Eui-Sung Choi

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.

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae.

Abstract In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium.

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.

Glycosylation of human α1‐antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Human α1‐antitrypsin (α1‐AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1‐AT secreted in the bakers yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1‐AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1‐AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1‐AT. The human α1‐AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1‐AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1‐AT in S. cerevisiae. The α1‐AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1‐AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1

Abstract To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APHgene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDHpromoter was serially deleted down to the −61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over l50 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.

Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis

A strain of Saccharomyces cerevisiae, KCCM50549, was found to efficiently ferment the inulin-containing carbohydrates in Jerusalem artichoke without acidic or enzymatic pretreatment prior to fermentation. S. cerevisiae KCCM50549 could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke (up to degree of polymerization (DP) of 15), in contrast to the other S. cerevisiae strain such as NCYC625 that fermented the fructo-oligosaccharides with DP of up to around six. Inulin-fermenting S. cerevisiae KCCM50549 produced c.a. 1.6 times more ethanol from Jerusalem artichoke compared with S. cerevisiae NCYC625. Direct ethanol fermentation of Jerusalem artichoke flour at 180 g/L without any supplements or pretreatments by S. cerevisiae KCCM50549 in a 5 L jar fermentor yielded 36.2 g/L of ethanol within 36 h. The conversion efficiency of inulin-type sugars to ethanol was 70% of the theoretical ethanol yield.

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.

Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3)

Abstract When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH.

Mutation of the homologue of GDP‐mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha

A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP‐mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP‐mannose, such as cell wall fragility and defects in N‐ and O‐glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum‐associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177. Copyright

A cell surface display system using novel GPI‐anchored proteins in Hansenula polymorpha

A cell surface display system was developed in yeast Hansenula polymorpha.The four genes HpSED1, HpGAS1, HpTIP1and HpCWP1, encoding glycosylphosphatidyl‐inositol (GPI)‐anchored cell surface proteins from H. polymorpha, were cloned, characterized and evaluated for their efficacies as cell surface display motifs of reporter proteins. Sequence analysis of these genes revealed that each encodes a typical GPI‐anchored protein that is structurally similar to a counterpart gene in S. cerevisiae. The genes showed a high content of serine‐threonine (alanine) and harboured a putative secretion signal in the N‐terminus and the GPI‐attachment signal in the C‐terminus. The surface anchoring efficiency of these putative cell surface proteins was tested by fusion to the C‐terminal of carboxymethylcellulase (CMCase) from Bacillus subtilis. In all cases, high CMCase activities were detected in intact cell fraction, indicating anchoring of CMCase to the cell surface. HpCwp1p, HpGas1p and the 40 C‐terminal amino acids of HpTip1p from H. polymorphaexhibited a comparatively high CMCase surface anchoring efficiency. When these proteins were used as anchoring motifs for surface display of the glucose oxidase (GOD) from Aspergillus niger, most enzyme activity was detected at the cell surface. Fluorescence activated cell sorter (FACS) analysis of cells displaying GOD on the cell surface demonstrated that GOD was well exposed on the cell surface. HpCwp1p showed the highest anchoring efficiency among others. Copyright