{"id":92233,"date":"2016-07-09T02:34:09","date_gmt":"2016-07-09T02:34:09","guid":{"rendered":"http:\/\/healthmedicinet.com\/i\/overexpression-of-membrane-bound-gluconate-2-dehydrogenase-to-enhance-the-production-of-2-keto-d-gluconic-acid-by-gluconobacter-oxydans\/"},"modified":"2016-07-09T02:34:09","modified_gmt":"2016-07-09T02:34:09","slug":"overexpression-of-membrane-bound-gluconate-2-dehydrogenase-to-enhance-the-production-of-2-keto-d-gluconic-acid-by-gluconobacter-oxydans","status":"publish","type":"post","link":"https:\/\/healthmedicinet.com\/i\/overexpression-of-membrane-bound-gluconate-2-dehydrogenase-to-enhance-the-production-of-2-keto-d-gluconic-acid-by-gluconobacter-oxydans\/","title":{"rendered":"Overexpression of membrane-bound gluconate-2-dehydrogenase to enhance the production of 2-keto-d-gluconic acid by Gluconobacter oxydans"},"content":{"rendered":"

Overexpression of ga2dh in G. oxydans DSM2003<\/em><\/h4>\n

Efficient expression of ga2dh<\/em> (gox1230-1232) was essential for enhancing the 2KGA production in G. oxydans DSM2003<\/em>. A well-characterized promoter is a prerequisite for the overexpression of an enzyme.
\n To achieve the optimum recombinant strains, three different promoters (G. oxydans_tufB<\/em>, G. oxydans_ gHp0169<\/em> and the native promoter of the ga2dh<\/em> gene) were introduced into the broad-host-range vector pBBR1MCS5 for expression of
\n the ga2dh<\/em> gene. G. oxydans_tufB<\/em> and G. oxydans_ gHp0169<\/em> were confirmed to be strong promoters for cloning and expression of homologous and
\n heterologous genes in G. oxydans<\/em>18<\/a>]. The three recombinant strains (G. oxydans_tufB_ga2dh G. oxydans_g2adh_ga2dh,<\/em> and G. oxydans_gHp0169_ga2dh<\/em>) and the control strain G. oxydans_<\/em>pBBR1MCS5 were cultured in shaking flasks, and the activities of the obtained resting
\n cells toward GA for 2KGA production were compared. Growth behaviors of the recombinant
\n strains were similar to that of G. oxydans_<\/em>pBBR1MCS5, but the biomass at the late-log phase was slightly lower than that of the
\n control strain (Table\u00a01<\/a>). During the biocatalysis of GA by resting cells, all of the ga2dh<\/em>-overexpressing strains produced concentrations of 2KGA higher than that of the control
\n strain. Amongst these strains, G. oxydans_tufB_ga2dh<\/em> and G. oxydans_gHp0169_ga2dh<\/em> exhibited the highest specific productivities of 2KGA (0.83 and 0.85\u00a0g\/g\/h), about
\n 100\u00a0% higher than that of G. oxydans_<\/em>pBBR1MCS5 (0.42\u00a0g\/g\/h).<\/p>\n

Table\u00a01.<\/strong><\/a> Cell growth of different G. oxydans<\/em> strains and their specific2KGA productivities\n <\/p>\n

Concerning the biomass and specific productivity of 2KGA production, the optimal strain
\n G. oxydans_tufB_ga2dh<\/em> was selected and batch bioconversion of GA by resting cells was conducted in a 7-L
\n fermenter with a GA concentration increased to 320\u00a0g\/L. During the bioconversion,
\n the agitation speed and aeration rate were controlled at 600\u00a0rpm and 8\u00a0L\/min, respectively.
\n pH was controlled at 5.8 using 4\u00a0mol\/L NaOH solution. As shown in Fig.\u00a01<\/a>, almost all the GA was converted to 2KGA by 30\u00a0g
\n (wet wt)<\/sub>
\n \/L cells of the engineered strain in about 25\u00a0h, generating 319\u00a0g\/L 2KGA at a productivity
\n of 12.76\u00a0g\/L\/h. In contrast, the control strain G. oxydans_<\/em>pBBR1MCS5 required 51\u00a0h to complete this reaction,generating 307\u00a0g\/L 2KGA at a productivity
\n of 6.02\u00a0g\/L\/h. The results show that enhanced expression of the ga2dh<\/em> gene in G. oxydans<\/em> under the control of tufB<\/em> promoter efficiently improved the production yield of 2KGA from GA.<\/p>\n

