Coupled fermentation-bioconversion process for production of chiral α-chlorohydrin with recombinant ketoreductase

Enzymatic production of chemicals typically includes fermentation of engineered bacteria, preparation of enzymes, and bioconversion processes. Here coupled fermentation-bioconversion process for production of chiral atazanavir intermediate α-chlorohydrin was established. In the fermentation step, the ketoreductase was released from recombinant E. coli cells into medium after high-temperature induction, and the enzyme activity reached 5960 U/mL in the fermentation supernatant. In the bioconversion step, the fermentation supernatant was used directly for conversion of 200 g/L α-chloroketone in heterogeneous reaction mixture, and the residual amount of chloroketone reached less than 0.1% after 24 h of reaction. After filtration and drying, powder α-chlorohydrin was obtained with total yield of 95.7%. Finally, the filtrate of the reaction mixture was used for 2nd batch conversion, and powder α-chlorohydrin was obtained with yield of 95.6%. The above coupled process simplified enzyme preparation procedure, allowed the bioproduction of α-chlorohydrin to be carried out smoothly with reduced waste water discharge, and was advantageous for industrial scale-up.     

Continuous production of β-galactosidase in repeated batch culture of Penicillium multicolor with a membrane bioreactor

In crossflow filtration (CFF) of a culture broth of Penicillium multicolor, several types of membranes were tested with respect to permeate flux and the permeability of β-galactosidase, an extracellular enzyme. Membranes with surface pore sizes of 0.5 and 0.08 μm were selected because of the high flux and high β-galactosidase permeability. They were combined with a 3 × 10⁻² m³ fermentor as a system of repeated batch culture with crossflow filtration. With this system, β-galactosidase was continuously produced for 6 d and its productivity was about 3 times higher than that in fed-batch culture.     

Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding

Summary The effect of the ratio of methanol to ammonia, in the feeding solution the C/N ratio, on microbial PHB production was investigated. A constant C/N ratio regulated both the PHB content and the specific rate of PHB production. The results indicated that to produce the maximum PHB effectively in a short time the C/N ratio should be controlled automatically according to the increase in PHB content. Variation of the PHB content was estimated by tracing the timecourse of CO₂ concentration in exhaust gas. When the cell concentration reached 70 g/l, C/N ratio was gradually increased from the initial C/N ratio of 10 (mol methanol/mol ammonia). At 121 h, concentration of PHB reached 136 g/l, which was the maximum level so far obtained. The reaction time was considerably shortened compared with our previous study (175 h). Furthermore, PHB concentration reached 149 g/l at 170 h and total cell concentration became 233 g/l. PHB yield from methanol was 0.20 (g PHB/g methanol), which was also superior to the previous result of 0.18.     

A whole-cell process for the production of ε-caprolactone in aqueous media

ε-Caprolactone is an industrially important intermediate produced in multi-10,000 ton scale annually with broad applications. We report on a whole-cell biocatalytic conversion of cyclohexanol to ε-caprolactone using the combination of alcohol dehydrogenase (ADH) with two stability-improved variants (QM and M15) of the Baeyer-Villiger monooxygenase CHMO with a special focus on process development at the 200 mM scale. Influence of parameters such as volumetric mass transfer co-efficient, stirrer speed and catalytic loading (amount of E. coli whole-cells expressing ADH and CHMO) on the process efficiency were studied and optimised. This resulted in over 98% conversion, a product titer of 20 g L^–1 and an isolated product amount of 9.1 g (80%). This corresponds to a space-time yield of 1.1 g L^–1 h⁻¹ and a reaction yield (mole of product per mole substrate) of 0.9. Comparing the two CHMO variants a significant difference in catalytic yield (weight of product to weight of catalyst; 0.6 vs 0.3) was observed without any inherent changes in the process. Hence, the reported process can accommodate in the future improved variants of the CHMO.     

Feeding strategies for the enhanced production of α-arbutin in the fed-batch fermentation of Xanthomonas maltophilia BT-112

To develop a cost-effective method for the enhanced production of α-arbutin using Xanthomonas maltophilia BT-112 as a biocatalyst, different fed-batch strategies such as constant feed rate fed-batch, constant hydroquinone (HQ) concentration fed-batch, exponential fed-batch and DO-control pulse fed-batch (DPFB) on α-arbutin production were investigated. The research results indicated that DPFB was an effective method for α-arbutin production. When fermentation with DO-control pulse feeding strategy to feed HQ and yeast extract was applied, the maximum concentrations of α-arbutin and cell dry weight were 61.7 and 4.21 g/L, respectively. The α-arbutin production was 394 % higher than that of the control (batch culture) and the molar conversion yield of α-arbutin reached 94.5 % based on the amount of HQ supplied (240 mM). Therefore, the results in this work provide an efficient and easily controlled method for industrial-scale production of α-arbutin.     

