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Aims: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization., Methods and Results: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l -1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l -1 , which is 5·6 times higher than before (15·6 g l -1 )., Conclusions: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH., Significance and Impact of the Study: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization., (© 2020 The Society for Applied Microbiology.)

In recent years, the wide application of exopolysaccharides (EPSs) in food, cosmetics, medicine, and other fields has drawn tremendous attention. In this study, an EPS produced by Pseudoalteromonas agarivorans Hao 2018 was isolated and purified, and its fermentation conditions were optimized. Its structure and biological functions were also studied. The purity and molecular weight of EPS were determined by high performance liquid chromatography (HPLC), and the EPS exhibited a number average of 2.26 × 105 and a weight average of 2.84 × 105. EPS has good adsorption for Cu2+ and Pb2+. The adsorption rates can reach up to 69.79% and 82.46%, respectively. The hygroscopic property of EPS was higher than that of chitosan, but slightly lower than that of sodium hyaluronate. However, the water-retaining activity of EPS was similar to that of chitosan and sodium hyaluronate. EPS has strong ability to scavenge free radicals, including OH radical and O2− radical. Further, its activity on O2− radicals has similarities with that of vitamin C. EPS has broad application prospects in many fields, such as cosmetics, environmental protection. [ABSTRACT FROM AUTHOR]

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica , where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB , VioC , and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica , and high deoxyviolacein production was found strongly associated with the weak expression of VioD . By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica . This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Bioactivities, such as freshness maintenance, whitening, and prebiotics, of marine neoagaro-oligosaccharides (NAOS) with 4-12 degrees of polymerization (DPs) have been proven. However, NAOS produced by most marine β-agarases always possess low DPs (≤6) and limited categories; thus, a strategy that can efficiently produce NAOS especially with various DPs ≥8 must be developed. In this study, 60 amino acid residues with no functional annotation result were removed from the C-terminal of agarase AgaM1, and truncated recombinant AgaM1 (trAgaM1) was found to have the ability to produce NAOS with various DPs (4-12) under certain conditions. The catalytic efficiency and stability of trAgaM1 were obviously lower than the wild type (rAgaM1), which probably endowed trAgaM1 with the ability to produce NAOS with various DPs. The optimum conditions for various NAOS production included mixing 1% agarose (w/v) with 10.26 U/ml trAgaM1 and incubating the mixture at 50°C in deionized water for 100 min. This strategy produced neoagarotetraose (NA4), neoagarohexaose (NA6), neoagarooctaose (NA8), neoagarodecaose (NA10), and neoagarododecaose (NA12) at final concentrations of 0.15, 1.53, 1.53, 3.02, and 3.02 g/L, respectively. The NAOS served as end-products of the reaction. The conditions for trAgaM1 expression in a shake flask and 5 L fermentation tank were optimized, and the yields of trAgaM1 increased by 56- and 842-fold compared with those before optimization, respectively. This study provides numerous substrate sources for production and activity tests of NAOS with high DPs and offers a foundation for large-scale production of NAOS with various DPs at a low cost., (Copyright © 2020 Qu, Wang, Wu, Chan, Di, Wang and Zeng.)

2-Hydroxyphenazine (2-OH-PHZ) is an effective biocontrol antibiotic secreted by Pseudomonas chlororaphis GP72AN and is transformed from phenazine-1-carboxylic acid (PCA). PCA is the main component of the recently registered biopesticide "Shenqinmycin". Previous research showed that 2-OH-PHZ was better in controlling wheat take-all disease than PCA; however, 2-OH-PHZ production was low under natural conditions. Herein, we confirmed that PCA induced reactive oxygen species in its host P. chlororaphis GP72AN and that the addition of DTT improved PCA production by 1.8-fold, whereas the supplementation of K 3 [Fe(CN) 6 ] and H 2 O 2 increased the conversion rate of PCA to 2-OH-PHZ. Finally, a two-stage fermentation strategy combining the addition of DTT at 12 h and H 2 O 2 at 24 h enhanced 2-OH-PHZ production. Taken together, the two-stage fermentation strategy was designed to enhance 2-OH-PHZ production for the first time, and it provided a valuable reference for the fermentation of other antibiotics.