\"thumbnail\"Fig.\u00a01.<\/strong><\/a> Comparison of 2KGA production by G. oxydans_<\/em>pBBR1MCS5 and G.oxydans_tufB_ga2dh.<\/em> The biotransformations were carried out in 7-L fermenterat 30\u00a0\u00b0C, pH 5.8, 600\u00a0rpm,
\n aeration rate 8\u00a0L\/min and cell concentration 30\u00a0g
\n (wet wt)<\/sub>
\n \/L. Gluconobacter oxydans_<\/em>pBBR1MCS5 (blank<\/em>), G. oxydans_tufB_ga2dh<\/em> (filled<\/em>)\n <\/p>\n

As expected, the transcriptional levels of the ga2dh<\/em> gene in the engineered strain (G. oxydans_tufB_ga2dh<\/em>) were significantly enhanced (Fig.\u00a02<\/a>). The ga2dh<\/em> expression level obtained was normalized in the control strain G. oxydans<\/em>_pBBR1MCS5. The transcriptional abundance of the three subunits (gox<\/em>1230, 1231 and 1232) of the ga2dh<\/em> gene in G. oxydans_tufB_ga2dh<\/em> were 180, 35 and 60-fold higher than those of the control strain G. oxydans<\/em>_pBBR1MCS5 respectively. But the transcriptional abundance of the gdh<\/em> gene (gox<\/em>0265) in G. oxydans_tufB_ga2dh<\/em> was about 85\u00a0% of that in G. oxydans<\/em>_pBBR1MCS5.<\/p>\n

\"thumbnail\"Fig.\u00a02.<\/strong><\/a> Relative transcriptional abundance of the ga2dh<\/em> and gdh<\/em> gene G. oxydans_<\/em>pBBR1MCS5 (blank<\/em>) and G. oxydans_tufB_ga2dh<\/em> (shadow<\/em>)\n <\/p>\n

Optimization of the biocatalysis conditions by resting G. oxydans_tufB_ga2dh<\/em> cells
\n <\/h4>\n

To explore the potential of G. oxydans_tufB_ga2dh<\/em> in 2KGA production and achieve a high production titer, the biocatalysis conditions
\n for 2KGA production from GA were optimized. In a 7-L fermenter, pre-experiments had
\n proven that the optimum reaction temperature and pH were 30\u00a0\u00b0C and 5.8, respectively.\n <\/p>\n

A suitable amount of cell content is necessary for high 2KGA production and the economic
\n feasibility of the bioprocess. To determine the effect of the cell content on 2KGA
\n production, various concentrations of resting G. oxydans_tufB_ga2dh<\/em> cells were used to catalyze 320\u00a0g\/L GA. As shown in Fig.\u00a03<\/a>, 2KGA production and the reaction rate increased with the cell concentration increasing
\n to 30\u00a0g
\n (wet wt)<\/sub>
\n \/L, and then were nearly constant as cell concentration continued to increase. In
\n the presence of 30\u00a0g\/L resting cells, 2KGA accumulation linearly increased and reached
\n a maximum after 25\u00a0h, at which time all GA had been converted to 2KGA, resulting in
\n the highest productivity of 12.76\u00a0g\/L\/h.<\/p>\n

\"thumbnail\"Fig.\u00a03.<\/strong><\/a> Effect of cell concentration on 2KGA production. The biotransformations were conducted
\n by 10, 20, 30, 40 and 60\u00a0g\/L G. oxydans_tufB_ga2dh<\/em> resting cells, respectively\n <\/p>\n

