Citric acid (CA), an important platform-compound, has attracted much attention because of its broad applications and huge market demand. To solve high residual sugar at the fermentation end, we put forwarded strategy of pre-saccharification and then fermentation. Results showed that the residual total sugar decreased by 10.4% and the productivity increased by 4.0% and initially high glucose inhibited cell growth. Furthermore, commercial glucoamylase with high low-pH stability was proposed to staged-add in the fermentation process, which timely compensated enzyme loss, ensuring the glucose supply rate. The fermentation productivity was evidently enhanced by 13.3% with residual total sugar decreasing by 31.3%, simplifying the subsequent product separation and extraction process. Our results confirmed that staged-addition glucoamylase strategy was feasible to effective production of CA.

•The thermostable isoamylase acts on terminal α-1,6 bonds.•The enzyme debranched specifically and efficiently.•Amylase and isoamylase were simultaneously used for resistant starch production.Debranching enzymes contribute to the enzymatic production of resistant starch (RS) by reducing substrate molecular weight and increasing amylose yield. In the present study, the action pattern of a thermostable isoamylase-type debranching enzyme on different types of starch was investigated. The molecular weight distribution, glycosidic bond composition and contents of oligosaccharides released were monitored by various liquid chromatography techniques and nuclear magnetic resonance spectroscopy (NMR). These analyses showed that the isoamylase could specifically and efficiently attack α-1,6-glucosidic linkages at branch points, leaving the amylose favored by other amylolytic enzymes. Its ability to attack side chains composed of 1–3 glucose residues differentiates it from other isoamylases, a property which is also ideal for the RS preparation process. The enzyme was used as an auxiliary enzyme in the hydrolytic stage. The highest RS yield (53.8%) was achieved under the optimized conditions of 70 °C and pH 5.0, using 7 U isoamylase per g starch and 2 NU amylase per g starch. These data also help us better understand the application of isoamylase for preparation of other products from highly branched starch materials.Download high-res image (154KB)Download full-size image

In this study, we constructed an l-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metAFbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMAFbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.

Citric acid (CA) as an extremely important platform compound has attracted intense attention due to wide applications and huge markets. Here, we proposed a novel method, using pellet inoculation to replace spores, and constructed the seed recycling cultivation process, effectively avoided the longtime (spore preparation 30 days) of seed culture (including spores germination 12 h) in the traditional batch-fermentation. On this basis, using pellet-dispersion strategy, the bottleneck caused by the mycelium structure was overcome, with the seed restoring high cell-viability with CA titer (11.0 g/L) even in the eighth batch compared to that in the control (4.6 g/L). The optimum morphology of these recycling cultured seeds for CA production was dispersed pattern rather than pellets. And the CA production was 130.5 g/L on average in 5 L five-conjoined-fermenters recycling eight batches, especially increasing 3.1 g/L compared with the control. To our knowledge, this is the first that reported the application of these strategies in effective production of CA. Our fermentation strategies not only significantly enhanced CA productivity, but also severed as a promising stepping-stone for other fermentations dominated with the filamentous fungi.

The aproteinogenic amino acid, L-phenylglycine, is an important side chain building block for some drugs. It would be of great commercial and environmental value to biocatalyse L-phenylalanine to L-phenylglycine, and thus replace the organic synthesis method. To produce L-phenylglycine from L-phenylalanine, an L-phenylglycine aminotransferase was screened and characterized. HpgTAO showed high homology to α-aminoadipate aminotransferase. The L-phenylalanine binding site was near the residues S26, R401, N201, and G46 in HpgTAO, and L-phenylalanine formed a hydrogen bond with Asn20, which was similar to the substrate binding mechanism of α-aminoadipate aminotransferase. HpgTAO showed increased activity in alkalescent environment below 40°C. The kinetic analysis showed that L-phenylalanine had the highest affinity to HpgTAO, which ensured the recycle biosynthesis of Lphenylglycine from L-phenylalanine. To date, it was the only aminotransferase using L-phenylalanine as an optimal amino donor. The L-phenylglycine biocatalysis operon was also constructed by co-expressing the hmaS, hmo and hpgT by a single plasmid. The first in vitro conversion of L-phenylalanine to L-phenylglycine was achieved by directly using the L-phenylalanine fermentation broth as the raw material.

Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages in glycogen, amylopectin and α/β-limit dextrins. A semi-rational design strategy was performed to improve catalytic properties of isoamylase from Bacillus lentus. Three residues in vicinity of the essential residues, Arg505, Asn513, and Gly608, were chosen as the mutation sites and were substituted by Ala, Pro, Glu, and Lys, respectively. Thermal stability of the mutant R505P and acidic stability of the mutant R505E were enhanced. The kcat/Km values of the mutant G608V have been promoted by 49 %, and the specific activity increased by 33 %. This work provides an effective strategy for improving the catalytic activity and stability of isoamylase, and the results obtained here may be useful for the improvement of catalytic properties of other α/β barrel enzymes.

β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADCC.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. The purified ADCC.g was optimal at 55 °C and pH 6 with excellent stability at 16–37 °C and pH 4–7. A pH–stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6–7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g l-aspartate/l and another three feedings of 0.5 % (w/v) l-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the Km and Vmax on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.

