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Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineered Pseudomonas taiwanensis VLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g L aq -1 methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g L aq in the in situ extraction phase) at 53% of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.-1 methyl ketones (corresponding to 69.3 g L org -1 in the in situ extraction phase) at 53% of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solvent-sensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis à la carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Besides Aspergillus terreus and Ustilago maydis, Ustilago cynodontis is also known as a natural itaconate producer. U. cynodontis was reported as one of the best itaconate producing species in the family of the Ustilaginaceae, featuring a relatively high pH tolerance in comparison to other smut fungi. However, in contrast to U. maydis, it readily displays filamentous growth under sub-optimal growth conditions. In this study, U. cynodontis is established as efficient pH-tolerant itaconic acid producer through a combination of morphological and metabolic engineering. Deletions of the genes ras2, fuz7, and ubc3 abolished the filamentous growth of U. cynodontis, leading to a stable yeast-like growth under a range of stress-inducing conditions. The yeast-like morphology was also maintained in a pulsed fed batch production of 21 g L -1 itaconic acid and 9.3 g L -1 (S)-2-hydroxyparaconate at a pH of 3.8. The genetic and metabolic basis of itaconic acid production in U. cynodontis was characterized through comparative genomics and gene deletion studies. A hyper-producer strain was metabolically engineered using this knowledge resulting in a 6.5-fold improvement of titer., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpE P290S , AroF-1 P148L , and PheA T310I , and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroG fbr , TyrA fbr ) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering., (© 2017 Wiley Periodicals, Inc.)

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs., Results: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed., Conclusions: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.

(13)C label-based metabolic flux analysis is a powerful technique for the determination of intracellular reaction rates and is used in such different research fields as quantitative physiology, metabolic engineering, and systems biology. Metabolic fluxes can be determined at high quality using (pseudo)-steady-state cultures and advanced mathematical models for data interpretation. Here, we describe a protocol for parallel metabolic flux analysis that consists of downsized microbial (yeast) cultivation, miniaturized sample preparation, and semiautomated analytics and data evaluation. With this protocol dozens of metabolic flux analyses can be carried out in 1 week, thereby enabling for example the analysis of genetic and environmental perturbations on the operation of metabolic networks.

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by (13)C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30-47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively.