conferred a severe growth disadvantage.