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Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l−1 in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.

Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.

Background: Assembly of heterologous metabolic pathways is commonly required to generate microbial cell factories for industrial production of both commodity chemicals (including biofuels) and high-value chemicals. Promoter-mediated transcriptional regulation coordinates the expression of the individual components of these heterologous pathways. Expression patterns vary during culture as conditions change, and this can influence yeast physiology and productivity in both positive and negative ways. Well-characterized strategies are required for matching transcriptional regulation with desired output across changing culture conditions. Results: Here, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production of trans-nerolidol, an acyclic sesquiterpene alcohol, in yeast. The choice of regulatory system significantly affected physiological features (growth and productivity) over batch cultivation. Use of constitutive promoters resulted in poor growth during the exponential phase. Delaying expression of the assembled metabolic modules using the copper-inducible CUP1 promoter resulted in a 1.6-fold increase in the exponential-phase growth rate and a twofold increase in productivity in the post-exponential phase. However, repeated use of the CUP1 promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineered GAL regulatory circuit and a set of four different GAL promoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L-1 using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through the GAL system; final nerolidol titres of 4-5.5 g L-1 were achieved, depending on the conditions. Conclusion: Direct comparison of different transcriptional r

Background: Predictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of "constitutive" and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter. Results: The "constitutive" promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the "constitutive" promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several "constitutive" promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift. Conclusions: The data provides a foundation for predictable and optimised control of gene expression l

To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome. Based on S. cerevisiae CEN. PK 113-5D, we constructed xylose-metabolizing strains where the promoter of xylulokinase gene XKS1 was replaced by TEF1 promoter, PGK1 promoter and HXK2 promoter on the chromosome. We quantitated the transcriptional level of XKS1 gene (accumulated mRNA) and measured the activity of xylulokinase in each stains. Furthermore, we also determined the intracellular level of ATP and evaluated the xylose-fermenting abilities of the engineered strains. The engineered strains exhibited higher expression of xylulokinase than the parental strain at both transcription and enzyme activity levels. The highest xylulokinase activity was observed in the strain whose XKS1 was controlled by PGKlp, and was decreasingly followed by the strains whose XKS1 was controlled by TEF1p, HXK2p and native promoter. The expression level of xylulokinase negatively correlated with intracellular level of ATP and positively correlated with ability of ethanol production from xylose. The highest ethanol yield was 0.35 g/g consumed sugars while the lowest xylitol yield, which was 0.18 g/g consumed xylose, was observed. By promoter replacement, xylulokinase was overexpressed at different levels. In this work, higher expressional level of xylulokinase improved the conversion of xylose to ethanol.