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Remarkably, a hydrolase from Ideonella sakaiensis 201-F6, termed PETase, exhibits great potential in polyethylene terephthalate (PET) waste management due to it can efficiently degrade PET under moderate conditions. However, its low yield and poor accessibility to bulky substrates hamper its further industrial application. Herein a multigene fusion strategy is introduced for constructing a hydrophobic cell surface display (HCSD) system in Escherichia coli as a robust, recyclable, and sustainable whole-cell catalyst. The truncated outer membrane hybrid protein FadL exposed the PETase and hydrophobic protein HFBII on the surface of E. coli with efficient PET accessibility and degradation performance. E. coli containing the HCSD system changed the surface tension of the bacterial solution, resulting in a smaller contact angle (83.9 ± 2° vs. 58.5 ± 1°) of the system on the PET surface, thus giving a better opportunity for PETase to interact with PET. Furthermore, pretreatment of PET with HCSD showed rougher surfaces with greater hydrophilicity (water contact angle of 68.4 ± 1° vs. 106.1 ± 2°) than the non-pretreated ones. Moreover, the HCSD system showed excellent sustainable degradation performance for PET bottles with a higher degradation rate than free PETase. The HCSD degradation system also had excellent stability, maintaining 73% of its initial activity after 7 days of incubation at 40°C and retaining 70% activity after seven cycles. This study indicates that the HCSD system could be used as a novel catalyst for efficiently accelerating PET biodegradation.

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BACKGROUND Blending d‐lactic acid (d‐LA) with l‐lactic acid can significantly improve the thermostability of polylactic (PLA). Although microbial production of d‐LA under acidic conditions is beneficial for the reduction of production costs, the yield is low due to the acidic toxicity of the source strains. Herein, an Issatchenkia orientalis glycosylphosphatidylinositol‐anchored protein IoGas1, which is required for resistance to low pH and salt stress, was expressed in the YIP‐J‐C‐D‐A1 yeast strain. This strain was integrated with Escherichia coli d‐lactate dehydrogenase gene and several attenuated key pathway genes, including pyruvate decarboxylases (PDC1, PDC6), JEN1 (a monocarboxylate transporter), d‐lactate dehydrogenase1 (DLD1), l‐lactate cytochrome‐c oxidoreductase (CYB2) and alcohol dehydrogenase 1(ADH1). RESULTS The results revealed that the production of d‐LA by the modified strain YIP‐I‐J‐C‐D‐A1 was remarkably improved and reached 85.3 g L–1 d‐LA, with a yield of 0.71 g g–1 and a productivity of 1.20 g L h–1 in batch‐fed fermentation. The d‐LA production of the YIP‐I‐J‐C‐D‐A1 strain (CGMCC2.5785) was further improved by attenuating the ethanol and glycerol pathways. The resulting strain YIP‐A15G12 (CGMCC2.5803) produced 92.0 g L–1 d‐LA with a yield of 0.70 g g–1 and a productivity of 1.21 g L h–1 in batch‐fed fermentation at a final pH of 3.58. CONCLUSION Taken together, the expression of the acid‐resistant gene IoGAS1 in a modified yeast strain can significantly improve the efficiency of producing d‐LA at low pH, which may prove beneficial for the industrial production of the biodegradable material, PLA.

BACKGROUND d‐Lactic acid (d‐LA) is gaining increased attention as it can be applied in the food, pharmaceutical, cosmetic, polylactic acid and textile industries. Acid‐tolerant Saccharomyces cerevisiae is often engineered to produce organic acids. For the pyruvate decarboxylases (PDCs) or alcohol dehydrogenase 1 (ADH1) disrupted strains, the productivity of d‐LA significantly decreases due to the accumulation of acetaldehyde and acetic acid. To overcome the problem, Escherichia coli acetylating acetaldehyde dehydrogenase (A‐ALD) enzyme genes (MHPF and EUTE) (EC 1.2.1.10) were integrated into the d‐LA producing strain YIP‐A15G12 strain (CGMCC2.5803). The function of these genes is to directly convert acetaldehyde into acetyl‐CoA without energy consumption. This strain was integrated with Escherichia coli d‐lactate dehydrogenase and Issatchenkia orientalis glycosylphosphatidylinositol‐anchored protein IoGas1gene and several attenuated key pathway genes, including PDC1, PDC6, ADH1, ADH5, JEN1 (a monocarboxylate transporter), d‐lactate dehydrogenase 1 (DLD1), l‐lactate cytochrome c oxidoreductase (CYB2) and glycerol‐3‐phosphate dehydrogenases (GPD1, GPD2). RESULTS The strain expressions of the A‐ALD enzymes effectively complemented the attenuated acetaldehyde dehydrogenase (ALD) acetyl‐CoA synthetase (ACS) pathway and significantly improved the levels of acetyl‐CoA and the productivity of d‐LA. Finally, the resulting strainYIP‐m‐ald6 (CGMCC2.5826) produced 86.9 g L–1 d‐LA with a yield of 0.77 g g–1 glucose and a volumetric productivity of 1.77 g L–1 h–1 at pH 3.58 under fed‐batch fermentation conditions. CONCLUSION Introducing the E. coli acetyl‐CoA synthesis pathway into the yeast strain can significantly enhance the growth of the strain and improve the d‐LA productivity, as a benefit for d‐LA production in industry. © 2021 Society of Chemical Industry (SCI).

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