Monoterpenoid biosynthesis in Saccharomyces cerevisiae

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis

Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine.     

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.     

Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae

Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l^?1; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up‐regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform.     

代谢改造酿酒酵母合成萜类化合物的研究进展

萜类化合物是一类种类繁多、功能多样的化合物,部分具有抗癌、增强免疫力等作用,具有良好的生物活性,在食品、保健品以及医疗等领域应用广泛.近年来,随着对萜类化合物生物合成途径研究的深入,研究人员采用代谢工程手段构建了多种萜类产物的高产酿酒酵母工程菌株,部分已经达到或者接近工业化生产水平.因此,采用合成生物学相关技术手段合成...     

Peroxisomes are involved in biotin biosynthesis in Aspergillus and Arabidopsis

Among the eukaryotes only plants and a number of fungi are able to synthesize biotin. Although initial events leading to the biosynthesis of biotin remain largely unknown, the final steps are known to occur in the mitochondria. Here we deleted the Aopex5 and Aopex7 genes encoding the receptors for peroxisomal targeting signals PTS1 and PTS2, respectively, in the filamentous fungus Aspergillus oryzae. In addition to exhibiting defects in the peroxisomal targeting of either PTS1 or PTS2 proteins, the deletion strains also displayed growth defects on minimal medium containing oleic acid as the sole carbon source. Unexpectedly, these peroxisomal transport-deficient strains also exhibited growth defects on minimal medium containing glucose as the sole carbon source that were remediated by the addition of biotin and its precursors, including 7-keto-8-aminopelargonic acid (KAPA). Genome database searches in fungi and plants revealed that BioF protein/KAPA synthase, one of the biotin biosynthetic enzymes, has a PTS1 sequence at the C terminus. Fungal ΔbioF strains expressing the fungal and plant BioF proteins lacking PTS1 still exhibited growth defects in the absence of biotin, indicating that peroxisomal targeting of KAPA synthase is crucial for the biotin biosynthesis. Furthermore, in the plant Arabidopsis thaliana, AtBioF localized to the peroxisomes through recognition of its PTS1 sequence, suggesting involvement of peroxisomes in biotin biosynthesis in plants. Taken together we demonstrate a novel role for peroxisomes in biotin biosynthesis and suggest the presence of as yet unidentified peroxisomal proteins that function in the earlier steps of biotin biosynthesis.     

Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering

In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.     

Biotin biosynthesis, transport and utilization in rhizobia

Biotin, a B-group vitamin, performs an essential metabolic function in all organisms. Rhizobia are alpha-proteobacteria with the remarkable ability to form a nitrogen-fixing symbiosis in combination with a compatible legume host, a process in which the importance of biotin biosynthesis and/or transport has been demonstrated for some rhizobia-legume combinations. Rhizobia have also been used to delimit the biosynthesis, metabolic effects and, more recently, transport of biotin. Molecular genetic analysis shows that an orthodox biotin biosynthesis pathway occurs in some rhizobia while others appear to synthesize the vitamin using alternative pathways. In addition to its well established function as a prosthetic group for biotin-dependent carboxylases, we are beginning to delineate a role for biotin as a metabolic regulator in rhizobia.     

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae

Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.     

Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae

Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (α₂β₂). In this work we evaluated the expression of different combinations of α and β peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group.     

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.     

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.     

酿酒酵母菌葡萄糖信号传导途径研究进展

简要概述了酿酒酵母细胞的葡萄糖信号传导途径的研究进展,总结了葡萄糖的抑制途径和诱导途径.     

酿酒酵母木糖发酵酒精途径工程的研究进展

途径工程(Pathway engineering),被称为第三代基因工程,改变代谢流向,开辟新的代谢途径是途径工程的主要目的。利用途径工程理念,对酿酒酵母（Saccharomyces cerevisiae）代谢途径进行理性设计,以拓展这一传统酒精生产菌的底物范围,使其充分利用可再生纤维质水解物中的各种糖分,是酿酒酵母酒精途径工程的研究热点之一。这里介绍了近年来酿酒酵母以木糖为底物的酒精途径工程的研究进展。     

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.