An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

Li, Kun-tai; Liu, Donghong; Chu, Ju; Wang, Yonghong; Zhuang, Yingping; Zhang, Siliang

doi:10.1007/s00449-008-0209-5

Cited by 51 publications

(25 citation statements)

References 24 publications

(20 reference statements)

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“…The different feeding compositions included glucose, hydrolyzed starch, and a concentrated mixture of hydrolyzed starch and soy peptone. Following application of these carbon sources, the pH of the fermentation medium increased, and fed-batch cultures controlled by pH change have been used for producing recombinant proteins and chemical products [ 28 , 30 , 31 ]. Based on changes in pH, the effect of different feeding compositions on the production of recombinant alkaline amylase in B. subtilis 168 mut-16# was investigated.…”

Section: Resultsmentioning

confidence: 99%

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Shen

Chen

et al. 2016

J Biol Eng

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BackgroundAlkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168.ResultsThe activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#.ConclusionThe results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

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Section: Resultsmentioning

confidence: 99%

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Shen

Chen

et al. 2016

J Biol Eng

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“…Our engineered vitamin B 12 producing E. coli strain overcomes some major limitations that currently face industrial producers of vitamin B 12 like long growth cycles. For example, the present industrial vitamin B 12 producing P. denitrifians strain needs about 180 h to complete fermentation 36 , 39 , 40 , whereas the recombinant E. coli strain constructed here only needs 24 h. Our successful demonstration of the engineering of the complete pathway for vitamin B 12 production E. coli offers a powerful illustration for how the dozens of genes of a complex heterologous pathway can be successfully introduced into new host organisms. This demonstration of our modular design concept should offer encouragement to other scientists and biotechnologists that are considering similar undertakings.…”

Section: Discussionmentioning

confidence: 90%

“…Although vitamin B 12 productivity of the engineered E. coli strain is much less than industrial strains such as P. denitrificans 36 (the highest vitamin B 12 production of 214.3 mg l −1 ) and P. freudenreichii 37 (the highest vitamin B 12 production of 206.0 mg l −1 ), it is comparable with some native vitamin B 12 producers, for example: wild-type Bacillus megaterium and an engineered strain produced 0.26 and 8.51 µg l −1 vitamin B 12 , respectively 38 , and wild-type P. denitrificans produced 2.75 µg g −1 DCW vitamin B 12 18 . Consider that the vitamin B 12 production of a single P. denitrificans strain was increased approximately 100-fold over a 10-year period via a great many rounds of random mutagenesis 4 ; however, the vitamin B 12 production of the E. coli strain we engineered in the present study was increased about 250-fold in several months, without random mutagenesis, and can very likely be improved further.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

et al. 2018

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The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea. Here, using genetic and metabolic engineering, we generate an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. In vitro and/or in vivo analysis of genes involved in adenosylcobinamide phosphate biosynthesis from Rhodobacter capsulatus suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increase the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg g−1 DCW via metabolic engineering and optimization of fermentation conditions. Beyond our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

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“…in the medium. Consequently, the pH might be a restricting factor for enzyme production, because pH strongly affects the growth of microbial strains and enzyme production [26]. Some reports have shown the importance of controlling pH in fed-batch fermentation processes to obtain high production of the target metabolites [27].…”

Section: Optimization Of Fed-batch Fermentationmentioning

confidence: 99%

Improved production of Pseudomonas sp. ECU1011 acetyl esterase by medium design and fed-batch fermentation

Pan

et al. 2011

Bioprocess Biosyst Eng

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We optimized culture medium and batch-fed fermentation conditions to enhance production of an acetyl esterase from Pseudomonas sp. ECU1011 (PSAE). This enzyme enantioselectively deacetylates α-acetoxyphenylacetic acid. The medium was redesigned by single-factor and statistical optimization. The addition of ZnSO(4) enhanced enzyme production by 37%. Yeast extract concentration was directly associated with the enzyme production. The fermentation was scaled up in a 5-l fermenter with the optimized medium, and the correlations between enzyme production and dissolved oxygen, pH, and feeding strategy were investigated. The fermentation process was highly oxygen-demanding, pH sensitive and mandelic acid-inducible. The fermentation pH was controlled at 7.5 by a pH and dissolved oxygen feedback strategy. Feeding mandelic acid as both a pH regulator and an enzyme inducer increased the enzyme production by 23%. The results of the medium redesign experiments were confirmed and explained in fed-batch culture experiments. Mathematical models describing the fermentation processes indicated that the enzyme production was strongly associated with cell growth. The optimized pH and dissolved oxygen stat fed-batch process resulted high volumetric production of PSAE (4166 U/l, 7.2-fold higher than the initial) without enantioselectivity decline. This process has potential applications for industrial production of chiral mandelic acid or its derivatives.

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An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

Cited by 51 publications

References 24 publications

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

Improved production of Pseudomonas sp. ECU1011 acetyl esterase by medium design and fed-batch fermentation

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