The deep-sea bacterium strain FA13 was isolated from the sediment of the South Atlantic Ocean and identified as Bacillus circulans based on 16S ribosomal DNA sequence. Through liquid fermentation with five media, the cell-free supernatant fermented with ISP2 showed the highest inhibition activities against mycelial growth of Aspergillus parasiticus mutant strain NFRI-95 and accumulation of norsolorinic acid, a precursor for aflatoxin production. Based on ISP2, uniform design was used to optimize medium formula and fermentation conditions. After optimization, the inhibition efficacy of the 20-time diluted supernatant against A. parasiticus NFRI-95 mycelial growth and aflatoxin production was increased from 0-23.1% to 100%. Moreover, compared to the original protocol, medium cost and fermentation temperature were significantly reduced, and dependence on seawater was completely relieved, thus preventing the fermentor from corrosion. This is the first report of a deep-sea microorganism which can inhibit A. parasiticus NFRI-95 mycelial growth and aflatoxin production.

The xylose operon is an efficient biological element used for the regulation of gene expression in Bacillus licheniformis. Although the mechanism underlying the xylose-mediated regulation of this operon has been elucidated, the transcriptional changes that occur under various fermentation conditions remain unclear. In this study, the effects of different conditions on xylose operon expression were investigated. Significant upregulation was observed during the transition from the logarithmic phase to the stationary phase (2.5-fold, n = 3, p < 0.01). Glucose suppressed transcription over 168-fold (n = 3, p < 0.01). Meanwhile, the inhibitory effect of glucose hardly strengthened at concentrations from 20 to 180 g/L. Furthermore, the transcription of the xylose operon increased at elevated temperatures (25–42 °C) and was optimal at a neutral pH (pH 6.5–7.0). Based on these findings, relevant fermentation strategies (delaying the induction time, using dextrin as a carbon source, increasing the fermentation temperature, and maintaining a neutral pH) were proposed. Subsequently, these strategies were validated through the use of maltogenic amylase as a reporter protein, as an 8-fold (n = 3, p < 0.01) increase in recombinant enzyme activity compared to that under unoptimized conditions was observed. This work contributes to the development of fermentation optimization and furthers the use of the xylose operon as an efficient expression element. [ABSTRACT FROM AUTHOR]

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.

In industrial biotechnology, microbial cell factories utilize renewable resources to produce energy, materials and chemicals. Industrial biotechnology plays an increasingly important role in solving the resource, energy and environmental problems. Systems biology has shed new light on industrial biotechnology, deepening our understanding of industrial microbial cell factories and their bioprocess from "Black-box" to "White-box". Systems-wide profiling of genome, transcriptome, proteome, metabolome, and fluxome has proven valuable to better unveil network operation and regulation on the genome scale. System biology has been successfully applied to create microbial cell factories for numerous products and derive attractive industrial processes, which has constantly expedited the development of industrial biotechnology. This review focused on the recent advance and applications of omics and trans-omics in industrial biotechnology, including genomics, transcriptomics, proteomics, metabolomics, fluxomics and genome scale modeling, and so on. Furthermore, this review also discussed the potential and promise of systems biology in industrial biotechnology.