The pulA1 gene, encoding a novel thermostable type I pullulanase PulA1 from Bacillus sp. CICIM 263, was identified from genomic DNA. The open reading frame of the pulA1 gene was 2655 base pairs long and encoded a polypeptide (PulA1) of 885 amino acids with a calculated molecular mass of 100,887 Da. The pulA1 gene was expressed in Escherichia coli and Bacillus subtilis. Recombinant PuLA1 showed optimal activity at pH 6.5 and 70 °C. The enzyme demonstrated moderate thermostability as PuLA1 maintained more than 88% of its acitivity when incubated at 70 °C for 1 h. The enzyme could completely hydrolyze pullulan to maltotriose, and hydrolytic activity was also detected with glycogen, starch and amylopection, but not with amylose, which is consistent with the property of type I pullulanase. PulA1 may be suitable for industrial applications to improve the yields of fermentable sugars for bioethanol production.

•Expression system based on inducible Bacillus promoters was constructed.•The expression of TreS was strictly controlled.•Conditions for the TreS production was optimized.Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12.1 U/mL). Furthermore, the effect of different cultural conditions on the inducible expression of BlsTs was investigated, and the optimal condition was as follows: 4% maltodextrin, 0.4% soybean powder, 1% xylose added after 10 h of growth and an induction time of 12 h at 37 °C. As a result, the maximal yield reached 24.7 U/mL. This study contributes to the industrial application of B. licheniformis, a GRAS workhorse for enzyme production.

•Sequence features for mandelate oxidase (MO) were summarized, and used in the screening of MO.•By using sequence-based screening, the HmoSC and HmoAO were screened.•The MO activities of HmoSC and HmoAO were identified, and characterized.•The HmoSC showed a better characterization than HmoAO.Sequence-based screening was carried out to find a type of cytosolic mandelate oxidase that converted l-mandelate to phenylglyoxylate using oxygen as the final electron acceptor. The sequence features of the cytosolic mandelate oxidase were summarized, and were used in the screening process. Mandelate oxidases from Streptomyces coelicolor (HmoSC) and Amycolatopsis orientalis (HmoAO) were screened and then they were heterologously expressed and characterized. At pH 7.3 40 °C, the HmoAO showed kcat and Km values of 140 min−1 and 10.2 mM, the HmoSC showed kcat and Km values of 105.1 min−1 and 2.06 mM. The HmoSC was thermal stable and retained its 90% activity at 60 °C for up to 5 h, while HmoAO lost most of its activity at this temperature. The HmoSC could effectively catalyze the conversion of l-mandelate to phenylglyoxylate at higher temperature using oxygen as the final electron acceptor.

•The bottleneck of the homoserine biosynthetic pathway was overcome by overexpressing the metL gene in Escherichia coli..•By modifying the exporter and transporter systems of homoserine, the growth rate and productivity were significantly improved.•A high-homoserine-producing strain was constructed from wild type E. coli W3110 using metabolic engineering strategies.L-homoserine as a nonessential amino acid, can be used as a feed additive due to its significant value as well as in many other applications in agriculture. In this study, Escherichia coli W3110 was engineered for the production of L-homoserine from glucose. The lysA, metA, and thrBC genes were inactivated to block the competing and degradation pathways of L-homoserine. To enhance the carbon flux to homoserine, the effects of the lysC, thrA, and metL genes on L-homoserine production and the impact of L-homoserine on the aspartokinases (AKs) AKI, AKII, and AKIII were studied. The results demonstrated that, the bottleneck of the homoserine biosynthetic pathway was overcome and carbon flux to homoserine was enhanced by overexpressing the metL gene in E. coli W3110. The rhtA gene was also overexpressed under the control of the pN25 promoter to improve the survivability under the stress of high concentration as well as the productivity of L-homoserine. Moreover, we verified that the inactivation of the TdcC transporter further increased L-homoserine production. A high-l-homoserine-producing strain was constructed from wild type E. coli W3110 using metabolic engineering strategies. This improved the production of L-homoserine from 0 to 39.54 g/L in fed-batch fermentation.Download full-size image

•l-Phenylglycine (l-Phg) was firstly produced from glucose by fermentation.•Three enzymes from actinomyces were expressed in E. coli and purified.•New cycle multi-enzyme cascade reactions were developed for l-Phg production.•Optimizing gene expression and transamination increased l-Phg yield by 224-fold.The aproteinogenic amino acid l-phenylglycine (l-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis l-Phg from glucose, an engineered Escherichia coli containing l-Phg synthetic genes was firstly developed by an l-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (l-4-hydroxymandelate synthase), Hmo (l-4-hydroxymandelate oxidase) and HpgT (l-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on l-Phg formation. HpgT conversing phenylglyoxylate to l-Phg uses an unusual amino donor l-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of l-Phg, the effects of attenuating l-phenylalanine transamination was investigated. After deletion of tyrB and aspC, l-Phg yield increased by 12.6-fold. The limiting step in the l-Phg biosynthesis was also studied; the l-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of l-phenylalanine transamination, the l-Phg yield was increased by 224-fold comparing with the original strain.Download high-res image (79KB)Download full-size image