Flux-Enabled Exploration of the Role of Sip1 in galactose yeast metabolism

13C metabolic flux analysis (13C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The SIP1 gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of SIP1 using a multi-scale variant of 13C MFA known as 2-Scale 13C metabolic flux analysis (2S-13C MFA). In this study, all strains have the galactose metabolism deactivated (gal1Δ background) so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D gal1Δ background, results in a substantial decrease in pentose phosphate pathway (PPP) flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of SIP1 in the CEN.PK113-7D gal1Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments near a critical sugar ratio that is known to allow galactose to enter the cell. Additionally, we report a number of fluxomic changes associated with these growth rate increases and unexpected flux profile redistributions resulting from deletion of SIP1 in glucose-only medium

Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B₂ Biosynthesis

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although β-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer β-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

Monoterpenes (C10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP production but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes. Biotechnol. Bioeng. 2017;114: 1703-1712. © 2017 Wiley Periodicals, Inc

Renewable production of high density jet fuel precursor sesquiterpenes from <i>Escherichia coli</i>

Abstract Background Aviation fuels are an important target of biofuels research due to their high market demand and competitive price. Isoprenoids have been demonstrated as good feedstocks for advanced renewable jet fuels with high energy density, high heat of combustion, and excellent cold-weather performance. In particular, sesquiterpene compounds (C15), such as farnesene and bisabolene, have been identified as promising jet fuel candidates. Results In this study, we explored three sesquiterpenes—epi-isozizaene, pentalenene and α-isocomene—as novel jet fuel precursors. We performed a computational analysis to calculate the energy of combustion of these sesquiterpenes and found that their specific energies are comparable to commercial jet fuel A-1. Through heterologous MVA pathway expression and promoter engineering, we produced 727.9 mg/L epi-isozizaene, 780.3 mg/L pentalenene and 77.5 mg/L α-isocomene in Escherichia coli and 344 mg/L pentalenene in Saccharomyces cerevisiae. We also introduced a dynamic autoinduction system using previously identified FPP-responsive promoters for inducer-free production and managed to achieve comparable amounts of each compound. Conclusion We produced tricyclic sesquiterpenes epi-isozizaene, pentalenene and α-isocomene, promising jet fuel feedstocks at high production titers, providing novel, sustainable alternatives to petroleum-based jet fuels

Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in engineered microbial systems since its formation is unavoidable in terpene biosynthesis. In this work, we develop an experimental platform to study IPP toxicity in isoprenol-producing Escherichia coli. We first characterize the physiological response to IPP accumulation, demonstrating that elevated IPP levels are linked to growth inhibition, reduced cell viability, and plasmid instability. We show that IPP toxicity selects for pathway "breakage", using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, using multi-omics data, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which slows nutrient uptake, decreases ATP levels, and perturbs nucleotide metabolism. We also observe the extracellular accumulation of IPP and present preliminary evidence that IPP can be transported by E. coli, findings that might be broadly relevant for the study of isoprenoid biosynthesis. Finally, we discover that IPP accumulation leads to the formation of ApppI, a nucleotide analog of IPP that may contribute to observed toxicity phenotypes. This comprehensive assessment of IPP stress suggests potential strategies for the alleviation of prenyl diphosphate toxicity and highlights possible engineering targets for improved IPP flux and high titer isoprenoid production

Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme

Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C-H activation and cyclization of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates