Engineering biology for sustainable 1,4-butanediol synthesis

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Biocatalysis is a cornerstone of green chemistry positioned to help tackle the environmental impact of the modern chemical industry. Compared with traditional chemical synthesis, biocatalysis bypasses energy intensive reaction conditions, toxic solvents, and downstream processing requirements to facilitate greener synthetic processes [

1.

  • Sheldon R.A.
  • et al.
The hitchhiker’s guide to biocatalysis: recent advances in the use of enzymes in organic synthesis.

]. Furthermore, when genetically encoded and interfaced with primary metabolic reactions within a microbial host, multistep chemical syntheses can be performed in one-pot via the fermentation of sustainable bio-based feedstocks, rather than from nonrenewable fossil fuels. Combined with the microbial recycling of ‘waste’ feedstock such as lignin and polyethylene terephthalate (PET) plastic into primary metabolic substrates, engineering biology for sustainable synthesis is kickstarting a Golden Age in industrial chemistry.

Previous work in the field targeted platform/bulk and fine chemicals that are known metabolites in nature by reconstituting and enhancing their native biosynthetic pathways in laboratory microbes. However, this approach is limited to a fraction of small molecules used by the chemical industry today and, therefore, engineering biological systems to produce valuable chemicals of non-natural origin is a critical challenge for the field moving forward.

Emerging strategies in this area include the use of refactored biosynthetic pathways, the evolution of enzymes to perform non-native chemistry, the computational design of new-to-nature enzymes, and the repurposing of non-enzymatic catalysts to operate in concert with engineered metabolic pathways (termed ‘biocompatible chemistry’). A seminal example by Yim et al. stands as one of the earliest successes in this field, combining in silico pathway design with metabolic engineering to achieve high-level bioproduction of the unnatural chemical 1,4-butanediol (BDO) from glucose [

2.

  • Yim H.
  • et al.
Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.

].

BDO is a prevalent petrochemical used to synthesise important materials (e.g., spandex) and industrial products (e.g., tetrahydrofuran) [

3.

  • Zhu Y.
  • et al.
Bio-based 1,4-butanediol and tetrahydrofuran synthesis: perspective.

]. Yet traditional manufacturing routes rely on unsustainable fossil resources (acetylene or n-butane) and energy-intensive chemical processes. Engineering biology to enable BDO production from sustainable feedstocks by fermentation is therefore an important challenge.

The study began by identifying suitable ‘reaction operators’ using in-house SimPheny Biopathway Predictor software, resulting in ca. 10 000 feasible routes to BDO via known metabolic chemistry (not necessarily catalysed by known enzymes) from central metabolic intermediates. Iterative ranking based on attributes, including pathway length, number of non-native steps, unfavourable thermodynamics, and maximum theoretical yield, consolidated the available pathway designs to around 10% of the initial hits. Manual curation based on known enzymatic chemistry targeting the shortest number of steps from central metabolism led the authors to two pathway designs pivoting around the intermediate 4-hydroxybutyrate (4-HB), which could be subsequently reduced to BDO by two successive NAD(P)H dependent enzymatic reductions through 4-hydroxybutyryl-CoA. Two routes to 4-HB via succinyl semialdehyde were proposed: from succinate, via succinyl-CoA catalysed by a succinyl-CoA synthase and CoA-dependent dehydrogenase; and from α-ketoglutarate, catalysed by a α-ketoacid decarboxylase (Figure 1). The 4-HB biosynthesis and subsequent reduction pathways were constructed and validated as two separate ‘modules’ in different Escherichia coli MG1655 lacIQ+ strains before being combined into a single strain. The result was 1.3 mM BDO after 40 h incubation, which, whilst low, marked the first instance of BDO biosynthesis entirely from central metabolites in a microorganism.

Figure 1

Figure 1Metabolic engineering of Escherichia coli for sustainable 1,4-butanediol synthesis from carbohydrate feedstocks.

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Abbreviations: C. beijerinckii, Clostridium beijerinckii; 4-HB, 4-hydroxybutyrate; M. bovis, Mycobacterium bovis; P. gingivalis, Porphyromonas gingivalis.

