Next-generation feedstocks methanol and ethylene glycol and their potential in industrial biotechnology.

Microbial fermentation processes are expected to play an important role in reducing dependence on fossil-based raw materials for the production of everyday chemicals. In order to meet the growing demand for biotechnological products in the future, alternative carbon sources that do not compete with human nutrition must be exploited. The chemical conversion of the industrially emitted greenhouse gas CO2 into microbially utilizable platform chemicals such as methanol represents a sustainable strategy for the utilization of an abundant carbon source and has attracted enormous scientific interest in recent years. A relatively new approach is the microbial synthesis of products from the C2-compound ethylene glycol, which can also be synthesized from CO2 and non-edible biomass and, in addition, can be recovered from plastic waste. Here we summarize the main chemical routes for the synthesis of methanol and ethylene glycol from sustainable resources and give an overview of recent metabolic engineering work for establishing natural and synthetic microbial assimilation pathways. The different metabolic routes for C1 and C2 alcohol-dependent bioconversions were compared in terms of their theoretical maximum yields and their oxygen requirements for a wide range of value-added products. Assessment of the process engineering challenges for methanol and ethylene glycol-based fermentations underscores the theoretical advantages of new synthetic metabolic routes and advocates greater consideration of ethylene glycol, a C2 substrate that has received comparatively little attention to date.

Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol.

Ethylene glycol is an attractive two-carbon alcohol substrate for biochemical product synthesis as it can be derived from CO2 or syngas at no sacrifice to human food stocks. Here, we disclose a five-step synthetic metabolic pathway enabling the carbon-conserving biosynthesis of the versatile platform molecule 2,4-dihydroxybutyric acid (DHB) from this compound. The linear pathway chains ethylene glycol dehydrogenase, D-threose aldolase, D-threose dehydrogenase, D-threono-1,4-lactonase, D-threonate dehydratase and 2-oxo-4-hydroxybutyrate reductase enzyme activities in succession. We screen candidate enzymes with D-threose dehydrogenase and D-threonate dehydratase activities on cognate substrates with conserved carbon-centre stereochemistry. Lastly, we show the functionality of the pathway by its expression in an Escherichia coli strain and production of 1 g L-1 and 0.8 g L-1 DHB from, respectively, glycolaldehyde or ethylene glycol.

In vivo implementation of a synthetic metabolic pathway for the carbon-conserving conversion of glycolaldehyde to acetyl-CoA.

Ethylene glycol (EG) derived from plastic waste or CO2 can serve as a substrate for microbial production of value-added chemicals. Assimilation of EG proceeds though the characteristic intermediate glycolaldehyde (GA). However, natural metabolic pathways for GA assimilation have low carbon efficiency when producing the metabolic precursor acetyl-CoA. In alternative, the reaction sequence catalyzed by EG dehydrogenase, d-arabinose 5-phosphate aldolase, d-arabinose 5-phosphate isomerase, d-ribulose 5-phosphate 3-epimerase (Rpe), d-xylulose 5-phosphate phosphoketolase, and phosphate acetyltransferase may enable the conversion of EG into acetyl-CoA without carbon loss. We investigated the metabolic requirements for in vivo function of this pathway in Escherichia coli by (over)expressing constituting enzymes in different combinations. Using 13C-tracer experiments, we first examined the conversion of EG to acetate via the synthetic reaction sequence and showed that, in addition to heterologous phosphoketolase, overexpression of all native enzymes except Rpe was required for the pathway to function. Since acetyl-CoA could not be reliably quantified by our LC/MS-method, the distribution of isotopologues in mevalonate, a stable metabolite that is exclusively derived from this intermediate, was used to probe the contribution of the synthetic pathway to biosynthesis of acetyl-CoA. We detected strong incorporation of 13C carbon derived from labeled GA in all intermediates of the synthetic pathway. In presence of unlabeled co-substrate glycerol, 12.4% of the mevalonate (and therefore acetyl-CoA) was derived from GA. The contribution of the synthetic pathway to acetyl-CoA production was further increased to 16.1% by the additional expression of the native phosphate acyltransferase enzyme. Finally, we demonstrated that conversion of EG to mevalonate was feasible albeit at currently extremely small yields.

A New Synthetic Pathway for the Bioproduction of Glycolic Acid From Lignocellulosic Sugars Aimed at Maximal Carbon Conservation.

Glycolic acid is a two-carbon α-hydroxy acid with many applications in industrial sectors including packaging, fine chemistry, cosmetics, and pharmaceutics. Currently, glycolic acid is chemically manufactured from fossil resources. This chemical mode of production is raising some concerns regarding its use in health for personal care. Microbial production of GA stands as a remarkable challenge to meet these concerns, while responding to the increasing demand to produce bio-sourced products from renewable carbon resources. We here report on the design and expression of a novel non-natural pathway of glycolic acid in E. coli. The originality of this new pathway, termed "glycoptimus" relies on two pillars. On the one hand, it requires the overexpression of three naturally occurring E. coli genes, namely kdsD encoding a D-arabinose-5-P isomerase, fsaA encoding a class 1 aldolase that cleaves D-arabinose-5-P into glyceraldehyde-3-P and glycolaldehyde, and aldA coding for an aldehyde dehydrogenase that oxidizes glycoladehyde in glycolate. These three genes constitute the "glycoptimus module." On the other hand, the expression of these genes together with a reshaping of the central carbon metabolism should enable a production of glycolic acid from pentose and hexose at a molar ratio of 2.5 and 3, respectively, which corresponds to 50% increase as compared to the existing pathways. We demonstrated the 'in vivo' potentiality of this pathway using an E. coli strain, which constitutively expressed the glycoptimus module and whose carbon flow in glycolysis was blocked at the level of glyceraldehyde-3-P dehydrogenase reaction step. This engineered strain was cultivated on a permissive medium containing malate and D-glucose. Upon exhaustion of malate, addition of either D-glucose, D-xylose or L-arabinose led to the production of glycolic acid reaching about 30% of the maximum molar yield. Further improvements at the level of enzymes, strains and bioprocess engineering are awaited to increase yield and titer, rendering the microbial production of glycolic acid affordable for a cost-effective industrial process.

Construction of a synthetic pathway for the production of 1,3-propanediol from glucose.

In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.

Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway.

A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s-1), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (kcat/Km) between natural and synthetic OHB substrates (84 and 31 s-1 mM-1, respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis.

Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering.

BACKGROUND: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. RESULTS: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C4) was isolated and characterized in 60% undetoxified HSSL. C4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C4. CONCLUSION: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C4 may represent a major advantage for the production of bioethanol using HSSL as substrate.