Process parameters study of α-amylase production in a packed-bed bioreactor under solid-state fermentation with possibility of temperature monitoring

Production of α-amylase in a laboratory-scale packed-bed bioreactor by Bacillus sp. KR-8104 under solid-state fermentation (SSF) with possibility of temperature control and monitoring was studied using wheat bran (WB) as a solid substrate. The simultaneous effects of aeration rate, initial substrate moisture, and incubation temperature on α-amylase production were evaluated using response surface methodology (RSM) based on a Box-Behnken design. The optimum conditions for attaining the maximum production of α-amylase were 37°C, 72% (w/w) initial substrate moisture, and 0.15 L/min aeration. The average enzyme activity obtained under the optimized conditions was 473.8 U/g dry fermented substrate. In addition, it was observed that the production of enzyme decreased from the bottom of the bioreactor to the top.     

A novel process of cyclodextrin production by the use of specific adsorbents: Part II. A new reactor system for selective production of α-cyclodextrin with specific adsorbent

We have developed a novel process of α-cyclodextrin (α-CD) production by using a new adsorbent that is characterized by its exceedingly powerful selectivity for α-CD compared with other CDs. α-CD production was carried out in a closed reactor system that was composed of a main reactor, wherein liquefied starch was converted to CDs by cyclodextrin glucosyltransferase (CGTase: EC 2.4.1.19), and a column packed with the adsorbent. While the reaction mixture was circulated in the system, α-CD was selectively adsorbed in the column and its concentration in the mixture of the main reactor was kept at a low level. This low concentration of α-CD stimulated the conversion of starch to CDs and as a result, enhanced its yield based on added starch. When 8.3 % (w/v) of liquefied starch was used in the reactor system, the yield of α-CD was 22.2% and α-CD occupied 58.7 % of the reaction mixture of total CDs synthesized. Meanwhile, in a batch system without the adsorbent, the yield of α-CD and its fraction were 10.8% and 45.0%, respectively. After the conversion reaction, and following the preliminary washing with water through the column. α-CD was easily eluted with hot water, resulting in a high purity of about 95%.     

Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor

Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40?U/ml. Then, a high-temperature batch culture of the thermophile in a 15?l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600?rpm and 15?l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79?U/ml and productivity of 19804?U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.     

Is increased male flower production a strategy for avoidance of predispersal seed predation in andromonoecious plants?

Floral gender in angiosperms often varies within and among populations. We conducted a field survey to test how predispersal seed predation affects sex allocation in an andromonoecious alpine herb Peucedanum multivittatum. We compared plant size, male and perfect flower production, fruit set, and seed predation rate over three years among nine populations inhabiting diverse snowmelt conditions in alpine meadows. Flowering period of individual populations varied from mid‐July to late August reflecting the snowmelt time. Although perfect flower and fruit productions increased with plant size, size dependency of male flower production was less clear. The number of male flowers was larger in the early‐flowering populations, while the number of perfect flowers increased in the late‐flowering populations. Thus, male‐biased sex allocation was common in the early‐flowering populations. Fruit‐set rates varied among populations and between years, irrespective of flowering period. Fruit‐set success of individual plants increased with perfect flower number, but independent of male flower number. Seed predation by lepidopteran larvae was intense in the early‐flowering populations, whereas predation damage was absent in the late‐flowering populations, reflecting the extent of phenological matching between flowering time of host plants and oviposition period of predator moths. Seed predation rate was independent of male and perfect flower numbers of individual plants. Thus, seed predation is a stochastic event in each population. There was a clear correlation between the proportion of male flowers and the intensity of seed predation among populations. These results suggest that male‐biased sex allocation could be a strategy to reduce seed predation damage but maintain the effort as a pollen donor under intensive seed predation.     

Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

A perfusion-control strategy based on cellular consumption rates of oxygen and glucose was established for the production of single-chain urokinase-type plasminogen activator (scu-PA). Employing this strategy, the influences of microcarrier types and the culture media on culture performances were evaluated. In the control perfusion culture, which used a solid microcarrier and a 1% fetal bovine serum (FBS) medium, viable cell density reached 3.1?×?10⁷?cells?ml^?1. However, formation of large, heterogeneous aggregates (500–1,000?μm) resulted in a gradual decrease in viable cell density to less than 1.0?×?10⁷?cells?ml^?1. Accordingly, declines in the production of urokinase-type plasminogen activator (u-PA) and in the scu-PA portion of u-PA were observed. In the serum-free media, cell growth and u-PA production were suppressed 2–3?times, but were significantly enhanced when a porous microcarrier, Cultispheer G, was used. The cell-growth profile showed a continuous increase in cell density, reaching 5.1?×?10⁷?cells?ml^?1, and the production of u-PA remained stable throughout the culture (1586?±?247?IU?ml^?1). The values of all the parameters associated with cell growth and u-PA production were fairly comparable to or even higher than those in the control culture. Moreover, a 13% higher scu-PA portion of u-PA was observed in the serum-free culture, regardless of the microcarrier type, compared with scu-PA portion of u-PA in the control culture.     

