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Additional file 1: Table S1. Primers design for site-directed mutagenesis. Table S2. Activity ratios of PulAR and its mutants at pH 5.0 to at pH 6.0. Fig. S1. SDS-PAGE analysis of WT-PulA and its mutants. M, Markers; Lane 1, purified WT-PulA; Lane 2, purified PulA-A365V; Lane 3, purified PulA-V401C; Lane 4, purified PulA-A365V/V401C; Lane 5, purified PulA-A365V/V401C/T504C; Lane 6, purified PulA-A365V/V401C/T504C/H499A. Fig. S2. Multiple sequence alignment of pullulanases from Anoxybacillus sp. AR-29, Anoxybacillus sp. LM18-11, Bacillus acidopullulyticus and Bacillus naganoensis. Conserved residues are indicated in frames.

Additional file 1: Table S1. Strains and plasmids used in this study. Table S2. Primers used in this study. Table S3. Annotated metabolites detected by LC–MS/MS in S. nodosus ZJB2016050 and S. nodosus ZJB20140315 among different groups and related pathways. Figure S1. Metabolic pathway analysis revealing metabolic impacts of metabolic pathway to the key fermentation of S. nodosus via comparison. Figure S2. The enrichment of metabolic pathway. Figure S3. The production of AmB for different metabolites addition with strain S. nodosus ZJB2016050. Figure S4. RT-qPCR analysis of transcriptional levels of single gene expression in S. nodosus ZJB2016050.

Additional file 1. Additional figures and tables.

Additional file 1: Supporting information 1: The single colonies, growth curve and active compounds contents in O. sinensis zjut; Supporting information 2 The details of iTRAQ quantitative proteomics; Supporting information 3: The identified results of the samples and DEPs in O. sinensis zjut; Supporting information 4: GO enrichment analysis of DEPs in O. sinensis zjut; Supporting information 5: The proteins involved in metabolic pathway of D-mannital, cordycepin and purine nucleotides in O. sinensis zjut, and purine nucleotides metabolic pathway; Supporting information 6: The metabolic pathways of D-mannose, D-galactose and D-glucose as well as the proteins involved in these pathways; Supporting information 7: The contents of sixteen types of amino acids in O. sinensis zjut; Supporting information 8: The metabolic pathways of the histidine, arginine, phenylalanine and tyrosine as well as the proteins involved in these pathways; Supporting information 9: The proteins involved in the biosynthetic pathway of fatty acids; Supporting information 10: The metabolic pathways of glycolysis/gluconeogenesis and citrate cycle as well as the proteins involved in these pathways; Supporting information 11: The proteins with anti-oxidant activity in O. sinensis zjut.

Additional file 1: Table S1. Primers for donor DNA amplification, pTarget construction and positive colony validation. Figure S1. Structures of plasmids used in this study. Detailed description on constructions of these plasmids is shown in the section.

Effect of metal ions on the activity of recombinant BgADH3. (DOCX 17Â kb)

Results of screening microorganisms for direct asymmetric reduction of ethyl 4-chloro-3-oxobutanoate. (DOCX 24Â kb)

Effect of metal ions on the activity of recombinant BgADH3. (DOCX 17Â kb)

GC and HPLC analysis of the ee or de values of products. (DOCX 19Â kb)

Results of screening stereoselective carbonyl reductases for direct asymmetric reduction of ethyl 4-chloro-3-oxobutanoate. (DOCX 18Â kb)

1H NMR of the isolated (R)-CHBE (500 MHz, CDCl3). Figures S3. 13C NMR of the isolated (R)-CHBE (126 MHz, CDCl3). (DOCX 1122 kb)

Primers from Burkholderia gladioli CCTCC M 2012379 for PCR amplification. (DOCX 18Â kb)

Amino acid sequence alignments of short-chain alcohol dehydrogenase/reductase using ESPript 3.0. Sequences are Burkholderia gladioli from this study (BgADH3, GenBank Accession No. AEA63541), Gluconobacter oxydans 621H (Gox2036, GenBank Accession No. AAW61772.1), Lactobacillus brevis (LbADH, GeneBank Accession No. CAD66648.1), Salmonella enteric (Ygha, PDB: 3R3S_A), and Bacillus anthracis (BaADH, PDB: 3I3O_A). The cofactor-binding motifs in the SDRs, TGXXXGXG and PG, are highlighted in orange and triangle while the residues of the catalytic tetrad (N, S, Y, and K) are highlighted in green. (DOCX 212Â kb)

