Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production

Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45 mg/L to 71 mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71 mg/L to 140 mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140 mg/L to 330 mg/L). Through fed-batch fermentation using resting cells, over 1.1 g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

Geranyl diphosphate synthase: An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli

The expression level of geranyl diphosphate synthase (GPPS) was suspected to play a key role for geraniol production in recombinant Escherichia coli harboring an entire mevalonate pathway operon and a geraniol synthesis operon. The expression of GPPS was optimized by using ribosomal binding sites (RBSs) designed to have different translation initiation rates (TIRs). The RBS strength in TIR window of 500 arbitrary unit (au)–1400 au for GPPS appears to be suitable for balancing the geraniol biosynthesis pathway in this study. With the TIR of 500 au, the highest production titer of geraniol was obtained at a level of 1119 mg/L, which represented a 6-fold increase in comparison with the previous titer of 183 mg/L. The TIRs of GPPS locating out of range of the optimal window (500–1400 au) caused significant decreases of cell growth and geraniol production. It was suspected to result from metabolic imbalance and plasmid instability in geraniol production by inappropriate expression level of GPP synthase. Our results collectively indicated GPPS as an important regulation point in balancing a recombinant geraniol synthesis pathway. The GPPS-based regulation approach could be applicable for optimizing microbial production of other monoterpenes.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways

Fatty acid short-chain esters (FASEs) are biodiesels that are renewable, nontoxic, and biodegradable biofuels. A novel approach for the biosynthesis of FASEs has been developed using metabolically-engineered E. coli through combination of the fatty acid and 2-keto acid pathways. Several genetic engineering strategies were also developed to increase fatty acyl-CoA availability to improve FASEs production. Fed-batch cultivation of the engineered E. coli resulted in a titer of 1008 mg/L FASEs. Since the fatty acid and 2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in a variety of microorganisms to produce various FASEs from cheap and readily-available, renewable, raw materials such as sugars and cellulose in the future.     

Improvement of ganoderic acid production by fermentation of Ganoderma lucidum with cellulase as an elicitor

Two-stage cultivation of Ganoderma lucidum was performed for the enhanced production of ganoderic acid (GA). Cellulase was identified to be an effective elicitor for the improvement of GA production, and GA titer reached 1334.5 mg/l compared to the control (779.6 mg/l) using lactose as the substrate without cellulase addition. Loading of 5 mg/l cellulase on day 3 resulted in the maximal GA titer of 1608 mg/l. To our knowledge, this is the first time that cellulase was used as the elicitor to enhance GA production. Submerged fermentation in a 2.0-l bioreactor was also conducted with cellulase as the elicitor, and as a result the maximal GA titer of 1252.7 mg/l was obtained on day 12. This is so far the best GA production obtained in submerged fermentation of G. lucidum.     

Identification of a marine NADPH-dependent aldehyde reductase for chemoselective reduction of aldehydes

A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was overexpressed in Escherichia coli. The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde reductase. The kinetic parameters K_m and k_cat of OsAR were 0.89 ± 0.08 mM and 11.07 ± 0.99 s⁻¹ for benzaldehyde, 0.04 ± 0.01 mM and 6.05 ± 1.56 s⁻¹ for NADPH, respectively. This enzyme exhibited high activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application potential of this marine enzyme in such selective reduction of synthetic importance.     

Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO₃ combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO₃, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.     

Production of C5 carboxylic acids in engineered Escherichia coli

Valeric acid and 2-methylbutyric acid serve as chemical intermediates for a variety of applications such as plasticizers, lubricants and pharmaceuticals. The commercial process for their production uses toxic intermediates like synthesis gas and relies on non-renewable petroleum-based feedstock. In this work, synthetic metabolic pathways were constructed in Escherichia coli for the renewable production of these chemicals directly from glucose. The native leucine and isoleucine biosynthetic pathways in E. coli were expanded for the synthesis of valeric acid and 2-methylbutyric acid (2MB) respectively by the introduction of aldehyde dehydrogenases and 2-ketoacid decarboxylases. Various aldehyde dehydrogenases and 2-ketoacid decarboxylases were investigated for their activities in the constructed pathways. Highest titers of 2.59 g/L for 2-mthylbutyric acid and 2.58 g/L for valeric acid were achieved in shake flask experiments through optimal combinations of these enzymes. This work demonstrates the feasibility of renewable production of these high volume aliphatic carboxylic acids.     

Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2 YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.     

Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae

Microbial production of higher alcohols from renewable feedstock has attracted intensive attention thanks to its potential as a source for next-generation gasoline substitutes. Here we report the discovery, characterization and engineering of an endogenous 1-butanol pathway in Saccharomyces cerevisiae. Upon introduction of a single gene deletion adh1Δ, S. cerevisiae was able to accumulate more than 120 mg/L 1-butanol from glucose in rich medium. Precursor feeding, ¹³C-isotope labeling and gene deletion experiments demonstrated that the endogenous 1-butanol production was dependent on catabolism of threonine in a manner similar to fusel alcohol production by the Ehrlich pathway. Specifically, the leucine biosynthesis pathway was engaged in the conversion of key 2-keto acid intermediates. Overexpression of the pathway enzymes and elimination of competing pathways achieved the highest reported 1-butanol titer in S. cerevisiae (242.8 mg/L).     

Direct bioconversion of d-xylose to 1,2,4-butanetriol in an engineered Escherichia coli

The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L⁻¹ BT from 10 g L⁻¹ d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability.     

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.     

Production of ω-hydroxyundec-9-enoic acid and n-heptanoic acid from ricinoleic acid by recombinant Escherichia coli-based biocatalyst

ω-Hydroxyundec-9-enoic acid and n-heptanoic acid are valuable building blocks for the production of flavors and antifungal agents as well as bioplastics such as polyamides and polyesters. However, a biosynthetic process to allow high productivity and product yield has not been reported. In the present study, we engineered an Escherichia coli-based biocatalytic process to efficiently produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid from a renewable fatty acid (i.e., ricinoleic acid). Expression systems for catalytic enzymes (i.e., an alcohol dehydrogenase of Micrococcus luteus, a Baeyer–Villiger monooxygenase of Pseudomonas putida KT2440, an esterase of Pseudomonas fluorescens SIK WI) and biotransformation conditions were investigated. Biotransformation during stationary growth phase of recombinant E. coli in a bioreactor allowed to produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid at a rate of 3.2 mM/h resulting in a final product concentration of ca. 20 mM. The total amount of ω-hydroxyundec-9-enoic acid and n-heptanoic acid produced reached 6.5 g/L (4.0 g/L of ω-hydroxyundec-9-enoic acid and 2.5 g/L of n-heptanoic acid). These results indicate that the high value carboxylic acids ω-hydroxyundec-9-enoic acid and n-heptanoic acid can be produced from a renewable fatty acid via whole-cell biotransformation.     

Engineering Escherichia coli to convert acetic acid to free fatty acids

Fatty acids (FAs) are promising precursors of advanced biofuels. This study investigated conversion of acetic acid (HAc) to FAs by an engineered Escherichia coli strain. We combined established genetic engineering strategies including overexpression of acs and tesA genes, and knockout of fadE in E. coli BL21, resulting in the production of ~1 g/L FAs from acetic acid. The microbial conversion of HAc to FAs was achieved with ~20% of the theoretical yield. We cultured the engineered strain with HAc-rich liquid wastes, which yielded ~0.43 g/L FAs using waste streams from dilute acid hydrolysis of lignocellulosic biomass and ~0.17 g/L FAs using effluent from anaerobic-digested sewage sludge. ¹³C-isotopic experiments showed that the metabolism in our engineered strain had high carbon fluxes toward FAs synthesis and TCA cycle in a complex HAc medium. This proof-of-concept work demonstrates the possibility for coupling the waste treatment with the biosynthesis of advanced biofuel via genetically engineered microbial species.