Competing pathways for carbon flux were eliminated using the OptKnock algorithm by identifying multiple knockouts (ΔadhE, Δpfl, Δldh, and Δmdh) that coupled maximum BDO production to cell growth, predicting 0.37 g/g yield under anaerobic conditions by blocking native fermentation pathways to ethanol, formate, lactate, and succinate and driving BDO production to balance cellular redox. In practice, the knockouts severely reduced cell fitness, with the ΔadhEΔpflΔldh intermediate strain unable to grow under anaerobic conditions; likely due to rediverted carbon flux through the native pyruvate dehydrogenase, which displayed reduced activity under the experimental conditions. Although overcome by replacing the limiting subunit (lpdA) with a functional homolog from Klebsiella pneumoniae, this barrier highlights a general limitation of in silico prediction models to account for off-pathway effects in complex metabolic systems yet how these can be mitigated via rational pathway engineering. The recovered strain displayed improved BDO production but only grew under microaerobic conditions and produced significant quantities of unwanted side-products such as acetate, pyruvate, ethanol, stalled pathway intermediates, and γ-butyrolactone (GBL) formed by spontaneous cyclisation of 4-hydroxybutyryl-CoA. To address this, the authors prioritised flux through BDO synthesis by knocking out arcA and mdh to alleviate transcriptional repression of several aerobically expressed oxidative TCA cycle genes and block entry into the reductive TCA cycle, respectively. Aided by expression of an NADH-insensitive citrate synthase mutant GltA(R163L) to increase TCA cycle intermediates, the resulting strain achieved 95% carbon flux to BDO and a titre of ca. 13 mM after 40 h. A final evaluation of the engineered strain by pulse-labelling with 13C-glucose indicated a late-stage 4-HB bottleneck resulting from inefficient alcohol and aldehyde dehydrogenase activity in the downstream pathway. A codon-optimised aldehyde dehydrogenase from Clostridium beijerinckii was implemented that displayed improved reduction of 4-HB-CoA to BDO in concert with endogenous E. coli reductase enzymes, with minimal competing background reduction of acetaldehyde to ethanol.

Finally, the authors expanded the pathway to encompass alternative carbohydrate feedstocks. By introducing a sucrose-utilisation operon into the E. coli genome, comparable BDO titres were achieved from sucrose, xylose, and impure mixed biomass hydrolysate, the latter providing attractive flexibility from a bioprocess perspective when considering the relatively high cost and environmental impact of using glucose as a feedstock at scale [

4.

  • Salim I.
  • et al.
Assessing the environmental sustainability of glucose from wheat as a fermentation feedstock.

].

The final engineered strain produced 18 g/l BDO from 20 g/l glucose over a 5-day fermentation. This achievement at such an early stage in the development of metabolic engineering, synthetic biology, and industrial biotechnology for sustainable chemical synthesis is remarkable and sets a benchmark for what can be achieved through predictive in silico metabolic modelling, rational metabolic engineering, and de novo biochemical pathway design. Titres achieved here also eclipsed those reported for the overproduction of known metabolites in E. coli. For example, isoprene production was achieved in 2011 by fermentation of glucose at only 314 mg/l scale [

5.

  • Zhao Y.
  • et al.
Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway.

].

Continued efforts have built upon this seminal work to design and commercialise an E. coli strain capable of producing 125 g/l BDO from renewable feedstocks [

6.

  • Burgard A.
  • et al.
Development of a commercial scale process for production of 1,4-butanediol from sugar.

]. Industrial use required extensive host strain and pathway engineering (>50 further genetic edits), in addition to genetic reconfigurations essential for scale-up. Consistent with the original report, new modifications were introduced by rational design aided by 13C flux and transcriptomic analyses. Knockouts ΔsadΔgabD prevented flux moving back into the TCA cycle by limiting succinate semialdehyde conversion to succinate at high 4-HB concentrations, increasing BDO titres from 18 g/l to 29 g/l [

7.

  • Barton N.R.
  • et al.
An integrated biotechnology platform for developing sustainable chemical processes.

]. Further strain improvements focussed on resolving competing side reactions and sources of both rate-limitation and by-product formation. Flux analysis pinpointed a rate-limiting bottleneck in the pathway during the final reduction steps from 4-HB to BDO once optimisations reached an 80–90 g/l scale. As such, Ald and Cat2 enzymes were engineered via directed evolution to suppress competing Ac-CoA reduction pathways and overcome product inhibition at high BDO titres, respectively. Once implemented, the evolved enzymes reduced 4-HB titres by 75% whilst increasing BDO titres by 20% to 110 g/l [

6.

  • Burgard A.
  • et al.
Development of a commercial scale process for production of 1,4-butanediol from sugar.

]. To address by-product formation, ppc expression was increased to improve overall TCA cycle flux from pyruvate. Additionally, deleting ATP inefficient electron transport chain components (ΔndhΔappBCΔcydAB), thioesterases (ΔybgCΔtesB), and acetate kinase/phosphotransacetylase (ΔackA-pta) genes decreased CO2, GBL, and acetate formation, respectively. Such modifications were responsible for reaching a final BDO titre of 125 g/l.

To improve industrial uptake, a final reconfiguration of genetic parts was required to improve genetic stability and reduce processing costs. This was achieved by chromosomal integration of the plasmid-encoded pathway genes under constitutive promoters and the removal of unstable cryptic prophage. Further genetic stability was conferred by mutating phage-associated receptor genes (tonA/lamb) and removing duplicated genomic regions. This stabilised strain was then subjected to final growth media and bioprocess optimisations to minimise downstream processing steps, affording the final BDO production strain for commercialisation.

The success of this work serves as a milestone in the field of microbial biocatalysis and demonstrates the feasibility of engineered de novo biosynthetic pathways at scale. Together with continued enzyme discovery, increasingly rapid enzyme engineering, and synthetic biology methods, there is undoubtedly a bright future ahead for this field as the need and demand for more sustainable chemical manufacturing technologies becomes a reality.

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