5-Aminolevulinic acid (5-ALA) is a key metabolic intermediate of the heme biosynthesis pathway, which has broad application prospects in agriculture and medicine. However, segregational instability of plasmid-based expression systems and low yield have hampered large-scale manufacture of 5-ALA. In this study, two important genes of the 5-ALA C5 biosynthesis pathway, hemA and hemL, were integrated into Escherichia coli MG1655 for chemically induced chromosomal evolution (CIChE). The highest hemA and hemL copy-number, 98 per genome, was obtained in CIChE strain MG136. The 5-ALA titer of this strain reached 2724 mg/L in optimized condition. Then, after undergoing adaptative evolution and the deletion of recA, strain MG136a ΔrecA::FRT could stably produce 4550 mg/L 5-ALA from glucose, 450 times the amount produced by hemA- hemL single copy strain MG1655-hemAL. This study constructed a plasmid-free E. coli strain for 5-ALA production, which will provide the basis for further manipulation of metabolic regulation and optimization of fermentation.

High yield recombinant protein production is highly desirable for biotechnological purposes. In the design of recombinant expression conditions, a number of essential central elements such as expression strain, type of medium, bioprocess optimization, and mathematical modeling should be considered. Well-designed industrial scale production of one recombinant protein with optimized influential parameters and yield can address the cost and production reproducibility issues. In the present study, statistical experimental design methodology was used to investigate the effect of fermentation conditions (dissolved oxygen, IPTG, and temperature) on rPDT production by Escherichia coli. rPDT is a recombinant fusion protein consisting of three different protein domains including the N-terminal 179 amino acid fragment of the S1 subunit of pertussis toxin, the full-length genetically detoxified diphtheria toxin (CRM197), and the 50 kDa tetanus toxin fragment C. A 15 Box-Behnken design augmented with center points revealed that IPTG and DO at the center point and low temperature will result in high yield. The optimal condition for rPDT production were found to be 100 µM IPTG, DO 30% and temperature 20 °C. [ABSTRACT FROM AUTHOR]

Background: 2-phenylethanol (2-PE) is an important aromatic compound with a lovely rose-like scent. Saccharomyces cerevisiae is a desirable microbe for 2-PE production but its natural yield is not high, and one or two crucial genes' over-expression in S. cerevisiae did not improve 2-PE greatly. Results: A new metabolic module was established here, in which, permease Gap1p for l-phenylalanine transportation, catalytic enzymes Aro8p, Aro10p and Adh2p in Ehrlich pathway respectively responsible for transamination, decarboxylation and reduction were assembled, besides, glutamate dehydrogenase Gdh2p was harbored for re-supplying another substrate 2-oxoglutarate, relieving product glutamate repression and regenerating cofactor NADH. Due to different promoter strengths, GAP1, ARO8, ARO9, ARO10, ADH2 and GDH2 in the new modularized YS58(G1-A8-A10-A2)-GDH strain enhanced 11.6-, 15.4-, 3.6-, 17.7-, 12.4- and 7.5-folds respectively, and crucial enzyme activities of aromatic aminotransferases and phenylpyruvate decarboxylase were 4.8- and 7-folds respectively higher than that of the control. Conclusions: Under the optimum medium and cell density, YS58(G1-A8-A10-A2)-GDH presented efficient 2-PE synthesis ability with ~ 6.3 g L−1 of 2-PE titer in 5-L fermenter reaching 95% of conversation ratio. Under fed-batch fermentation, 2-PE productivity at 24 h increased 29% than that of single-batch fermentation. Metabolic modularization with promoter strategy provides a new prospective for efficient 2-PE production. [ABSTRACT FROM AUTHOR]

FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No. 968 up to 19 mg/L in a pilot industry-scale batch fermentation. Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228. The genome of B. thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C. violaceum No. 968, and the bacterium indeed produces authentic FK228. By simple fermentation in shaking flasks in a preferred M8 medium, B. thailandensis MSMB43 produced FK228 up to 67.7 mg/L; by fed-batch fermentation in a 20-L fermentor in M8 medium, B. thailandensis MSMB43 produced FK228 up to 115.9 mg/L, which is 95 fold higher than that of C. violaceum No. 968 under the same laboratory fermentation conditions. RT-PCR analysis indicated that the high FK228 yield of B. thailandensis MSMB43 was due to high expression of biosynthetic genes, represented by Bth&#95;depA , during the fermentation process. Further genetic manipulation resulted in a recombinant strain, B. thailandensis MSMB43/pBMTL3- tdpR , which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR . This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium. Therefore, the wild-type B. thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.