Amino acid sequence alignments of short-chain alcohol dehydrogenase/reductase using ESPript 3.0. Sequences are Burkholderia gladioli from this study (BgADH3, GenBank Accession No. AEA63541), Gluconobacter oxydans 621H (Gox2036, GenBank Accession No. AAW61772.1), Lactobacillus brevis (LbADH, GeneBank Accession No. CAD66648.1), Salmonella enteric (Ygha, PDB: 3R3S_A), and Bacillus anthracis (BaADH, PDB: 3I3O_A). The cofactor-binding motifs in the SDRs, TGXXXGXG and PG, are highlighted in orange and triangle while the residues of the catalytic tetrad (N, S, Y, and K) are highlighted in green. (DOCX 212Â kb)

GC and HPLC analysis of the ee or de values of products. (DOCX 19Â kb)

Results of screening stereoselective carbonyl reductases for direct asymmetric reduction of ethyl 4-chloro-3-oxobutanoate. (DOCX 18Â kb)

1H NMR of the isolated (R)-CHBE (500 MHz, CDCl3). Figures S3. 13C NMR of the isolated (R)-CHBE (126 MHz, CDCl3). (DOCX 1122 kb)

Additional file 1: Figure S1. Amino acid sequences multiple alignment of 2-HADH from P. aeruginosa CCTCC M 2011394, B. xenovorans LB400, P. putida ATCC 12633, P. aeruginosa NUST, P. fluorescens strain EBC191. Figure S2. Optimization of biooxidation reaction conditions by resting cells of recombinant E. coli BL21(DE3)/pET28b-HADH. (A) Effect of temperature on the biooxidation. The optimum temperature was determined over the range from 25 and 65 °C. The recombinant E. coli BL21(DE3)/pET28b-HADH showed high activity at 35–55 °C, and at higher temperatures the activity began to decrease significantly. (B) Effect of pH on the biooxidation; The optimum pH on the oxidation reaction was determined over the range from 6.0 and 9.0. When the pH was below 7.5 or over 8.5, the enzyme activity decreased dramatically. Figure S3. SDS-PAGE analysis of the expressed (R)-2-KAR in recombinant E. coli BL21(DE3)/pET28b-KAR. 1. Markers; 2. Cell-free extract of recombinant E. coli BL21(DE3)/pET28b-KAR; 3. Purified (R)-2-KAR from E. coli BL21(DE3)/pET28b-KAR (~32 kDa). Figure S4. SDS-PAGE analysis of the expressed GDH in recombinant E. coli BL21(DE3)/pET28b-GDH and coexpressed (R)-2-KAR and GDH in recombinant E. coli BL21(DE3)/pCDFDuet-KAR-GDH. 1. E. coli BL21(DE3)/pCDFDuet-KAR-GDH with 0.1 mM IPTG. The upper arrow indicated (R)-2-KAR (~32 kDa) and the lower arrow represented GDH (~28 kDa).2. E. coli BL21(DE3)/pCDFDuet-KAR-GDH with 0 mM IPTG; 3. E. coli BL21(DE3)/pET28b-GDH with 0.1 mM IPTG; 4. E. coli BL21(DE3)/pET28b-GDH with 0 mM IPTG; 5.Empty plasmid of pET28b with 0 mM IPTG. 6. Markers. Figure S5. SDS-PAGE analysis of the coexpressed (S)-2-HADH, (R)-2-KAR and GDH in recombinant E. coli BL21(DE3)/pET28b-HADH/pCDFDuet-KAR-GDH. 1. Markers; 2. Cell-free extract of recombinant E. coli BL21(DE3)/pET28b-HADH/pCDFDuet-KAR-GDH. The upper arrow indicated (S)-2-HADH (~42 kDa), the middle arrow indicated (R)-2-KAR (~32 kDa) and the lower arrow represented GDH (~28 kDa).

