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Whole-cell systems offer many benefits for biochemical production, such as relatively easy enzyme control and higher tolerance toward harsh environments, than purified enzymes. These systems can be applied to many bioconversion reactions, but they sometimes require cofactor regeneration units to support reactions at high substrate concentrations. Here, we examined l-glutamate oxidase (GOX) from Streptomyces sp. X119-6, which produces α-ketoglutarate (α-KG) from l-glutamate, and catalase (KatE) from Escherichia coli, which removes hydrogen peroxide generated by GOX. After optimizing the expression vector, pH, strains, culture conditions, and isopropyl β-d-1-thiogalactopyranoside concentration, we compared their efficiency to that of a previously reported GOX from Streptomyces mobaraensis. Our results indicated that GOX from Streptomyces sp. X119-6 and KatE increased α-KG production by 2.76-fold. This GOX required high levels of α-KG as an amino donor to convert 5-aminovaleric acid to glutaric acid. Performing the reaction at pH 8 enabled us to avoid the exogenous addition of catalase, but severe substrate inhibition was observed, resulting in the production of 287 mM glutaric acid. This α-KG regeneration system has potential for improving production in various aminotransferase systems.

In this study, the conversions of 1-dodecanoic, ω-hydroxydodecanoic acid and α,ω-dodecanedioic acid using whole cell biotransformation of Escherichia coli BW25113ΔfadD expressing CAR and ADH enzymes were demonstrated. First 13 CAR enzymes were examined for 1-dodecanoic acid reduction, and CAR encoded by mab4714 from Mycobacterium abscessus showed the highest conversion of 53.1% in single cells of heterologous CAR and endogenous ADH. For a better conversion, the host cells were engineered to simultaneously express Yarrowia lipolytica ADH2 with the GroES/EL-DnaK/J/E chaperone in a single host system. In addition, two-cell system using two strains of E. coli expressing CAR-Sfp and ADH-GroES/EL-DnaK/J/E was also investigated. In results, additional ADH expression was not effective in a single host system, whereas two cell system significantly increased α,ω-dodecanedioic acid conversion by total 71.3%; α,ω-dodecanediol (68.2%) and ω-hydroxydodecanoic acid (3.1%), respectively. Interestingly, the MAB4714 CAR enzyme could converted ω-hydroxydodecanoic acid into α,ω-dodecanediol up to 97.2% conversion in 17 h (12.4 mg/L/h). Finally, structural understanding of the higher activity against ω-hydroxydodecanoic was understood by docking simulations which suggested hydrogen-bonding interactions between ω-hydroxyl group and polar residues such as Gln434 and Thr285 were holding the substrate tightly with more stable positioning in the active site.

This paper reviews the recent advances in the production of industrially valuable biopolymer building blocks, such as ω-hydroxy fatty acids, ω-amino fatty acids, diamines, and dicarboxylic acids, using monooxygenase enzymes of cytochrome P450 monooxygenase (CYP) or Baeyer–Villiger monooxygenase (BVMO). In particular, we highlight the use of CYP and BVMO in the oxidation of fatty acids derived from vegetable oils. Dodecanoic acid (lauric acid), (9Z)-octadec-9-enoic acid (oleic acid), (9Z,12Z)-octadeca-9,12-dienoic acid (linoleic acid), and 12-hydroxy-9-cis-octadecenoic acid (ricinoleic acid) are the most abundant saturated or monounsaturated fatty acids found in vegetable oils, such as coconut, palm, sunflower, and caster oils, respectively. The introduction of hydroxyl functional groups by CYP enzymes and ester structures by BVMO enzymes increases the hydrophilicity of the fatty acid. The subsequent conversion into other meaningful functional groups, such as keto, amino, and acidic groups, can increase the potential of the fatty acid to serve as biopolymer building blocks. Thus, the monooxygenase enzyme reaction has a relatively high potential and can contribute to the production of various high value-added and platform biochemicals, including biopolymers, biolubricants, and biosurfactants. In this review article, we emphasize the versatility of the CYP and BVMO enzymes and present several examples of their biochemical applications.