The effect of initial GA concentration on 2KGA production was also investigated. Reactions
\n with four different GA concentrations (320, 380, 440 and 480\u00a0g\/L) were conducted with
\n 30\u00a0g
\n (wet wt)<\/sub>
\n \/L resting cells at pH 5.8 and 30\u00a0\u00b0C. Almost all the GA at different concentrations
\n were converted to 2KGA with yields close to 100\u00a0%, but the productivity decreased
\n with increasing GA concentration, because of the extension of reaction time when the
\n substrate concentration was increased (Fig.\u00a04<\/a>a). The 2KGA productivities were 12.76, 9.04, 5.93 and 4.93\u00a0g\/L\/h, at initial GA concentrations
\n of 320, 380, 440 and 480\u00a0g\/L, respectively. At a high initial GA concentration of
\n 480\u00a0g\/L, an enhanced concentration of resting cells (60\u00a0g
\n (wet wt)<\/sub>
\n \/L) had no effect on the productivity for 2KGA production (Fig.\u00a04<\/a>b). Both GA conversion rate and 2KGA production rate with 60\u00a0g
\n (wet wt)<\/sub>
\n \/L resting cells were identical with those at 30\u00a0g
\n (wet wt)<\/sub>
\n \/L resting cells.<\/p>\n

\"thumbnail\"Fig.\u00a04.<\/strong><\/a> Effect of initial GA concentration on 2KGA production. a<\/strong> The initial GA concentration were 320, 380, 420 and 480\u00a0g\/L, respectively. Cell concentration
\n was 30\u00a0g
\n (wet wt)<\/sub>
\n \/L. GA (open<\/em>); 2KGA (filled<\/em>), b<\/strong> Initial GA concentration 480\u00a0g\/L; cell concentration 60\u00a0g
\n (wet wt)<\/sub>
\n \/L. GA (open<\/em>); 2KGA (filled<\/em>). c<\/strong> DO profile during the batch bioconversion, cell concentration 30\u00a0g
\n (wet wt)<\/sub>
\n \/L\n <\/p>\n

Given that the formation of 2KGA from GA requires oxygen as the final acceptor of
\n electrons formed during the oxidation of GA, oxygen conditions were maintained via
\n a high agitation speed (600\u00a0rpm) and air flow rate (8\u00a0L\/min) throughout the batch
\n bioconversion period. However, as shown in Fig.\u00a04<\/a>c, it was observed that when the reaction started, dissolved oxygen (DO) sharply decreased
\n to 0\u00a0% air saturation, and then remained constant (less than 0\u00a0%) until the latter
\n stages of the reaction (about 20\u00a0% of GA remaining). This may imply that DO is an
\n important factor influencing the conversion of GA to 2KGA.\n <\/p>\n

The main factors controlling DO concentration during biotransformation are the degree
\n of agitation, gas flow rate and oxygen partial pressure in the supplied gas. Because
\n of the limitations of the fermenter design, the agitation speed and gas flow rate
\n could not be increased further. Thus, to increase DO levels, oxygen instead of air
\n was supplied continuously to support the oxidation of 480\u00a0g\/L GA by 30\u00a0g
\n (wet wt)<\/sub>
\n \/L resting cells. Under this condition, the agitation speed and oxygen flow rate were
\n controlled at 600\u00a0rpm and 1\u00a0L\/min, respectively. The DO level in the reaction mixture
\n was maintained above 100\u00a0% throughout the process. The time course for GA consumption
\n and 2KGA production are shown in Fig.\u00a05<\/a>a. During the first 24\u00a0h of batch bioconversion, approximately 50\u00a0% of GA was linearly
\n reduced and produced 199.42\u00a0\u00b1\u00a020.34\u00a0g\/L 2KGA. Both 2KGA production and the conversion
\n rate of GA to 2KGA were higher than those in bioconversion experiments under continuous
\n air supply, in which a 2KGA titer of 129.42\u00a0\u00b1\u00a03.43\u00a0g\/L and conversion rate of 27.4\u00a0%
\n were obtained at 24\u00a0h (Fig.\u00a04<\/a>a). After 24\u00a0h, a continuous rise in 2KGA titer was accompanied by a gradual decrease
\n in GA levels, as observed by declines in the product formation and substrate consumption
\n rates. By 108\u00a0h, all GA was completely transformed into 2KGA at a level of 461.09\u00a0g\/L
\n with a productivity of 4.27\u00a0g\/L\/h. To determine the reasons for this behavior, samples
\n of resting cells in the reaction mixture were taken at different reaction times and
\n the relative activities toward GA were determined. The catalytic activities of the
\n samples at the beginning of the reactions from air supply experiments or oxygen supply
\n experiments were set at 100\u00a0%. As shown in Fig.\u00a05<\/a>b, the catalytic activities of the resting cells in these two experiments decreased
\n with the extension of reaction time; however, there was a more marked loss of activity
\n in the oxygen supply experiment than in the air supply experiment. Catalytic activities
\n of resting cells in the oxygen supply experiment decreased by 40\u00a0% in 24\u00a0h, and showed
\n 33\u00a0% activity at the end of bioconversion. The results clearly reveal that high oxygen
\n levels suppressed the oxidative activity of resting cells toward GA, and an excess
\n of oxygen during bioconversion may result in decreasing productivity. Therefore, an
\n optimal oxygen level is important for high 2KGA productivity.<\/p>\n