As a platform chemical, acetoin has a great potential of application in medicine and food industries. In order to improve the efficiency of acetoin production, Bacillus amyloliquefaciens was treated by atmospheric and room temperature plasma and gamma rays. Two-round screening was adopted for obtaining positive mutants, and the best mutant B. amyloliquefaciens H-5 produced acetoin up to 68.2 g/L in shake flask. Then, culture conditions were optimized in 5-L fermentor to enhance acetoin production. Finally, 85.2 g/L acetoin was produced by B. amyloliquefaciens H-5, which was increased by 26.8% compared with that of the original strain B. amyloliquefaciens FMME088. These results indicated that the high-producing strain can be obtained efficiently by compound mutagenesis, which has a promising prospect for commercial scale process.

Collagenases are the most important group of commercially-produced enzymes. However, even though biological resources are abundant in the sea, very few of these commercially popular enzymes are from marine sources, especially from marine bacteria. We optimized the production of marine collagenases by Pseudoalteromonas sp. SJN2 and investigated the antioxidant activities of the hydrolysates. Media components and culture conditions associated with marine collagenase production by Pseudoalteromonas sp. SJN2 were optimized by statistical methods, namely Plackett-Burman design and response surface methodology (RSM). Furthermore, the marine collagenases produced by Pseudoalteromonas sp. SJN2 were seen to efficiently hydrolyze marine collagens extracted from fish by-products, and remarkable antioxidant capacities of the enzymatic hydrolysates were shown by DPPH radical scavenging and oxygen radical absorbance capacity (ORAC) tests. The final optimized fermentation conditions were as follows: soybean powder, 34.23 g·L -1 ; culture time, 3.72 d; and temperature, 17.32 °C. Under the optimal fermentation conditions, the experimental collagenase yield obtained was 322.58 ± 9.61 U·mL -1 , which was in agreement with the predicted yield of 306.68 U·mL -1 . Collagen from Spanish mackerel bone, seabream scale and octopus flesh also showed higher DPPH radical scavenging rates and ORAC values after hydrolysis by the collagenase. This study may have implications for the development and use of marine collagenases. Moreover, seafood waste containing beneficial collagen could be used to produce antioxidant peptides by proteolysis., Competing Interests: The authors declare no conflicts of interest.

In order to produce hyaluronate lyase of high yield, we optimized the fermentation Arthrobacter globiformis A152 in quadruple fermentation of 5 L, and studied the kinetics of fermentation. Both the highest biomass and enzyme activity could be achieved when the rotation speed was 400 r/min and the ventilation volume was 3.5 L/min. In addition, digital models of cell growth, product synthesis and substrate consumption were built by equation of logistic, luedeking-piret, product synthesis and substrate consumption. Nonlinear fitting and estimation of optimal parameters were obtained by MATLAB. The model correlated well between prediction and experimental data, and reflected the change rules of cell growth, hyaluronidase synthesis and substrate consumption during the process of producing hyaluronate lyase. The establishment of fermentation kinetics digital models could provide basis for controlling and prediction of the production process.

Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P. pastoris GS115-EG Ⅱ, was selected on G-418 resistant plates, followed by shake-flask cultivation. Enzyme characterization showed that the recombinant Cel5A reacted optimally at pH 4.5 and 60 ℃, with 50 kDa of molecular weight, preferentially degrading amorphous cellulose. Recombinant Cel5A was not significantly different from the native T. reesei Cel5A. Moreover, a shake-flask fermentation of the recombinant strain was optimized as below: incubation temperature 28 ℃, initial pH 5.0, inoculum volume 2%, methanol addition (per 24 h) 1.5% (V/V), sorbitol addition (per 24 h) 4 g/L and Tween 80 4 g/L. Under above optimized condition, the recombinant produced 24.0 U/mL of the Cel5A after 192 h fermentation. When incubated in a 5 L fermentation, Cel5A enzyme activity reached 270.9 U/mL at 180 h, with 4.16 g/L of the total protein. The study indicates that the recombinant strain P. pastoris GS115-EG Ⅱ is extremely suitable for heterologous expression of T. reesei cellulase Cel5A. And the recombinant Cel5A can be used as an alternative to the native T. reesei Cel5A in development of a commercially relevant enzyme based biorefinery process.

Background: Alkaline 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., Results: The 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#., Conclusion: The 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.

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.

Production of mycophenolic acid (MPA) by submerged fermentation using the microfungus Penicillium brevicompactum MTCC 8010 is reported here. Screening experiments were used to identify: the suitable media composition; the optimal initial pH; and the optimal incubation temperature to maximize the production of MPA in batch cultures. The initial concentrations of the selected sources of carbon (glucose), nitrogen (peptone) and the precursors (methionine, glycine) were then optimized by: (1) one-at-a-time variation of factors; and (2) a central composite design (CCD) of experiments, in a 12-day batch culture at an initial pH of 5.0, an incubation temperature of 25 °C, and an agitation speed of 200 rpm. The medium optimized using the one-at-a-time variation yielded a peak MPA titer of 1232 ± 90 mg/L. The medium optimized by the CCD method yielded a 40% higher MPA titer of 1737 ± 55 mg/L. The latter value was nearly 9-fold greater than the titer achieved prior to optimization.

Exopolysacharides (EPS) produced by Lactobacillus acidophilus play an important role in food processing with its well-recognized antioxidant activity. In this study, a L. acidophilus mutant strain with high-yielding EPS (2.92±0.05 g/L) was screened by chemical mutation (0.2 % diethyl sulfate). Plackett-Burman (PB) design and response surface methodology (RSM) were applied to optimize the EPS fermentation parameters and central composite design (CCD) was used to optimize the EPS extraction parameters. A strain with high-yielding EPS was screened. It was revealed that three parameters (Tween 80, dipotassium hydrogen phosphate and trisodium citrate) had significant influence (P < 0.05) on the EPS yield. The optimal culture conditions for EPS production were: Tween 80 0.6 mL, dipotassium hydrogen phosphate 3.6 g and trisodium citrate 4.1 g (with culture volume of 1 L). In these conditions, the maximum EPS yield was 3.96±0.08 g/L. The optimal extraction conditions analyzed by CCD were: alcohol concentration 70 %, the ratio of material to liquid (M/L ratio) 1:3.6 and the extraction time 31 h. In these conditions, the maximum EPS extraction yield was 1.48±0.23 g/L. It was confirmed by the verification experiments that the EPS yield from L. acidophilus mutant strains reached 5.12±0.73 g/L under the optimized fermentation and extraction conditions, which was 3.8 times higher than that of the control (1.05±0.06 g/L). The results indicated that the strain screening with high-yielding EPS was successful and the optimized fermentation and extraction conditions significantly enhanced EPS yield. It was efficient and industrially promising.

Metabolic engineering strives to develop microbial strains that are capable of high-titer production of a variety of industrially significant pharmaceuticals, nutraceuticals, commodity, and high-value compounds. Despite extensive success with many proof-of-concept systems there is still the need for optimization to achieve industrially relevant titers, yields, and productivities. The field of metabolic pathway optimization and balancing has formed to address this need using a scientific and systematic approach. In this chapter, we aim to outline various pathway optimization and system balancing strategies while giving insights and tips into the systems and procedures that have demonstrated recent success in the peer-reviewed literature., (© 2016 Elsevier Inc. All rights reserved.)