Engineering Saccharomyces cerevisiae with enhanced supply of precursor metabolites for efficient production of fuels and chemicals

Saccharomyces cerevisiae has been widely established as a platform microorganism for industrial production of a wide variety of products including but not limited to ethanol, organic acids, amino acids, enzymes, and therapeutic proteins, owing to its high tolerance to harsh industrial conditions, such as low pH, high sugar concentration and growth inhibitors in the biomass hydrolysate, as well as resistance to phage infection. However, one of the major challenges for high level production of value-added compounds other than ethanol is the strong fluxes for ethanol formation even under aerobic condition, a phenomenon known as Crabtree effect. Notably, a wide variety of products with industrial interest are derived from a few precursor metabolites, such as 2,3-butanediol (BDO) and iso-butanol from pyruvate, n-butanol, polyhydroxybutyrate, and isoprenoids from acetyl-CoA, and free fatty acids, fatty alcohols, and fatty acid ethyl esters from long chain acyl-CoAs. Therefore, metabolic engineering and synthetic biology approaches were applied to engineer S. cerevisiae with enhanced supply of these precursor metabolites for efficient production of fuels and chemicals. More importantly, these engineering efforts can be integrated to construct a platform yeast cell factory, since pyruvate is the direct precursor for acetyl-CoA generation and enhanced acetyl-CoA levels will provide a driving force for acyl-CoAs pool engineering. To construct a pyruvate overproducing yeast strain, three structural genes encoding pyruvate decarboxylases, PDC1, PDC5, and PDC6 were deleted to completely eliminate ethanol production. Followed by overexpression of MTH1 and adaptive evolution, the resultant Pdc- yeast strain grew on glucose as the sole carbon source with pyruvate as the major product. Subsequent introduction of a BDO biosynthetic pathway resulted in the production of BDO at a yield over 70% of the theoretical value and a titer higher than 100 g/L using fed-batch fermentation. To engineer acetyl-CoA pool in S. cerevisiae, alternative acetyl-CoA biosynthesis routes were characterized using synthetic biology approaches and metabolic engineering was applied to redirect the metabolic fluxes towards acetyl-CoA biosynthesis. Acetyl-CoA biosynthetic pathways from Escherichia coli (pyruvate dehydrogenase, PDH) and Yarrowia lipolytica (ATP-dependent citrate lyase, ACL) were found to enable the growth of the Acs- (acs1Δ acs2Δ) strain on glucose as the sole source. To construct a functional PDH in the cytosol of yeast, different lipoylation pathways were introduced and engineered. Besides the naturally existing scavenging pathway and de novo biosynthetic pathway, we also designed a semi-synthetic lipoylation pathway based on the acyl-ACP synthetase (AasS). The scavenging pathway resulted in a functional PDH that enabled the growth of Acs- strain to a similar level of the wild-type strain. The de novo biosynthetic lipoylation pathway was hindered by the difficulty in reconstituting a functional type II fatty acid synthase (FAS) in the cytosol to provide the precursor (octanoyl-ACP) for protein lipoylation. The introduction of the semi-synthetic lipoylation pathway (VhAasS-cytoACP1-cytoPPT2) resulted in functional PDH and rescued the growth of the Acs- strain when octanoic acid was supplementated. Based on these results, a de novo biosynthetic lipoylation pathway was re-designed and proposed. Then these heterologous biosynthetic pathways were combined with host engineering to design and construct acetyl-CoA overproducing yeast strains. By deleting ADH1, ADH4, GPD1, and GPD2 involved in ethanol and glycerol formation, the glycolytic flux was redirected towards this precursor metabolite, resulting in a 4 fold improvement in n-butanol production. Subsequent introduction of alternative acetyl-CoA biosynthetic pathways, the production of n-butanol was further increased. Although significant improvement of n-butanol production was achieved, the final titer was still much lower than that of ethanol and the resultant yeast strains suffered from accumulation of the toxic intermediates such as acetaldehyde and acetate. Therefore, more efforts were put into the engineering of acetyl-CoA pools in the Pdc- strain, where ethanol production was completely eliminated and pyruvate was accumulated to high levels. To engineer acyl-CoAs levels in S. cerevisiae, a new biosynthesis platform based on the reversal of β-oxidation cycle was constructed using synthetic biology approaches. Compared with the conical FAS, which is ATP, ACP, and NADPH dependent, the reversed β-oxidation pathway is featured for its ATP and ACP independence and CoA and NADH dependence. The energetic benefits of ATP independence and availability of CoA and NADH versus ACP and NADPH confer advantages for acyl-CoAs biosynthesis. Reversed β-oxidation pathways were constructed and found to produce n-butanol, decanoic acid, and ethyl decanoate, indicating the functional reversal of β-oxidation cycle at least 4 turns. To facilitate the engineering in S. cerevisiae, new synthetic biology tools was also developed. First, plasmids with step-wise increased copy numbers (20-100 copies per cell) were constructed by engineering the expression level of selection marker proteins, including both auxotrophic and dominant markers. More importantly, the copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, a new and simplest synthetic biology approach named induced pathway optimization by antibiotic doses (iPOAD) was developed to optimize the performance of multi-gene biosynthetic pathways by different combination of antibiotic concentrations in S. cerevisiae. To demonstrate this approach, iPOAD was applied to optimize the lycopene and n-butanol biosynthetic pathways, with the production of lycopene and n-butanol increased by 10- and 100-fold, respectively. Finally, the iPOAD optimized pathway was integrated to chromosomes to increase the strain stability and eliminate the requirement of antibiotic supplementation, by taking advantage of the iPOAD and CRISPR-Cas9 technologies for multiplex pathway integration

