Mold-inhibitory activity of different yeast species during airtight storage of wheat grain

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (<10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C. fennica and C. silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.     

Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens

The yeast Pichia anomala inhibits the spoilage mold Penicillium roqueforti in laboratory experiments with high-moisture wheat in malfunctioning airtight storage. The ability of P. anomala to prevent mold growth during 14 months of grain storage was evaluated in outdoor silos with different air permeabilities. Freshly harvested wheat in 160-kg portions was inoculated with 10(2) colony-forming units (cfu) g(-1) P. roqueforti, alone or together with 10(4) cfu g(-1) P. anomala. During the first month P. anomala increased to about 10(6) cfu g(-1) in the treated silos to reach 10(7) cfu g (-1) after 9 months. Naturally occurring P. anomala in the untreated silos increased from 10(2) to about 10(3) cfu g(-1) during the first month and reached the same level as the treated silos after 9 months. Oxygen levels were reduced below the detection limit within 1 day, while carbon dioxide levels increased to 80-90% during the first month. P. roqueforti did not grow in wheat treated with P. anomala, regardless of silo permeability, but had increased to 10(5) cfu g(-1) in the untreated silos after 14 months of storage.     

Nutrient effects on biocontrol of Penicillium roqueforti by Pichia anomala J121 during airtight storage of wheat

The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of P. roqueforti. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts.     

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.     

A cost-effective cane molasses medium for enhanced cell-bound phytase production by Pichia anomala

AIM: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala. METHODS AND RESULTS: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media. The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1). Different cultural parameters were optimized in cane molasses medium in batch fermentation. The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass). In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium. The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U). CONCLUSIONS: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium. Furthermore, the production in cane molasses medium is cost-effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced. This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution.     

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.     

Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound

The antifungal activity spectrum of Lactobacillus coryniformis subsp. coryniformis strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds Aspergillus fumigatus, A. nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, F. graminearum, F. culmorum, and F. sporotrichoides. A weaker activity was observed against the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. The yeasts Rhodotorula glutinis, Sporobolomyces roseus, and Pichia anomala were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121 degrees C for 15 min. Maximum activity was observed at pH values of between 3. 0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH(4))(2)SO(4) precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from L. coryniformis subsp. coryniformis.     

Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes

The cheese-associated fungi Penicillium commune , P. roqueforti , P. solitum , P. discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar. Volatiles were collected by headspace solid-phase microextraction. Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d. In a mixed culture of P. roqueforti and P. commune , inoculated in a ratio of 1000 : 1, volatiles from both fungi could be detected within 3 d, making identification possible.     

Physiological characteristics of the biocontrol yeast Pichia anomala J121

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.     

Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.     

Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.     

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.     

Metabolite profiles of lactic acid bacteria in grass silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml(-1) for two of the three test organisms).     

Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Improving Biocontrol Activity of Pichia guillermondii Against Post-harvest Decay of Oranges in Commercial Packing-houses by Reduced Concentrations of Fungicides

Three commercial tests were conducted in 2000-2001 in two commercial packing-houses (Muravera and Villacidro) located in Sardinia, Italy, to evaluate the efficacy of biological, chemical and integrated treatments against Penicillium digitatum and P. italicum on naturally inoculated orange fruit. Damage caused by the packing-house processing line was also assessed. Treating orange fruits with the yeast Pichia guilliermondii (strain 5A) in the processing line generally led to a significant reduction of post-harvest decay compared to the processed control, while the commercial product Aspire®, based on Candida oleophila , was ineffective in inhibiting the pathogen when applied alone. The integrated application of thiabendazole or imazalil with the biocontrol agents significantly improved the control of fruit decay. Using thiabendazole at concentrations of 0.1 and 1.2 g L -1 , led to similar inhibition of fruit decay in two trials. Both yeasts were equally able to colonize the fruit actively during storage. Passing fruits through the packing line caused a significant increase in fruit decay.     

Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Antagonistic activity of the food-related filamentous fungus Penicillium nalgiovense by the production of penicillin.

Defined strains of the genus Penicillium used as starter cultures for food and strains isolated from mold-fermented foods were analyzed for their ability to inhibit the growth of Micrococcus luteus DSM 348 used as an indicator organism. Most of the strains belonging to the species Penicillium nalgiovense showed antagonistic activity in agar diffusion assays. Penicillium camemberti and Penicillium roqueforti strains proved to be inactive in these tests. The inhibitory substance excreted by P. nalgiovense strains was totally inactivated when treated with beta-lactamase (penicillinase), indicating that a beta-lactam antibiotic is produced by these strains. This observation was verified by PCRs with primer sets specific to the [delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine] synthetase gene (pcbAB), the isopenicillin-N-synthase gene (pcbC), and the acyl coenzyme A:6-aminopenicillanic acid acyltransferase gene (penDE) from Penicillium chrysogenum using chromosomal DNA of the fungal strains as a template. These results indicate that penicillin biosynthesis is a characteristic often found in strains of P. nalgiovense. No specific PCR signal could be identified with DNA from P. camemberti and P. roqueforti.     

Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae

Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.