Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone–butanol–ethanol production

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n -butanol production. The combined expression of StSUT1 , encoding a sucrose transporter from potato ( Solanum tuberosum ), and SUC2 , encoding a sucrose invertase from Saccharomyces cerevisiae , remarkably enhanced n -butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium . The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

De novo biosynthesis of C-arabinosylated flavones by utilization of indica rice C-glycosyltransferases

Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

De Novo Biosynthesis of C-Arabinosylated Flavones by Utilization of Indica Rice C-Glycosyltransferases

Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyltransferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20~110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

The Role of YggS in Vitamin B6 Homeostasis in Salmonella enterica Is Informed by Heterologous Expression of Yeast SNZ3

ABSTRACT YggS (COG0325) is a pyridoxal 5′-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli. Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5′-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5′-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae. The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence. IMPORTANCE Pyridoxal 5′-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.

Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.

In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

Background: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway.Results: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologousβ-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11~8.51 folds and 3.0~9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg/g DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains.Conclusions: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

Background : Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganisms is still limited by the poor efficiency of the heterologous pathway. Results: To address the bottleneck of astaxanthin yield in microbes, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modules of β-carotene ketolase ( crtW ) and hydroxylase ( crtZ ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05 mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination. Conclusions: Recombination of heterologous modules by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high-throughput screening of heterologous pathways by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW , but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathways.

In vitro and In vivo recombination of heterologous modular for improving biosynthesis of astaxanthin in yeast

Bacground: Astaxanthin is a kind of tetraterpene with strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganism is still limited by the poor efficiency of heterologous pathway.Results: To address the bottleneck of astaxanthin yield in microbe, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modular ofβ-carotene ketolase (crtW) and hydroxylation (crtZ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination.Conclusions: Recombination of heterologous modular by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high throughput screening of heterologous pathway by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW, but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathway.