Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Production of nisin with continuous adsorption to Amberlite XAD-4 resin using<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> N8 and<Emphasis Type="Italic"> L. lactis</Emphasis> LAC48

The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48. Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5×10⁶ IU g dcw^–1. The nisin concentration remained high (2.0×10⁶ IU g dcw^–1) after the logarithmic growth phase (10–22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase. The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth. In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation. The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin. Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

Effects of pH profiles on nisin fermentation coupling with foam separation

Online foam separation was proposed to recover nisin during fermentation of Lactococcus lactis subsp. lactis ATCC 11454. Firstly, the optimal pH profile of nisin fermentation was investigated including different realkalization set values and pH drop gradients. Then the selected pH profiles of 5.75 ± 0.05 and 6.25–5.75 (±0.02) were used to perform nisin fermentation coupling with foam separation. The results showed that pH profile of 5.75 ± 0.05 was better than that of 6.25–5.75 (±0.02) for online foam separation. With the optimal pH profile, an aeration of 20 ml min⁻¹ that started at 8 h of incubation and lasted for 2 h resulted in 6.6 times higher specific productivity than that of the fermentation without aeration. Nisin synthesis was therefore prolonged with low sucrose concentration in the culture broth, which indicated that the feedback inhibition of nisin is more influential than the substrate limitation of sucrose in the late phase of nisin fermentation. Total nisin production (4,870 ± 180 IU ml⁻¹) was increased by 30.3% with online foam separation. This effective online recovery method for nisin production could be easily scaled up due to the facile operation of foaming process.     

Cycle changing the medium results in increased nisin productivity per cell in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

High specific cellular nisin production was aimed by cycle changing the medium of Lactococcus lactis N8 and LAC48. The highest level of nisin production was reached with the 120 min cycles but maximal production was unstable. In shorter cycles (30 and 60 min) cells could be maintained in a high production state up to the end of the fermentation (28 and 14 cycles). N8 produced 19-fold and LAC48 15-fold more nisin with cycle changing the medium than without cycle incubation.     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 g_CDW L^?1) resulting in a recombinant protein titer of about 6.5 g L^?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used ¹³C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h^?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g^?1 h^?1 compared to 0.7 mmol g^?1 h^?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc.     

Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

Nisin‐induced expression of pediocin in dairy lactic acid bacteria

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml^?1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega‐3 fatty acids

Schizochytrium mangrovei strain PQ 6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω‐3, DHA ) and squalene using a 30‐L bioreactor with a working volume of 15 L under various batch and fed‐batch fermentation process regimes. The fed‐batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L^?1, 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g^?1 · L^?1, respectively, after a 96 h fed‐batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g^?1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial‐scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium .     

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l^–1 h^–1, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml^–1, by constant addition of sucrose at a feeding rate of 7 g l^–1 h^–1; an increase in 58% over that of the batch culture (2,658 IU ml^–1). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Influence of growth conditions on the nisin production of bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

Metabolic engineering of Escherichia coli for agmatine production

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ?speC ?speF ?speB ?argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223‐3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed‐batch fermentations of the AUX5 strain were conducted in a 3‐L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13 g agmatine L^?1 with the yield of 0.11 g agmatine g^?1 glucose in the batch fermentation and the fed‐batch fermentation of AUX5 allowed the production of 15.32 g agmatine L^?1 with the productivity of 0.48 g agmatine L^?1 h^?1, demonstrating the potential of E. coli as an industrial producer of agmatine.     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

Lactate removal by anionic-exchange resin improves nisin production by Lactococcus lactis

When lactate was removed from sucrose fermentation in situ, using the anionic-exchange resin Amberlite IRA-67, by Lactococcus lactis growing in batch culture, nisin production increased by two-fold when compared to the alkali pH-controlled fermentation. In comparison to sucrose, lactate removal increased nisin production 1.5-fold and 0.3-fold when galactose and glucose were used as carbon sources respectively.     

Acetate formation during recombinant protein production in Escherichia coli K‐12 with an elevated NAD(H) pool

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild‐type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady‐state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h^?1. Batch and fed‐batch processes using MEC697 were examined for the production of β‐galactosidase as a model recombinant protein. Fed‐batch culture of MEC697/pTrc99A‐lacZ compared to MG1655/pTrc99A‐lacZ at a growth rate of 0.22 h^?1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A‐lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A‐lacZ and a two‐fold increase in recombinant protein production.