Sang-Hwal Yoon

An update on microbial carotenoid production: application of recent metabolic engineering tools

Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity.

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for beta-carotene production in recombinant Escherichia coli harboring beta-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of beta-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and beta-carotene synthesis genes produced high amount of beta-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains - MG1655, DH5alpha, S17-1, XL1-Blue and BL21 - the DH5alpha was found to be the best beta-carotene producer. Using glycerol as the carbon source for beta-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5alpha harboring the whole MVA pathway and beta-carotene synthesis genes produced beta-carotene of 465mg/L at glycerol concentration of 2% (w/v).

Metabolic engineering of Escherichia coli for α-farnesene production.

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.

Increased β-Carotene Production in Recombinant Escherichia coli Harboring an Engineered Isoprenoid Precursor Pathway with Mevalonate Addition

When pT‐LYCm4 containing lycopene synthetic genes was co‐transformed with pSUcrtY or pSHcrtY containing crtY gene of Pantoea ananatis (P. ananatis) or Pantoea agglomerans (P.agglomerans), β‐carotene productions of 36 and 35 mg/L were obtained, respectively. No lycopene was detected in the β‐carotene production culture. pT‐HB, constructed by addition of P. ananatis crtY gene into pT‐LYCm4, was used for co‐transformation with pSdxs and pSSN12Didi, which increased isopentenyl diphosphate and dimethylallyl diphosphate synthesis. β‐Carotene production significantly increased 1.5‐fold (51 mg/L) with the amplification of the dxs gene through pSdxs and 4‐fold (135 mg/L) with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT‐DHB, constructed by integrating the dxs gene into pT‐HB, was used for cotransformation of Escherichia coli (E. coli) harboring pSSN12Didi, resulting in β‐carotene production of 141 mg/L. Recombinant E. coli harboring pT‐DHB and pSSN12Didi was used to maximize β‐carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, β‐carotene production of 503 mg/L in concentration and 49.3 mg/g DCW in content was obtained at 144 h, which was the highest level of carotenoid production in E. coli ever reported in the literature.

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two‐phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway‐only control. Biotechnol. Bioeng. 2010;107: 421–429.

Directing Vanillin Production From Ferulic Acid by Increased Acetyl-CoA Consumption in Recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl‐CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5α (pTAHEF‐gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5α (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl‐CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD‐2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli. Biotechnol. Bioeng. 2009;102: 200–208.

Engineering Escherichia coli for selective geraniol production with minimized endogenous dehydrogenation.

Geraniol, a monoterpene alcohol, has versatile applications in the fragrance industry, pharmacy and agrochemistry. Moreover, geraniol could be an ideal gasoline alternative. In this study, recombinant overexpression of geranyl diphosphate synthase and the bottom portion of a foreign mevalonate pathway in Escherichia coli MG1655 produced 13.3mg/L of geraniol. Introduction of Ocimum basilicum geraniol synthase increased geraniol production to 105.2mg/L. However, geraniol production encountered a loss from its endogenous dehydrogenization and isomerization into other geranoids (nerol, neral and geranial). Three E. coli enzymes (YjgB, YahK and YddN) were identified with high sequence identity to plant geraniol dehydrogenases. YjgB was demonstrated to be the major one responsible for geraniol dehydrogenization. Deletion of yjgB increased geraniol production to 129.7mg/L. Introduction of the whole mevalonate pathway for enhanced building block synthesis from endogenously synthesized mevalonate improved geraniol production up to 182.5mg/L in the yjgB mutant after 48h of culture, which was a double of that obtained in the wild type control (96.5mg/L). Our strategy for improving geraniol production in engineered E. coli should be generalizable for addressing similar problems during metabolic engineering.

Production of vanillin by metabolically engineered Escherichia coli

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l−1 was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.

Enhanced Vanillin Production from Recombinant E.coli Using NTG Mutagenesis and Adsorbent Resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl‐CoA synthase) and ech (enoyl‐CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5α harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin‐resistant mutant of NTG‐VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD‐2 resin absorption. The vanillin production of NTG‐VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild‐type strain. When 50% (w/v) of XAD‐2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG‐VR1 was 2.9 g/L, which was 2‐fold higher than that obtained with no use of the resin.

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the worlds principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with PBAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.