Primers from Burkholderia gladioli CCTCC M 2012379 for PCR amplification. (DOCX 18Â kb)

A method to determine dihydroxyacetone (DHA) and glycerol in fermentation broth was developed on the basis of pyrolytic methylation–gas chromatography (GC) using a vertical microfurnace pyrolyzer. With the on-line pyrolytic methylation in the presence of an organic alkali, tetramethylammonium hydroxide ((CH 3 ) 4 NOH, TMAH), DHA and glycerol were converted into their corresponding methyl ethers. Thus, fermentation broth samples containing DHA and glycerol could be well determined simultaneously by this pyrolytic methylation–GC without using any pretreatment procedures. Important factors such as reaction temperature, the amount of TMAH to DHA and glycerol were investigated in order to obtain the optimum methylation conditions. Thus, obtained results showed that 0.4 μl 25% TMAH aqueous solution at 400 °C enabled the highly sensitive determination of DHA and glycerol contained in 0.2 μl aqueous samples. The achieved calibration curves exhibit good linearity with regression coefficients of 0.996 and 0.998 for DHA and gelycerol at the concentration range from 1 to 100 g/l, respectively. The precision was quite high with the R.S.D. within 5.0% for DHA at 30 g/l and glycerol at 15 g/l concentration levels, and the limit of detection reached 0.016 g/l. The potential of the proposed method was assessed by applying it to determination of both the analytes in fermentation broth during the bioconversion of glycerol to DHA by Acetobacteria sp. The results proved that this on-line pyrolytic methylation–GC technique was a rapid, convenient and highly sensitive method for monitoring the bioprocesses of DHA.

Bacillus subtilis AS1.398 was cultivated in a 11.5-L total volume external-loop airlift bioreactor with a low height-to-diameter ratio of 2.9 and a riser-to-downcomer diameter ratio of 6.6 for the production of protease from crude substrates with dregs. The influence of aeration rate, liquid volume, and sparger hole diameter on protease production was investigated. An average of 8197 u/mL protease activity was obtained after a total fermentation time of 32 h in the external-loop airlift bioreactor with a liquid volume of 8.5 L, air flow rate of 1.5 vvm, and sparger hole diameter of 1.5 mm. The addition of one stainless steel sieve plate in the riser of the airlift bioreactor increased productivity of protease. After 32 h of fermentation, an average of 8718 u/mL protease activity was achieved in the external-loop airlift bioreactor with one sieve plate and an air flow rate of 1.2 vvm, liquid volume of 8.5 L, and gas sparger hole diameter of 1.5 mm. This was 9.0% higher than the typical averages of about 8000 u/mL protease activity in the mechanically stirred tank bioreactors of the enzyme factory using the same microorganism. It is possible to make a scale-up of the external-loop airlift bioreactor and feasible to operate it for production of protease from crude substrate with dregs.

An external-loop airlift bioreactor, with a low ratio 2.9 of height-to-diameter of the riser and a ratio 6.6 of riser-to-downcomer diameter, was used to produce alpha-amylase from fermentation with dregs by Bacillus subtilis. The effects of gas flow rate and liquid volume on alpha-amylase production were investigated. After a 36-h fermentation time, an average of 432.3U/ml alpha-amylase activity was obtained under the conditions of liquid volume 8.5l and gas flow rate 1.2vvm for the first 12h of fermentation, 1.4vvm from 12 to 27h, and 1.2vvm from 27h to the end. The activity was higher than that obtained in shaking flasks (409.0U/ml) and in a mechanically stirred tank bioreactor (397.2U/ml) under optimized operating conditions. The fermentation cycle of the airlift bioreactor was shorter than the 48h required for the shaking flasks and close to the 36h of the mechanically stirred tank bioreactor. It was demonstrated that the external-loop airlift bioreactor could substitute for the traditional mechanically stirred tank bioreactor to produce alpha-amylase from fermentation by Bacillus subtilis with dregs.

An external-loop airlift bioreactor (11.5 l), with a ratio of height to diameter of the riser of 2.9, was used for the production of citric acid from the mash of dried sweet potato with its dregs by Aspergillus niger . The effects of air flow rate, liquid volume and sparger hole diameter on citric acid production were investigated. A comparison of citric acid fermentation was made between the external-loop airlift bioreactor and the mechanically stirred tank bioreactor. After a total fermentation time of 65 h, an average of 10.6 mg/ml citric acid concentration was obtained in the external-loop airlift bioreactor under an air flow rate of 1.3 vvm, liquid volume of 8.5 l as compared to 9.6 mg/ml citric acid concentration in the 10 l mechanically stirred tank bioreactor under optimised operation conditions: agitation rate 200 rpm, air flow rate 1.0 vvm and liquid volume 6.5 l. It was demonstrated conclusively that the external-loop airlift bioreactor could be used to produce citric acid economically using A. niger from the mash of dried sweet potato with its dregs.