\"thumbnail\"Fig.\u00a05.<\/strong><\/a> Effect of DO control strategy on bioconversion of GA to 2KGA. a<\/strong> Time course of GA consumption and 2KGA production with oxygen supply b<\/strong> Relative activities of resting cells during bioconversion\n <\/p>\n

In the case of enhanced oxygen levels via continuous supply of oxygen (Fig.\u00a06<\/a>), cell content in the reaction mixture was also increased to 60\u00a0g
\n (wet wt)<\/sub>
\n \/L to make up for the loss of activities of cells. As expected, the conversion time
\n was considerably shortened. By 45\u00a0h, 480\u00a0g\/L GA was completely exhausted, and the
\n 2KGA titer reached about 453.3\u00a0g\/L, generating a productivity of 10.07\u00a0g\/L\/h, which
\n is 135.8\u00a0% higher than that achieved using 30\u00a0g\/L resting cells.<\/p>\n

\"thumbnail\"Fig.\u00a06.<\/strong><\/a> Bioconversion of GA to 2KGA with oxygen supply and enhanced cell content (60\u00a0g
\n (wet wt)<\/sub>
\n \/L)\n <\/p>\n

Bioconversion of glucose to 2KGA by G. oxydans_tufB_ga2dh<\/em><\/h4>\n

Overexpression of the ga2dh<\/em> gene in G. oxydans<\/em> could significantly improve the productivity and 2KGA production from GA, and enhance
\n the product formation rate from glucose. As shown in Fig.\u00a07<\/a>, the profiles for 2KGA production from glucose by G. oxydans_tufB_ga2dh<\/em> and the control strain G. oxydans<\/em>_pBBR1MCS5 were similar, but the glucose conversion rates and 2KGA formation rates
\n were evidently different. As shown in Fig.\u00a07<\/a>a, 200\u00a0g\/L glucose was consumed rapidly by 60\u00a0g\/L resting G. oxydans_tufB_ga2dh<\/em> cells, which was accompanied by an increase in 2KGA and GA accumulation. After all
\n glucose was fully depleted at 12\u00a0h, GA accumulation reached a maximum of 102.42\u00a0g\/L,
\n with a glucose conversion rate of 16.67\u00a0g\/L\/h. However, using the same amount of resting
\n cells of the control strain G. oxydans_<\/em>pBBR1MCS5, the glucose conversion time was extended to 24\u00a0h, corresponding to a lower
\n glucose conversion rate of 7.96\u00a0g\/L\/h (Fig.\u00a07<\/a>b). During the second period of GA conversion to 2KGA, all GA produced was further
\n converted to 2KGA by G. oxydans_tufB_ga2dh<\/em> within 12\u201321\u00a0h (i.e. elapsed 9\u00a0h) with a GA conversion rate of 11.38\u00a0g\/L\/h, which
\n was about fivefold higher than that obtained using G. oxydans<\/em>_pBBR1MCS5. 150.0\u00a0g\/L GA produced was gradually decreased by G. oxydans<\/em>_pBBR1MCS5 cells and was fully consumed over a long period of time (24\u2013102\u00a0h, elapsed
\n 78\u00a0h), resulting in a low GA conversion rate of 1.92\u00a0g\/L\/h. It was also demonstrated
\n that overexpression of the ga2dh<\/em> gene improves the conversion of GA to 2KGA. Unexpectedly, overexpression of the ga2dh<\/em> gene significantly enhanced the conversion of glucose to GA. Overall, the final 2KGA
\n titer reached 234.6\u00a0g\/L at 21\u00a0h during the batch bioconversion of glucose by the engineered
\n strain, corresponding to a productivity of 11.17\u00a0g\/L\/h, which amounted to a 407\u00a0%
\n increase compared with that obtained using the control strain G. oxydans<\/em>_pBBR1MCS5.<\/p>\n

\"thumbnail\"Fig.\u00a07.<\/strong><\/a> Comparison of 2KGA production from glucose. The biotransformations were carried out
\n in 7-L fermenter at 30\u00a0\u00b0C, pH 5.8, 600\u00a0rpm, aeration rate 8\u00a0L\/min, initial Glu concentration
\n 200\u00a0g\/L and cell concentration 60\u00a0g
\n (wet wt)<\/sub>
\n \/L. a<\/strong>G. oxydans_tufB_ga2dh<\/em>b<\/strong>G. oxydans<\/em>_ pBBR1MCS5\n <\/p>\n

When the glucose concentration was increased to 270\u00a0g\/L, full conversion of glucose
\n was observed at 15\u00a0h by 60\u00a0g\/L resting cells of G. oxydans_tufB_ga2dh<\/em>, and the 2KGA titer reached a maximum of 318\u00a0g\/L at 48\u00a0h, giving a productivity of
\n 6.63\u00a0g\/L\/h (Fig.\u00a08<\/a>a). During the overall reaction process, we also found that DO levels in the fermenter
\n remained below 0\u00a0%. Because sufficient oxygen supply could enhance 2KGA productivity
\n during GA conversion, oxygen instead of air was supplied continuously to support the
\n oxidation of 270\u00a0g\/L glucose at the same cell mass (Fig.\u00a08<\/a>b). As expected, the reaction time under oxygen supply was significantly decreased
\n compared with that when air was used as the electron acceptor. The glucose conversion
\n rate during the first period of GA formation and the GA conversion rate in the second
\n period of GA conversion to 2KGA were increased by 400 and 268.8\u00a0%, respectively. All
\n of the supplied glucose was converted to 321\u00a0g\/L 2KGA over 18\u00a0h by the constructed
\n strain G. oxydans_tufB_ga2dh<\/em>, corresponding to a productivity of 17.83\u00a0g\/L\/h. Both the glucose concentration during
\n batch biotransformation and the 2KGA productivity in this study were relatively high
\n compared with those achieved by Pseudomonas fluorescens<\/em>5<\/a>], 19<\/a>], 20<\/a>].<\/p>\n

\"thumbnail\"Fig.\u00a08.<\/strong><\/a> Comparison of 2KGA production from glucose by G. oxydans_tufB_ga2dh.<\/em> Initial Glu concentration 270\u00a0g\/L; Cell concentration 60\u00a0g
\n (wet wt)<\/sub>
\n \/L. a<\/strong> Air b<\/strong> O
\n 2<\/sub><\/p>\n","protected":false},"excerpt":{"rendered":"

Overexpression of ga2dh in G. oxydans DSM2003 Efficient expression of ga2dh (gox1230-1232) was essential for enhancing the 2KGA production in G. oxydans DSM2003. A well-characterized promoter is a prerequisite for the overexpression of an enzyme. To achieve the optimum recombinant strains, three different promoters (G. oxydans_tufB, G. oxydans_ gHp0169 and the native promoter of the Read More<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-92233","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/92233","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/comments?post=92233"}],"version-history":[{"count":0,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/92233\/revisions"}],"wp:attachment":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/media?parent=92233"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/categories?post=92233"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/tags?post=92233"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}