Preparative Scale Production of Functional Mouse Aquaporin 4 Using Different Cell-Free Expression Modes

The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration

Improving advanced biofuels production in Saccharomyces cerevisiae via protein engineering and synthetic biology approaches

Saccharomyces cerevisiae has been widely established as a platform microorganism for industrial production of fuels and chemicals from lignocellulosic biomass. However, we are still encountering several challenges to achieve cost-effective production of cellulosic biofuels, such as the sequential utilization of sugar mixtures caused by glucose repression and the low efficiency of synthesizing fatty acid derived advanced biofuels. In this thesis, we aim to improve the production of advanced biofuels in S. cerevisiae using protein engineering and synthetic biology approaches. To overcome glucose repression, a cellobiose utilization pathway consisting of a cellodextrin transporter and a β-glucosidase was introduced into S. cerevisiae to allow co-fermentation of mixed sugars. However, the utilization of cellobiose was still much lower than that of glucose, and the uptake of cellobiose was considered the rate-limiting step for cellobiose fermentation. Therefore, directed evolution of the cellodextrin transporter (CDT2) was carried out to improve the uptake activity and thus cellobiose fermentation. After three rounds of directed evolution, both the specific activity and transporter expression level of CDT2 were increased, leading to 2.15 fold improvement of the cellobiose uptake activity. Using high cell density fermentation under anaerobic conditions, the best mutant conferred 2.67 fold and 4.96 fold increase in the cellobiose consumption rate and ethanol productivity, respectively. Besides bioethanol, we are also interested in advanced biofuels that have similar properties to current transportation fuels. Since most of the advanced biofuels are derived from fatty acids, efficient production is limited by the low efficiency and high energy input of the fatty acid biosynthesis pathways. Reversal of β-oxidation cycles has been engineered to produce a series of fatty acid derived fuels and chemicals in Escherichia coli. Thus, another goal of this thesis is to construct a new fatty acid biosynthesis platform based on the reversal of β-oxidation cycles for advanced biofuel production in S. cerevisiae. Using synthetic biology approaches, reversed β-oxidation pathways were constructed and characterized to be able to produce butanol, indicating the functional reversal of β-oxidation cycles. Future work will focus on expanding this platform to produce other fatty acid derived advanced biofuels such as biodiesel (fatty acid ethyl ester, FAEE)

Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli

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Reversal of the β‑Oxidation Cycle in <i>Saccharomyces cerevisiae</i> for Production of Fuels and Chemicals

Functionally reversing the β-oxidation cycle represents an efficient and versatile strategy for synthesis of a wide variety of fuels and chemicals. However, due to the compartmentalization of cellular metabolisms, reversing the β-oxidation cycle in eukaryotic systems remains elusive. Here, we report the first successful reversal of the β-oxidation cycle in <i>Saccharomyces cerevisiae</i>, an important cell factory for large-scale production of fuels and chemicals. After extensive gene cloning and enzyme activity assays, a reversed β-oxidation pathway was functionally constructed in the yeast cytosol, which led to the synthesis of <i>n</i>-butanol, medium-chain fatty acids (MCFAs), and medium-chain fatty acid ethyl esters (MCFAEEs). The resultant recombinant strain provides a new broadly applicable platform for synthesis of fuels and chemicals in <i>S. cerevisiae</i>

Functional Reconstitution of a Pyruvate Dehydrogenase in the Cytosol of Saccharomyces cerevisiae through Lipoylation Machinery Engineering

Acetyl-CoA is a key precursor for the biosynthesis of a wide range of fuels, chemicals, and value-added compounds, whose biosynthesis in Saccharomyces cerevisiae involves acetyl–CoA synthetase (ACS) and is energy intensive. Previous studies have demonstrated that functional expression of a pyruvate dehydrogenase (PDH) could fully replace the endogenous ACS-dependent pathway for cytosolic acetyl-CoA biosynthesis in an ATP-independent manner. However, the requirement for lipoic acid (LA) supplementation hinders its wide industrial applications. In the present study, we focus on the engineering of a <i>de novo</i> synthetic lipoylation machinery for reconstitution of a functional PDH in the cytosol of yeast. First, a LA auxotrophic yeast strain was constructed through the expression of the Escherichia coli PDH structural genes and a lipoate–protein ligase gene in an ACS deficient (<i>acs1</i>Δ <i>acs2</i>Δ) strain, based on which an <i>in vivo</i> acetyl-CoA reporter was developed for following studies. Then the <i>de novo</i> lipoylation pathway was reconstituted in the cytosol of yeast by coexpressing the yeast mitochondrial lipoylation machinery genes and the E. coli type II fatty acid synthase (FAS) genes. Alternatively, an unnatural <i>de novo</i> synthetic lipoylation pathway was constructed by combining the reversed β-oxidation pathway with an acyl-ACP synthetase gene. To the best of our knowledge, reconstitution of natural and unnatural <i>de novo</i> synthetic lipoylation pathways for functional expression of a PDH in the cytosol of yeast has never been reported. Our study has laid a solid foundation for the construction and further optimization of acetyl-CoA overproducing yeast strains

Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS) has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors) to speed up design-build-test (DBT) cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biolog