Kinetics of α-amylase production in a batch and fed-batch culture of Bacillus subtilis with caseinate as nitrogen source and starch as carbon source

Summary Analysis of a large number of experimental data from the cultivation of Bacillus subtilis formed the basis for a kinetic model of the process explaining the effect of composition of the culture medium and of the growth rate on the rate of enzyme production. The resulting rate of formation of -amylase (EC 3.2.1.1) reflects the sum of the rate of enzyme production and the rate of its degradation as affected by the environment. The kinetic dependence confirms the previously described mechanism of regulation of enzyme biosynthesis. The mathematical model of the process served here to determine the optimal conditions for enzyme biosynthesis which were then verified in a fed-batch cultivation. The production of the enzyme in fed-batch culture was found to be twice that found in a batch cultivation.Symbols X biomass concentration, g·l^-1 - t time, h - S ₁ caseinate concentration, g·l^-1 - S ₂ starch concentration, g·l^-1 - P product concentration, U·ml^-1 - r _P specific rate of product formation, U·g^-1·h^-1 - R _P total rate of product formation, U·l^-1·h^-1 - Y yield coefficient - specific growth rate, h^-1     

Factors affecting fruit and seed production of Paeonia ostii “Feng Dan”, an economically important oil tree

Many factors may affect reproduction of animal-pollinated species. In this study, the effects of pollen limitation, attractive traits (flower number, plant height and flower width) and flowering phenological traits (flowering onset, duration and synchrony) on female reproduction, as well as the patterns of variation in fruit and seed production within plants, were investigated in Paeonia ostii “Feng Dan” over two flowering seasons (2018 and 2019). Fruit set was very high (90%), and pollen supplementation did not increase fruit and seed production in either year, indicating no pollen limitation. Fruit set, ovule number per fruit and mean individual seed weight per fruit were not affected by any of the six attractive and phenological traits in either year, whereas seed number per fruit was related to the three attractive traits in one or both years. Seed number per plant was positively affected by the three attractive traits and best explained by flower number in both years, but the effect of each of the three phenological traits on seed number per plant differed between years. Within plants, the fruit set, ovule number, seed set and seed number per fruit declined from early- to late-opening flowers, presumably because of resource preemption, but the mean individual seed weight did not vary across the flowering sequence. Our study shows that attractive traits of Paeonia ostii “Feng Dan” are more important than flowering phenological traits in the prediction of total seed production per plant.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.     

The “epimerring” highlights the potential of carbohydrate epimerases for rare sugar production

Rare sugars can find applications in various industrial sectors and, therefore, hold significant economic value. Due to their low natural abundance, efficient production processes are needed to enable their commercial exploitation. About a decade ago, the available biosynthetic routes were summarized in the so-called “Izumoring”, which mainly comprised reactions catalysed by keto-aldol isomerases and oxidoreductases. Although just a single epimerase specificity (acting on the 3-position of ketoses) was included, these enzymes hold the potential to truly revolutionize the field as they offer shortcuts in conversion processes. For example, C2-epimerases could replace double isomerization reactions, whereas C4/5-epimerases could form a new bridge between d- and l-sugars as alternative to the current two-step oxidoreduction reaction. Here, we present the “Epimerring” to highlight the potential of new epimerases that can still be discovered and/or engineered, which may open doors to new and improved synthesis routes for rare sugars. Several efforts and options in the search of such biocatalysts are shortly summarized.     

The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Recovery of plants from “Near-Lethal” stress

This study reports on the dieback and recovery of red-osier dogwood, Cornus sericea L. plants from near-lethal (NL, sublethal) stress after varying lengths of post-stress environment (PSE). Intact dormant stems were subjected to 47° C for one hour during either October, November or December, and then placed into either constant 0° C or 23° C (dark condition) or kept under natural conditions at Corvallis, OR. Plants exposed to NL-heat stress in October died prior to 9 weeks of 0° C PSE, while none of the plants from other PSE treatments showed signs of injury. For plants exposed to NL-heat stress during November and December, stemdieback occurred at 0° C after 12 and 15 weeks, respectively. None of the plants from the other PSE treatments were injured. Post-stress temperatures of 0° or 5° C following NL-heat in October were lethal while temperatures above 10° C allowed recovery. Post-stress exposure to 0° C injured excised stems within 48 h, whereas irreversible damage to whole plants occurred by two weeks. Dormant plants exposed in October to other stresses, e.g., freezing temperature and hydrogen cyanamide, at NL dosages showed that these stresses also caused plant dieback at 0° C and little or no dieback at 23° C PSE.Abbreviations NL Near-Lethal - PSE Post-Stress Environment     

Induction of cocklebur seed germination by anaerobiosis: A question about the “inhibitor hypothesis” of seed dormancy

Summary Germination of non-dormant small cocklebur (Xanthium pennsylvanicum Wallr.) seeds was improved by immersing them in water, suggesting that during their germination endogenous germination inhibitors are leached out. However, the same effect could be obtained by the quasi-anaerobic pre-incubation of the seeds. When seeds were fully imbibed, moreover, water immersion could no longer potentiate them to germinate, and only anaerobiosis increased the germination potential, thus raising a question against the inhibitor hypothesis of seed dormancy.