Heart failure (HF) is a coronary disease that affects people worldwide and has a high mortality rate. N-terminal pro-brain natriuretic peptide (NT-proBNP) has been proven to be a useful and accurate biomarker for diagnosing systolic HF. Here, we report a strategy for the high-level production of recombinant (r)NT-proBNP in Escherichia coli. An Fh8 tag with six histidines was fused to the N terminus of NT-proBNP along with the recognition site of tobacco etch virus (TEV) protease; the 6HFh8-NT-proBNP fusion peptide was expressed in flask cultures of E. coli in almost completely soluble form. The peptide was purified by HisTrap affinity chromatography, and the N-terminal tag was cleaved by TEV protease. After a second round of HisTrap affinity chromatography to remove the TEV protease and N-terminal tag, rNT-proBNP was isolated with high purity (≥ 98%) by carboxymethyl cation exchange chromatography. The final yield of purified rNT-proBNP (97.5 mg/l of bacterial culture; 3.25 mg/g of wet cell) was 55-fold higher than that reported in previous studies (0.5–1.75 mg/l of bacterial culture). Furthermore, the high cell density E. coli fed-batch culture enabled high-level production of rNT-proBNP in the order of grams per liter. The purified rNT-proBNP was detected by enzyme-linked immunosorbent assay and chemiluminescence enzyme immunoassay using commercial monoclonal antibodies recognizing different epitopes, showing a linear dose-response relationship in the range of tested concentrations (slope = 3.58 and r2 = 0.995). These results demonstrate the efficiency of our process for mass producing (gram-to-liter level) rNT-proBNP with acceptable analytical performance.

Sustainable manufacture of dicarboxylic acids (DCAs), which are used as raw materials for multiple commercial products, has been an area of considerable research interest in recent years. Traditional chemical-based manufacture of DCAs suffers from limitations such as harsh operational conditions and generation of hazardous by-products. Microbiological methods involving DCA production depend on the capability of alkane-assimilating microorganisms, particularly α, ω-oxidation, to metabolize alkanes. Alkanes are still used as the most common substrates for this method, but the use of renewable resources, such as vegetable oil-derived fatty acid methyl esters (FAMEs), offers multiple advantages for the sustainable production of DCA. However, DCA production using FAME, unlike that using alkanes, still has low productivity and process stability, and we have attempted to identify several limiting factors that weaken the competitiveness. This review discusses the current status and suggests solutions to various obstacles to improve the biotransformation process of FAMEs.

Various chemicals, i.e., furfural, vanillin, 4-hydroxybenzaldehyde and acetate produced during the pretreatment of biomass affect microbial fermentation. In this study, effect of vanillin, 4-hydroxybenzaldehyde and acetate on antibiotic production in Streptomyces coelicolor is investigated. IC 50 value of vanillin, 4-hydroxybenzaldehyde and acetate was recorded as 5, 11.3 and 115 mM, respectively. Vanillin was found as a very effective molecule, and it completely abolished antibiotic (undecylprodigiosin and actinorhodin) production at 1 mM concentration, while 4-hydroxybenzaldehyde and acetate have little effect. Microscopic analysis with field emission scanning electron microscopy (FESEM) showed that addition of vanillin inhibits mycelia formation and increases differentiation of S. coelicolor cells. Vanillin increases expression of genes responsible for sporulation (ssgA) and decreases expression of antibiotic transcriptional regulator (redD and actII-orf4), while it has no effect on genes related to the mycelia formation (bldA and bldN) and quorum sensing (scbA and scbR). Vanillin does not affect the glycolysis process, but may affect acetate and pyruvate accumulation which leads to increase in fatty acid accumulation. The production of antibiotics using biomass hydrolysates can be quite complex due to the presence of exogenous chemicals such as furfural and vanillin, and needs further detailed study. Electronic supplementary material The online version of this article (doi:10.1007/s13205-016-0539-y) contains supplementary material, which is available to authorized users.

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.